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Erratum: Expression of a large family of POU-domain regulatory genes in mammalian brain development
Abstract
Nature 34O, 35-42 (1989). IN this Article there is an error in the amino acid sequence encoded by Tst-1 gene (hybridizing to 7.6, 4.4, 3.2 and 2.4Kb brain mRNAs) reported in Fig. 16, two residues before the end of POU-specific domain. The correct sequence is ... WLEETD... instead ... WLEE AD.... D
... Moreover, the question arises why all other OCT factors do not have reprogramming function even though they harbor profound similarities with OCT4 at the level of both primary and secondary structure. Like all OCT factors, OCT4 harbors a DNA binding domain (DBD) and two intrinsic TADs (13)(14)(15). Few available studies have clearly attributed the importance of OCT4 DBD toward the reprogramming process, which is required to exert its pioneering function by binding to target gene loci in closed chromatin and determine its reprogramming competence (3,5,(16)(17)(18)(19). However, how transactivation of OCT4's target genes is conferred is largely unknown. ...
... Intrinsic properties within POU III factors that are essential or detrimental to reprogramming Eight proteins within the POU (Pit-Oct-Unc) family have been classified as OCT proteins (Fig. 4A) (13,14), of which only OCT4 is highly expressed in both naïve and primed pluripotent stem cells (Fig. 4B). The class III POU factors OCT6, OCT7 (also known as POU3F2 and BRN2), OCT8 (also known as POU3F3 and BRN1), and OCT9 (also known as POU3F3 and BRN4) are all expressed in brain cells (13-15, 36, 38) and have been shown to display functional redundancy in vivo (34,35,37). ...
... Total RNA was isolated using the RNeasy kit (Qiagen). First-strand complementary DNA (cDNA) was synthesized using Oligo(dT) [12][13][14][15][16][17][18] and M-MLV Reverse Transcriptase (USB). qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad). ...
OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.
... OCT proteins comprise eight members and are a subgroup of the POU (Pit-Oct-Unc) transcription factor family [12][13][14] . OCT proteins are highly conserved between humans and mice and specifically bind to the octamer motif (consensus sequence ATGCAAAT) via a bipartite DNA-binding domain (DBD) [12][13][14] . ...
... OCT proteins comprise eight members and are a subgroup of the POU (Pit-Oct-Unc) transcription factor family [12][13][14] . OCT proteins are highly conserved between humans and mice and specifically bind to the octamer motif (consensus sequence ATGCAAAT) via a bipartite DNA-binding domain (DBD) [12][13][14] . All OCT proteins harbor two transactivation domains (TADs) located on each side of the DBD, and these are responsible for transcriptional stimulation of target genes by interacting with basal transcription machineries and other factors [12][13][14] . ...
... OCT proteins are highly conserved between humans and mice and specifically bind to the octamer motif (consensus sequence ATGCAAAT) via a bipartite DNA-binding domain (DBD) [12][13][14] . All OCT proteins harbor two transactivation domains (TADs) located on each side of the DBD, and these are responsible for transcriptional stimulation of target genes by interacting with basal transcription machineries and other factors [12][13][14] . Despite these similarities, only OCT4 has been shown to be able to induce pluripotency [15][16][17][18] . ...
Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.
... The sim ilarities th a t exist w ithin the POU-domains of the dif proteins have suggested their classification into five classes; POU-I-V (He et al., 1989). The POU-I class is composed of Pit-1, POU-II of Oct-1, Oct-2 and Oct-11 (Goldsborough et al., 1993), POU-III of SCIP/Tst-1/Oct-6, Brn-1, Brn-2 and Cfl-a, POU-IV of Unc-86, Brn-3a and the Drosophila I-POU and tl-POU, and the POU-V class is composed of Oct-3/4 (see Figure 7, Chapter 3). ...
... Pit-1) and negative (eg. SCIP) regulators of genes expression, and in DNA replication (the functions of individual POU-domain proteins are discussed in later chapters), and are expressed in tem porally and spatially restricted p atterns during embryonic development (He et al., 1989). In the adult the expression of POUdomain proteins is also usually spatially restricted, except for Oct-1 which is expressed in most cell types. ...
... The pellet was resuspended in sterile H2O, at a final concentration of 1 p g/pl. F ig u re 4. P rim e rs s e q u e n c e s : P rim e rs w ere d e g e n e ra te , corresponding to the DNA sequences of known POU-domain proteins (He et al., 1989). ...
The mature nervous system is comprised of an enormous diversity of neurons and glial cells that arise from the apparently homogeneous population of pluripotent precursor cells of the embryonic neural tube. The mechanism by which these neuroepithelial precursor cells choose their differentiated fates is not understood. We have approached this problem by studying the development of one particular glial cell lineage, the oligodendrocyte lineage. Oligodendrocytes, the myelinating cells of the central nervous system, develop from glial progenitor cells known as 0-2A progenitor cells. 0-2A progenitor cells proliferate during embryonic development and first start to give rise to post-mitotic oligodendrocytes around the day of birth in the rat. 0-2A progenitor cells express receptors for platelet derived growth factor (PDGF), and divide in response to PDGF. When an 0-2A progenitor cell takes the decision to differentiate in vivo or in vitro it still expresses functional PDGF receptors but loses the ability to divide in response to PDGF. If we could understand the nature of the molecular switch involved in the loss of PDGF-responsiveness, we might begin to understand how the timing of oligodendrocyte differentiation is controlled during development. Transcription factors must have some role in differentiation since genes are expressed in differentiated cells that are not expressed in their undifferentiated precursor cells, and vice versa. For example, oligodendrocytes express genes encoding structural myelin proteins that are not expressed in 0-2A progenitor cells. Part of this Thesis involves experiments in which I investigate the involvment of transcription factors in oligodendrocyte development. I chose to concentrate on POU-domain transcription factors because their prototypic members appear to be lineage- specific, and have been shown to have a role in regulating the expression of differentiation specific genes. Using a strategy based on the polymerase chain reaction I identified several members of this family that are expressed in 0-2A progenitor cells, and investigated how they are regulated at the RNA level when 0-2A progenitors differentiate into oligodendrocytes in vitro. These experiments suggest that a subset of POU-transcription factors may be closely coupled to the transition from proliferation to differentiation in the oligodendrocyte lineage. During these experiments I also isolated cDNAs encoding the DNA binding region of two novel members of the POU-domain family that are closely related to the unc-86 gene of the nematode worm, Caenorhabditis elegans, and to a mammalian POU-factor gene known as brn-3. Together these define a brn-3 subfamily of POU-factors whose vertebrate members we refer to as brn-3a, brn-3b and brn-3c. I have investigated the patterns of expression of this subfamily in embryonic rat by in situ hybridisation. All members are expressed exclusively in the nervous system, notably by sensory neurons in the dorsal root ganglia (DRG), and by some deep dorsal horn neurons of the spinal cord. brn-3c is expressed in tiny subpopulations of neurons in the DRG and spinal cord suggesting that brn-3 family members may be involved in specifying particular subclasses of neurons that have a sensory phenotype. Unc-86 is involved in determining the differentiated fate of cells in many neuroblast lineages and also has a function in mature neurons. Brn-3a and Brn-3c the mammalian members of the same subfamily may have similar functions since they are expressed initially in dividing neurons in the DRG and then later in post-mitotic neurons in the DRG and spinal cord. These mammalian POU-factors may therefore be involved in the regulation of development or function of subpopulations of neurons.
... The original members of the family were; the anterior pituitary-specific Pit-1 factor (also named GHF-1) Bodner et al. 1988), the largely B-cell specific Oct-2 factor (also named OTF-2, NF-A2) Scheidereit et al. 1988;Muller et al. 1988), the ubiquitous Oct-1 factor (also named OTF-1, NFIII, NF-A1) ) and the 0. elegans unc86 gene product (Finney et al. 1988). Many more members the POU domain family have been subsequently identified in mammals (He et al. 1989), Drosophila (Johnson and Hirsh, 1990) and 0. elegans (Burglin et al. 1989 Verrijzer and Van der Vliet, 1993;Ruvkun and Finney, 1991;Rosenfeld, 1991). Details of these studies of structure and function will be discussed below. ...
... The application of a degenerate PGR method utilising primers based on 9 amino acid residues conserved among Pit-1, Oct-1, Oct-2 and unc-86 led to the cloning of mRNAs encoding four new POU family members (He et al. 1989). Three of the four clones were isolated from brain complementary DNA (cDNA): Brn-1 and Brn-2 from human, and Brn-3 from rat; whilst Tst-1 was isolated from rat testes cDNA. ...
... Three of the four clones were isolated from brain complementary DNA (cDNA): Brn-1 and Brn-2 from human, and Brn-3 from rat; whilst Tst-1 was isolated from rat testes cDNA. Studies of conservation of amino acids within the POU domain led to the classification of the then known eight POU family members into four sub families (He et al. 1989). These are referred to as POU-I (Pit-1), POU-II (Oct-1, Oct-2), POU-III (Brn-1, Brn-2, Tst-1) and POU-IV (Brn-3a, unc-86). ...
The Oct-2 and Brn-3 transcription factors show restricted spatial and temporal expression during neuronal development and in the adult organism. They are known to play important roles in regulating gene expression in neuronal cells. To date, however, only a small number of genes directly regulated by these factors have been identified and studied. Previous studies of the modular structure of the Oct-2 protein have identified three distinct repression domains. The mechanism of transcripitional repression employed by these three domains has not been investigated, although it has been proposed that each repressor domain interacts with components of the basal transcription complex. It was therefore proposed to investigate the mechanism of action of each of these three domains. An assay system was employed to study the ability of each domain to interfere with transcriptional activation by a range of different transcriptional activators. As a result of these studies it was found that each of the domains function by alternate mechanisms. These findings reveal Oct-2 to be a complex and precise regulator of gene expression, the characteristic of which are discussed. The most recently identified gene directly regulated by Oct-2 is the neuronal nitric oxide synthase (nNOS) gene. Studies of the promoter of the highly related inducible nitric oxide synthase (iNOS) gene have revealed the presence of an octamer DNA binding site. Since Oct-2 mediates transcriptional activation via the octamer site in other gene promoters it was proposed to determine whether the Oct-2 transcription factor regulates iNOS gene expression in a similar manner as seen for nNOS. The work presented in this thesis shows that both Oct-2 and the Brn3 transcription factors activate the iNOS gene promoter. As a result we proposed to investigate the mechanism of action and the relevance of the octamer sequence in the regulation of this gene. These studies have revealed that the octamer motif is essential for the activation of the iNOS gene by these transcription factors although depending upon cell type and thus cell specific factors the iNOS gene may be activated by Brn-3 in the absence of a functional octamer motif. The ability of these factors to interact with other transcription factors was also investigated. The results of these investigations is presented and discussed in this thesis.
... Both the PCR products of different 5′-deletion constructs and the [28][29][30][31][32][33][34][35][36][37][38] basic reporter vectors were digested with their respective restriction enzymes (New England) and ligated (T4 DNA ligase) to generate the above-mentioned constructs for each basic reporter vector (pMet Luciferase) in parallel. All plasmid DNA constructs were isolated using Endo-free Plasmid Mini Kit (Qiagen) to have high quality plasmid for transfection. ...
... Further analysis of this element indicated that it shared a number of consensus transcription factor binding sites, such as binding sites for MyoD, Oct-1, HSF and SRY. Recent reports have shown that MyoD is a transcription factor involved in regulating muscle differentiation, whereas both Oct-1 and Cdx are involved in regulation of the genes essential for growth and embryonic development [37,38]. The sex-determining region Y (SRY) is responsible for the initiation of male sex determination in humans. ...
... The putative polyadenylation (aataaa) signal is in bold and underlined.H.Chi, et al. Fish and Shellfish Immunology 93 (2019)[28][29][30][31][32][33][34][35][36][37][38] ...
Eomesodermin (Eomes) is a member of T-box transcription factor family and plays an important role in the regulation of a wide variety of developmental processes and immune response in animals. Here we report cloning and characterization of the full-length cDNA of Atlantic cod Eomes (GmEomes), which possesses a TBOX_3 domain similar to its counterpart in mammals. The regulated expression was observed in head kidney and spleen in response to live Vibrio anguillarum infection in vivo, and spleen leukocytes in vitro after PMA and poly I:C stimulation. Furthermore, we determined a 694 bp sequence, upstream of the transcriptional start site (TSS), to contain a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. The results showed that the basal promoters and positive transcriptional regulator activities of GmEomes were dependent by sequences located from -694 to -376 bp upstream of TSS. Furthermore, we found that some Eomes binding sites were present in the 5'-flanking regions of the cod IFNγ gene predicted by bioinformatics. However, Co-transfection of eomesodermin overexpression plasmids with INFγ reporter vector into CHSE-214 cells determined that Atlantic cod eomesodermin played a minor role in activation of the INFγ promoter.
... The data for the HTH proteins is adapted from Table I in He et al (1989). Brain regions indicate lo�ation where mRNA coding for an HTH protein is enriched compared with some other regions. ...
... similar role can be postulated for these proteins in mammals. Second, mRNAs encoding the mammalian POD domain proteins have a restricted distribution in the nervous system, which suggests specialized roles in different populations of neurons (He et al 1989). ...
... Oct-I is ubiquitous, and Oct-2 is present in both brain and spleen. Pit-1, present in restricted regions in the developing nervous system, is only present in the pituitary in the adult (He et al 1989). On the assumption that structurally related POD transcription factors might be important regulators of brain development, novel factors were sought and four new mammalian factors were identifi ed. ...
... The transcription factor POU3F4 (also known as Brain4, Brn4, etc.) belongs to the third family of POU proteins (POU III) together with Brn1 and Brn2. Studies have shown that POU3F2, POU3F3, and POU3F4 appear in the nervous system in the early embryo and are widely distributed [11][12][13], suggesting that these factors may participate in the development of the early nervous Graphical abstract system. Recent reports have shown that POU3F4 plays an important role in the reprogramming of somatic cells into neural cells. ...
... POU3F2, POU3F3, and POU3F4 belong to the POU protein family of transcription factors. They appear at an early embryonic stage and are widely distributed in the nervous system [11][12][13], suggesting that these factors may be involved in the development of the early nervous system. Zhang et al. established a denervated hippocampus model by cutting the fimbria fornix of adult rats to block the cholinergic fibers projecting from the septum to the hippocampus, and then transplanted NSCs from the subventricular zone (SVZ) into the hippocampus. ...
Background
Neural stem cells (NSCs) are considered to be the most promising cell type for cell replacement therapy in neurodegenerative diseases. However, their low neuronal differentiation ratio impedes their application in such conditions. Elucidating the molecular mechanism of NSC differentiation may provide the necessary experimental basis for expanding their application. Previous studies have indicated that POU3F4 can induce neuronal differentiation of NSCs, this study aims to underly the possible exact mechanism of POU3F4 on the NSC differentiation and development.
Methods
NSCs were isolated and cultured from the hippocampus of neonatal mice. The frozen hippocampal sections were prepared for immunohistochemical staining. Synaptic development was assessed using electron microscopy. High-throughput sequencing was employed to analyze the gene expression profile following the overexpression of Brn4. Gene expression levels were determined through Western blotting and qRT-PCR. Cell cycle and differentiation were evaluated using flow cytometry and immunofluorescent staining.
Results
It was found that POU3F4 promoted the neuronal differentiation of hippocampal NSCs and synapse development, and inhibited NSC proliferation. POU3F4-deficient mice exhibited impairments in learning and memory. RNA sequencing and ChIP assays confirmed that Gli1 was downstream of POU3F4. Loss and gain function experiments indicated that Gli1 mediated POU3F4 promoting neuronal differentiation and synapse development. Forced expression of Gli1 in hippocampus improved learning and memory function of animal models.
Conclusions
The results suggest that POU3F4 and Gli1 promote neuronal differentiation and synaptic development of NSCs, and that Gli1 partially mediates the effects of POU3F4.
Graphical abstract
... POU III factors (Oct6, Oct7, Oct8 and Oct9) are prominently expressed in cells and tissues of the central nervous system (CNS) 1,71,72 . They play critical roles in neurogenesis and gliogenesis [73][74][75][76][77] . ...
... Oct6 (also known as SCIP, Tst-1 and Pou3f1) is expressed in Schwann cells, oligodendrocyte progenitor cells (OPCs) and keratinocytes 71,[73][74][75][76]78,81 . Furthermore, Oct6 play an important role in the specification and differentiation of neuroectodermal lineages [82][83][84][85] . ...
Ectopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences.
... including Pit-2, OCT-2, BRN-1, BRN-2, BRN-3 and Testes-1 (Tst-1), but BRN-2 is the most strongly expressed as determined by in situ hybridisation. With the exception of Pit-1, which is expressed biphasically in the pituitary during development, these proteins are also expressed throughout the adult mouse brain (Xi He et al. 1989). In the mouse brain, BRN-2 cDNA hybridizes particularly strongly to transcripts in the hypothalamus and hippocampus and the hybridisation signal for BRN-2 was significantly stronger than for BRN-1. ...
... BRN-1 and BRN-2 are the most widely expressed class III POU domain proteins in the CNS. They were also localised to the cerebral cortex (layers II-V), the Purkinje cells of the cerebellum, the forebrain cholinergic system, midbrain dopaminergic system and the paraventricular nucleus of the hypothalamus (PVN) (Xi He et al. 1989). ...
NF-κB is a transcription factor family, which includes the proteins p65, p50 and p52. Inducible in most cell types, NF-κB (p65) has been demonstrated to be present in the nuclei of cortical neurones. This work demonstrates that p65, p50 and p52 are also present in striatal neurones, and that p52 localises to neuronal nuclear bodies. Administration of the excitotoxin quinolinic acid (QA) or the glial activator ciliary neurotrophic factor (CNTF) leads to an increase of protein binding to NF-κB DNA oligonucleotides in a biphasic temporal manner. PAGE revealed no significant change in expression of known NF-κB proteins after either treatment, but a 35 kD protein of unknown identity is recognised by anti-p50, being transiently expressed following both QA and CNTF administration. Furthermore, p52 and p65 can be localised within glial cells following QA treatment. Endogenous levels of striatal CNTF are also seen to increase significantly at 168 hours post-lesion. These results implicate NF-κB in the regulation of the glial response to striatal lesion. Polyglutamine repeats can bind via hydrogen bonding in vitro, and may lead to aggregation of proteins if mutation leads to increased trinucleotide repeat length. A preliminary study of the DNA binding, expression and localisation of the polyglutamine repeat transcription factors Brain-2 (BRN-2), glucocorticoid receptor and TATA binding protein (TBP) were investigated in the CNS of R6/2 transgenic mice expressing abnormal huntingtin protein (htt) possessing expanded polyglutamine repeats. These mice develop neuronal nuclear inclusions of aggregated htt and demonstrate symptoms similar to juvenile onset Huntington's disease. Increased protein binding to OCT consensus site DNA was found in the cortex of transgenes corresponding to BRN-2. This work provides no evidence that abnormal htt induces symptoms via transcriptional dysregulation, as has been proposed. However, decreased expression of BRN-2 is seen in the hypothalamic paraventricular nucleus in transgenic animals.
... We found brain-specific transcription factor POU3F2 to be one of the key regulators co-expressed with many genes within the SCZ-related module. POU3F2 is expressed primarily in the central nervous system and has an essential role in brain development [11]. However, the target genes of POU3F2 and their relationship with SCZ remain largely unknown. ...
... POU3F2 involves neuronal functions that favor the neurodevelopmental hypothesis of SCZ. For example, POU3F2 regulates layer production and cortical neuron migration in the developing neocortex [11,12], while POU3F2-deficient mice exhibit impaired hippocampal neurogenesis [13]. Further, GWAS and brain imaging analyses have identified SCZ-related brain activity affected by POU3F2 [14]. ...
Schizophrenia (SCZ) is a neuropsychiatric disorder with aberrant expression of multiple genes. However, identifying its exact causal genes remains a considerable challenge. The brain-specific transcription factor POU3F2 (POU domain, class 3, transcription factor 2) has been recognized as a risk factor for SCZ, but our understanding of its target genes and pathogenic mechanisms are still limited. Here we report that POU3F2 regulates 42 SCZ-related genes in knockdown and RNA-sequencing experiments of human neural progenitor cells (NPCs). Among those SCZ-related genes, TRIM8 (Tripartite motif containing 8) is located in SCZ-associated genetic locus and is aberrantly expressed in patients with SCZ. Luciferase reporter and electrophoretic mobility shift assays (EMSA) showed that POU3F2 induces TRIM8 expression by binding to the SCZ-associated SNP (single nucleotide polymorphism) rs5011218, which affects POU3F2-binding efficiency at the promoter region of TRIM8. We investigated the cellular functions of POU3F2 and TRIM8 as they co-regulate several pathways related to neural development and synaptic function. Knocking down either POU3F2 or TRIM8 promoted the proliferation of NPCs, inhibited their neuronal differentiation, and impaired the excitatory synaptic transmission of NPC-derived neurons. These results indicate that POU3F2 regulates TRIM8 expression through the SCZ-associated SNP rs5011218, and both genes may be involved in the etiology of SCZ by regulating neural development and synaptic function.
... The POU proteins constitute a large family of putative transcription factors, named from the first 3 members to be described (pit, oct and une). They are most closely related in their DNA binding homeobox domains (a helix-tum-helix motif) and in a contiguous POU-specific domain immediately upstream of this domain (Herr et al , 1988;He et al , 1989). In these regions, the degree of amino acid sequence conservation between SCIP and the other class m POU proteins, Bm-1 and -2, is striking. ...
... Oct-3 is expressed by the embryonic stem cell line pl9 only under conditions that permit cell proliferation but prevent differentiation (Okamoto et al., 1990). Nearly all the POU proteins analysed to date are expressed at high levels in regions of the early embryo, such as the ventricular zone of the neural tube, that are populated by rapidly proliferating progenitor cells (He et al , 1989). POU proteins may therefore act as transiently expressed regulators in proliferating progenitors. ...
This thesis concerns the role of neuropeptides and cytokines in regeneration of injured peripheral nerve. A novel experimental technique is described for the rapid preparation of pure cell populations from rat sciatic nerve, involving immunoselection by "panning". Forskolin elevates Schwann cell cAMPi in vitro, permitting growth factor-induced proliferation and expression of nerve growth factor (NGF) and of the transcription factor suppressed cAMP inducible POU (SCIP). The neuropeptide calcitonin gene-related peptide (CGRP) acts through cAMPi in other systems and is upregulated in regenerating motor axons following peripheral nerve injury. I show that CGRP and isoprenaline elevate cAMPi acutely in non-neuronal cells derived from rat sciatic nerve, but that this cAMPi elevation rapidly attenuates due, probably, to the desensitisation of receptors by cAMP-dependant protein kinase (PKA) and the (β-adrenoceptor kinase (βARK), respectively. CGRP fails to promote Schwann cell proliferation, in vitro, due to the time course of desensitisation rather than lack of signalling downstream of cAMPi, since CGRP elevates Schwann cell SCIP, and inhibits fibroblast proliferation. Degradation of CGRP by Schwann cell endopeptidase 24.11. may reduce desensitisation in vivo. Supraphysiological magnesium (Mg++) concentrations enhance receptor / G protein coupling, allowing maintenance of cAMPi elevation in response to CGRP. Consequently, previously desensitising levels of CGRP and isoprenaline are now mitogenic and increase NGF synthesis. In the second section, I show that interleukin-1β (IL-lβ) promotes Schwann cell proliferation and survival in culture and, with tumour necrosis factor a (TNFa), additively increases NGF production. These responses are lost with time and thus are not apparent in cells purified by conventional methods. IL-1β is active alone and stimulates several distinct intracellular signals: these probably include PKA, a pertussis toxin sensitive G protein, prostaglandin E2 (PGE2) and a tyrosine kinase or protein kinase C (PKC). Inhibiting any of these transduction signals reduces IL-1β action. Gamma-interferon (γ-IFN) dramatically enhances IL-lβ stimulated NGF synthesis, yet inhibits proliferation. When PGE2 synthesis or PKA are downregulated, IL-1β becomes weakly mitogenic for fibroblasts but no longer promotes NGF synthesis. Transforming growth factor type β (TGFβ), a macrophage product, does not influence quiescent Schwann cells, but inhibits mitogen induced proliferation. A model is proposed whereby the responses of non-neuronal cells after axotomy are regulated by a combination of axon and macrophage-derived signals, with a priming action by T cells. Clinical ramifications of the work are then discussed.
... A similar situation exists for the class IV POU domain proteins, Bm-3a and Bm-3b Degenerate oligonucleotides representing codons for amino acids conserved among POU domain proteins have been used to identify mRNAs encoding POU proteins in different tissue types (He et al., 1989;. The Bm-3 family of POU domain proteins was first identified in 1989 (He et al., 1989). ...
... A similar situation exists for the class IV POU domain proteins, Bm-3a and Bm-3b Degenerate oligonucleotides representing codons for amino acids conserved among POU domain proteins have been used to identify mRNAs encoding POU proteins in different tissue types (He et al., 1989;. The Bm-3 family of POU domain proteins was first identified in 1989 (He et al., 1989). Members of the Bm-3 family of transcription factors are expressed in neurons in both the central and peripheral nervous systems where they regulate neuronal differentiation Lakin et al., 1995). ...
Brn-3a and Brn-3b are closely related members of the POU domain-containing transcription factors. While Brn-3a is associated with neuronal differentiation and Brn-3b with neuronal proliferation, their expression is not strictly limited to the nervous system. It has been shown that these Brn-3 proteins are expressed in non-neuronal tissues including cervical epithelium, testis and breast. Moreover, these Brn-3 proteins functionally interact with the estrogen receptor and in association with the estrogen receptor have differing transactivation potentials. The p160 steroid receptor coactivators (Srcs) are also able to interact with the estrogen receptor in a ligand dependent manner and are able to enhance estrogen dependent transcription. This work describes a functional interaction between Brn-3 proteins and members of the steroid receptor coactivator family. In affinity chromatography and coimminoprecipitation experiments, Src-1 proteins were shown to physically interact with Brn-3a and Brn-3b. In addition, the transactivation potential of the Brn-3/Src complexes was tested on two promoters in three different cell lines. The steroid receptor coactivators potentiated the transcriptional effects of Brn-3 a and Brn-3b. The Src proteins, however, differed in their ability to potentiate the transcriptional activity of Brn-3 proteins on different promoters in transiently transfected cells. In addition, maximal activation of the Brn-3/Src complex differed between cell lines, indicating that additional cell-type specific proteins are important for maximal activation by the complex. It has recently been shown that Brn-3b may play a role in regulating BRCA-1 in mammary tumors as its expression is enhanced in primary breast tumors with reduced BRCA-1 expression. Moreover, this elevated Brn-3b expression is not seen in normal mammary cells, benign tumors, or malignant tumors which do not have reduced levels of BRCA-1. This work also describes the effects of stable Brn-3b overexpression and reduction in the estrogen receptor positive breast adenocarcinoma cell line MCF7. Overexpression of Brn-3b resulted in increased growth rate, saturation density, proliferation, and ability to form anchorage independent colonies when compared to mock transfected cells. MCF7 cells with reduced Brn-3b levels exhibited a significant decrease in growth rate, saturation density, proliferation, and ability to form anchorage independent colonies when compared to mock transfected controls. Differential gene expression in these cell lines was examined using both specific western immunodetection as well as comprehensive DNA array technology. Immunodetection revealed increases in ER, HSP-27, and Brn-3a in Brn-3b overexpressing cells as well as a decrease in these proteins in the Brn-3b reduced cell lines. The DNA array including 1200 cancer related genes revealed several genes differentially expressed when the Brn-3b overexpressing and reduced cell line mRNAs were compared. Finally, the results of three of the genes shown to be differentially regulated in the cDNA array differential display were corroborated by semi-quantitative RT-PCR.
... Also, the upstream A/T-rich block of DNA that is conserved among the salmonid sGnRH-encoding genes does possess recognition sequences that may be important for GnRH regulation in both the brain and the gonads. For example, consensus elements are present in each promoter that potentially could bind members of the POU homeodomain family of transcription regulators that are involved in morphogenesis and neurogenesis (38). Each promoter in this conserved region contains two elements that strongly resemble recognition sequences for mammalian Brn-2 (CATnTAAT) and at least one centrally positioned element that could be engaged by Oct-type factors (ATGCAAAT) (39). ...
... Each promoter in this conserved region contains two elements that strongly resemble recognition sequences for mammalian Brn-2 (CATnTAAT) and at least one centrally positioned element that could be engaged by Oct-type factors (ATGCAAAT) (39). Importantly, members of this family of regulators have been isolated in both the mammalian brain and gonad (38). ...
The GnRH gene is transcribed in both the brain and gonads. GnRH in the brain is critical for reproduction, but the function and importance of GnRH in the ovary and testis is not clear. In this study we examine whether regulation of the GnRH gene is distinct in the brain and gonads, whether the regulation of the GnRH gene in the gonads is altered after genome duplication, and whether the regulatory region of the GnRH gene is tightly conserved in vertebrates. From ovary and testis, we isolated and sequenced for the first time two different genes and their complementary DNAs that encode the identical peptide known as salmon GnRH. Rainbow trout were selected because they are tetraploid due to genome duplication.
A downstream promoter is used in the brain and gonads by salmon GnRH messenger RNA1 (mRNA1) and mRNA2, but mRNA2 also uses an upstream promoter only in the gonads. Two types of long mRNA2 transcripts in ovary and testis both use an alternative start site at position −323; one of these types also retains intron 1. This long 5′-untranslated region is a likely site for distinct regulation of mRNA in the gonad. Additional evidence for separate regulation is that a different expression pattern exists in brain and gonads for GnRH mRNAs during development and maturation. Gene duplication did not alter the encoded peptide, but changed the expression pattern and resulted in complete divergence of the promoter sequence from position −215. A comparison of the mammalian and trout GnRH genes reveals that the promoters are without sequence identity except for a few consensus sites in both regulatory regions. The duplicated trout genes provide a model to study a critical gene whose product controls reproduction in all vertebrates.
... Expression of the cyclin dependent kinase inhibitors p27 kip1 and p57 kip2 in the optic nerve and cortex intrinsically regulate oligodendrocyte differentiation through a counting mechanism whereby protein levels increase during successive divisions and oligodendrocyte differentiation occurs when a critical concentration of inhibitor is reached (Gao et al., 1997;Dugas et al., 2007). Astrocytes play multiple roles in the central nervous system, including regulation of synaptogenesis and maintenance of the blood brain barrier (reviewed in He and Sun, 2007). Astrocytes are derived from dorsal progenitor cells that initially give rise to excitatory neurons (Voigt, 1989;Mission et al., 1991;Gorski et al., 2002). ...
... In Sall1-deficient, animals no alteration in the pattern of Ctip2 expression was observed at E18.5 (n=4) ( Figure 16D), which suggests appropriate specification of deep cortical plate layers. Brn2 is expressed by layer V and layer II/III cells, as well as cells in the progenitor populations and intermediate zone at E18.5 (He et al., 1989;McEvilly et al., 2002) ( Figure 16E). No alteration in the position of Brn2-positive cells was observed in Sall1-deficient animals, which suggests appropriate specification of layer V and II/III cells (n=4) ( Figure 16F). ...
Sall1 is a zinc finger containing putative transcription factor that is robustly expressed during mammalian embryogenesis. In humans, the developmental disorder Townes Brocks Syndrome is associated with mutations in the SALL1 gene. Sall1-deficient animals die at birth due to kidney deficits; however, its function in the nervous system has not been characterized. During embryonic development Sall1 is expressed by cortical progenitor cells. In late embryonic and postnatal stages Sall1 is expressed by oligodendrocytes and glia, as well as regions of adult neurogenesis. In the absence of Sall1 forebrain derived structures, the cerebral cortex and olfactory bulbs, were specifically decreased in size, while no gross alterations in midbrain development was observed. This study investigated the cellular mechanisms of Sall1 function in the developing cortex. Alterations in progenitor cell number and the rate of neuronal differentiation were observed during cortical development in Sall1-mutant animals compared to controls. In the absence of Sall1 more cells are committed to early-born cortical structures, at the expense of the progenitor population. In addition, from mid-neurogenesis fewer cells are committed to later-born structures. Together, these findings suggest that early in development Sall1 promotes a progenitor state, and from mid-neurogenesis Sall1 promotes a neural fate. Furthermore, I propose that Sall1 regulates the transition from an early cortical progenitor cell to an intermediate progenitor cell. These findings suggest that Sall1 regulates cortical neurogenesis and progenitor cell maturation in the developing cortex. Sall1 is also expressed by peripheral and central components of the developing olfactory system. Alterations in neurogenesis and mitral cell production were observed in Sall1-deficient olfactory bulbs. In addition, the olfactory nerve failed to extend past the ventral-medial region of the olfactory bulb in Sall1-mutant animals. Intrinsic patterns of neurogenesis were observed during olfactory development in control animals and in Sall1-mutant animals, these patterns of neurogenesis were disrupted. These findings suggest a role for Sall1 in regulating neuronal differentiation and maturation in the developing olfactory system. Together, these findings suggest a conserved role for Sall1 in regulating neurogenesis and cellular maturation in the developing cortex and olfactory bulb.
... To further characterize CN development, we employed established markers of different CN components. Antibodies for BRN2, a POU-domain transcription factor (He et al., 1989), label the LAT and INT (not the MED) during late gestation embryogenesis (Fink et al., 2006). We performed dual immunofluorescence for BRN2 and tdT at E18.5 on cerebellar sections from litters treated with Tam at different embryonic stages. ...
The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate‐mapping study characterizing rhombic lip‐derived glutamatergic neurons of the nuclei, the most conspicuous family of long‐range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral‐to‐medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.
... Pou3f2/Brn2 belongs to the Pit-Oct-Unc (POU) family transcription factors, which play critical roles in cell-fate determination and maintenance in the nervous system. [76][77][78][79] In the mouse NCx, Pou3f2 is expressed by pallial progenitor cells and newborn excitatory neurons and, together with Pou3f3/Brn1, is required for the fate specification and migration of superficial layer excitatory neurons. [80][81][82] Recent work suggests that Pou3f2 regulates the patterning of the ganglionic eminences in primates. ...
... A combined principal component analysis (PCA) of the current and original RNA-seq dataset 21 confirmed high similarity of the deep sequencing gene expression profiles in all three differentiation stages (hESC, NPC, and NEU) with their corresponding cell types in the original study and show separation of libraries according to differentiation stages (Supplemental Fig. S1a). We further confirmed that both IsoHD NPCs and neurons showed decreased expression of stem cell marker gene POU5F1 (OCT3/4) 48 and increased expression of neuronal marker genes MAP2, 49 and POU3F2 (OCT7) 50 (Supplemental Fig. S1b-d). NPCs also increased expression of the NPC marker PAX6 51 whereas neurons showed increased expression of neuronal FOXP2 52 and forebrain neuron marker genes BCL11B, 53 CUX1, 54 and PPP1R1B (DARPP32) 55 Differential gene expression analysis identified differentially expressed genes (DEGs) across CAG lengths (45Q/81Q vs control, n = 3 per group) in each differentiation stage independently (|logFC|≥1 and BHadjusted P-value <0.01) (Supplemental Table S3). ...
Background:
In Huntington's disease (HD), a CAG repeat expansion mutation in the Huntingtin (HTT) gene drives a gain-of-function toxicity that disrupts mRNA processing. Although dysregulation of gene splicing has been shown in human HD post-mortem brain tissue, post-mortem analyses are likely confounded by cell type composition changes in late-stage HD, limiting the ability to identify dysregulation related to early pathogenesis.
Methods:
To investigate gene splicing changes in early HD, we performed alternative splicing analyses coupled with a proteogenomics approach to identify early CAG length-associated splicing changes in an established isogenic HD cell model.
Findings:
We report widespread neuronal differentiation stage- and CAG length-dependent splicing changes, and find an enrichment of RNA processing, neuronal function, and epigenetic modification-related genes with mutant HTT-associated splicing. When integrated with a proteomics dataset, we identified several of these differential splicing events at the protein level. By comparing with human post-mortem and mouse model data, we identified common patterns of altered splicing from embryonic stem cells through to post-mortem striatal tissue.
Interpretation:
We show that widespread splicing dysregulation in HD occurs in an early cell model of neuronal development. Importantly, we observe HD-associated splicing changes in our HD cell model that were also identified in human HD striatum and mouse model HD striatum, suggesting that splicing-associated pathogenesis possibly occurs early in neuronal development and persists to later stages of disease. Together, our results highlight splicing dysregulation in HD which may lead to disrupted neuronal function and neuropathology.
Funding:
This research is supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Nanyang Assistant Professorship Start-Up Grant, the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 1 (RG23/22), the BC Children's Hospital Research Institute Investigator Grant Award (IGAP), and a Scholar Award from the Michael Smith Health Research BC.
... The group of "common" R-loop genes contained 7127 genes, representing 66.98% and 84.54% of active genes with R-loops in ESC and NSPCs, respectively. 1303 genes (15.46%) were unique to NSPCs, and 3514 genes (33.02%) were unique to ESCs ( Fig. 2A). Figure 2B shows examples, with mitochondrial ribosomal protein 9 (MrpS9) being actively transcribed and forming R-loops in ESCs and Pou3f3 (also known as Brain-1), a gene with roles in brain development 30 and intellectual disability 31 being unique to NSPCs. Core ESC transcriptional factors such www.nature.com/scientificreports/ ...
Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called “R-loops” in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break “hotspots”. We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.
... Interestingly the Brain-3 (Brn-3) POU domain proteins have been shown to play a key role in the regulation of neuronal cell differentiation. Bm-3 is expressed in the mammalian central and peripheral nervous systems during development (He et al., 1989). Three distinct Bm-3 proteins have been discovered, each coded for by a separate gene, called Bm-3 a (Brn-3.0), ...
p> Pax-3 is a member of the Pax gene family of embryonic transcription factors that possess a conserved paired box motif coding for a 128 amino acid DNA binding domain called the paired domain. Pax proteins play important roles in vertebrate development. Pax-3 is expressed from embryonic day 8.5 in the developing central and peripheral nervous systems, as well as in developing limb muscle. Mutations in the Pax-3 gene give rise to developmental defects, characterised by the splotch phenotype in mice and Waardenburg Syndrome in humans. Abnormalities include spina bifida and exencephaly, limb deformities, pigmentation defects and loss of hearing. To further define the role that Pax-3 plays during embryogenesis, the regulation of Pax-3 activity was studied in the sensory neuron-derived cell line ND7. Attempts were also made to uncover novel molecular targets for Pax-3 in neuronal cells.
Expression of Pax-3 is restricted to undifferentiated ND7 cells, and is dependent on serum factors. Removal of serum leads to a rapid fall in Pax-3 mRNA expression, followed by cell cycle arrest and morphological differentiation. In the presence of 0.5% and 1% serum Pax-3 promoter activity was shown to decrease by the same amount as it did in the complete absence of serum, despite the fact that only 0% and 0.5% serum resulted in a decreased rate of ND7 cell proliferation. Therefore in neuronal cells the extent of cell proliferation may not be directly dependent on the level of Pax-3 transcription. In addition to peptide growth factors, serum lipids were shown to be necessary for the induction of Pax-3 promoter activity. The promoter may also undergo auto-regulation, as suggested by the fact that low levels of Pax-3 stimulated promoter activity whereas higher levels of Pax-3 repressed it. Pax-3 promoter activity was also modulated by Brn-3 proteins, which play important roles in neuronal cell differentiation.</p
... The copyright holder for this preprint (which this version posted July 24, 2020. . https://doi.org/10.1101/2020.07.23.218768 doi: bioRxiv preprint (Bloodgood et al., 2013;He et al., 1989;Lin et al., 2008;Spiegel et al., 2014;Wurst et al., 1994;Zhu et al., 2014) , and a general transcriptional activator and repressor ( FOXK1 ) known to have the ability to translocate genes from the lamina to the nucleus (Wang et al., 2015) . In comparison, ESC T1-LADs compared to CM T1-LADs were enriched for multiple cardiac-related transcription factor genes, including DLX1 and 2 (Table 7) . ...
Three-dimensional genome organization, specifically organization of heterochromatin at the nuclear periphery, coordinates cell type-specific gene regulation. While defining various histone modifications and chromatin-associated proteins in multiple cell types has provided important insights into epigenetic regulation of gene expression and cellular identity, peripheral heterochromatin has not been mapped comprehensively and relatively few examples have emerged detailing the role of peripheral heterochromatin in cellular identity, cell fate choices, and/or organogenesis. In this study, we define nuclear peripheral heterochromatin organization signatures based on association with LAMIN B1 and/or dimethylation of lysine 9 on H3 (H3K9me2) across thirteen human cell types encompassing pluripotent stem cells, intermediate progenitors and differentiated cells from all three germ layers. Genomic analyses across this atlas reveal that lamin-associated chromatin is organized into at least two different compartments, defined by differences in genome coverage, chromatin accessibility, residence of transposable elements, replication timing domains, and gene complements. Our datasets reveal that only a small subset of lamin-associated chromatin domains are cell type invariant, underscoring the complexity of peripheral heterochromatin organization. Moreover, by integrating peripheral chromatin maps with transcriptional data, we find evidence of cooperative shifts between chromatin structure and gene expression associated with each cell type. This atlas of peripheral chromatin provides the largest resource to date for peripheral chromatin organization and a deeper appreciation for how this organization may impact the establishment and maintenance of cellular identity.
... POU dom ain proteins now constitute a large family of transcriptional regulators, expressed in distinct spatio-tem poral expression patterns during developm ent (Wegner et al, 1993;He et al, 1989). Sequence conservation across the POU dom ain has been used repeatedly in the hom ology cloning of new family members, including the Brn-3 genes (see Chapter 3) which appear to be the m am m alian homologues of unc-86, sharing extensive hom ology both in the POU dom ain and in an am ino-term inal dom ain characteristic of class IV POU domain proteins. ...
Genetic studies of simple multicellular organisms have defined a number of transcriptional regulators involved in neuronal specification, and in some cases mammalian homologues have been identified. In this thesis I have focused on two such mammalian transcription factors which may play regulatory roles in sensory neurone development and function. Mammalian Brn-3c is a homologue of the C. elegans POU domain factor, unc-86, which is required for cell fate determination and for the postmitotic development of many neurones. Brn-3c is expressed in a restricted pattern within the nervous system, including a subset of dorsal root ganglion (DRG) neurones and may therefore be involved in the specification or later development of these neurones. To investigate this possibility, I decided to generate a Brn-3c null mutant mouse, using homologous recombination in embryonic stem (ES) cells, followed by injection of genetically altered ES cells into mouse blastulae. Control and knockout genomic DNA constructs were generated. Transfection of ES cells with these constructs, followed by sequential screening techniques, allowed the identification firstly of control cell lines and subsequently of four cell lines containing a null deletion in one allele at the Brn-3c locus. Parallel work by another group has shown, via the generation of a Brn-3c null mutant mouse, that Brn-Sc is essential for hair cell development in the inner ear. A second line of research involved the LIM-homeodomain transcription factor, Islet-1 (Isl1). Isl1 is required at an early stage in the generation of motor neurones, and its later expression in motor neurone subsets suggests roles in neuronal subtype generation. Its Drosophila homologue, Is1, is required for axonal pathfinding and neurotransmitter production and in the vertebrate pancreas Isl1 is involved in the regulation of at least three genes for secreted polypeptides. Isl1 is expressed in developing and adult DRG and may therefore play roles in the development and maintenance of trunk sensory neurones. I have shown that Isl1 is expressed in both SD and L neurones; that all or almost all nociceptors express Isl1, but Isl1 is not restricted to nociceptive neurones; and that the neurotransmitter CGRP is expressed exclusively by a subset of Isl1 positive neurones. I have also demonstrated that Isl1 is first expressed at around the time of the last mitotic division in sensory neurones, with some cells initiating Isl1 expression prior to their final mitosis. Using Isl1 null mutant mice I have shown that neural crest cells are formed in the absence of Isl1 and are able to migrate to their correct target areas, including the prospective DRG, and begin to coalesce into ganglia. I have putatively identified SCGIO expression in the location of cervical DRG in embryos lacking Isl1, which suggests that the initial stages of sensory neurone formation do not require Isl1. However, expression of other early neuronal markers could not be detected and there is evidence for increased cell death in the region of prospective DRG. Analysis of later stages of sensory neurone development have been hindered by the early death of the null mutant embryos. In addition to these studies, I attempted to use sequence homology to define conserved cis-acting elements within the regulatory regions of a number of genes expressed only, or selectively in sensory neurones. Putative cis-acting regions were analysed functionally using EMSAs to determine whether these regions were bound by DRG specific nuclear factors. On the basis of these experiments, three cis-acting sequences have been identified which bind to proteins present within DRG. These elements could therefore be involved in neuronal specific expression of adjacent genes.
... POU proteins bind the consensus sequence ATGCAAAT with the POU-specific domain contacting the initial 4 bases and the POU homeodomain contacting the last four bases (Klemm et al, 1994). Oct-6 was isolated from three different systems concurrently; sciatic nerve, testes and embryonic stem cells of the central nervous system (Monuki et al, 1989, He et al, 1989, Meijer et al, 1990and Suzuki et al, 1990). Oct-6 mRNA and protein have been detected in Schwann cell precursors at E12 ...
Myelin forming and non-myelin forming Schwann cells are the major glia within peripheral nerves. Recent studies have revealed the importance of the transcription factors Sox10, Pax3, Krox-20 and Oct-6 in Schwann cell development. This work describes one novel Schwann cell zinc-finger transcription factor, Zfp-57, and investigates the phenotype of Schwann cells deficient in another, Krox-24, with a view to discovering a function for these genes in Schwann cells. Zfp-57 is expressed in nerves from embryo day 12 to adult and is present in Schwann cell nuclei. To investigate whether Zfp-57 plays a role in myelination, Zfp-57 cDNA was overexpressed in Schwann cells. No effects were detected on Schwann cell differentiation towards a myelin phenotype, suggesting that Zfp-57 may not be involved in this process I undertook a detailed investigation of nerves of Krox-24 null mutant and heterozygous mice in which expression of the LacZ gene is controlled by the Krox-24 promoter. Krox-24 activation occurs in late embryonic/early postnatal peripheral nerve development. In nerves, mRNA levels of typical Schwann cell molecules are normal, proliferation and the ultrastructural morphology is not affected. In regenerating nerves Krox-24 deficient Schwann cells down-regulate myelin genes and up-regulate molecules required for regeneration as efficiently as wildtype cells except for p75NTR mRNA, which is increased in Krox-24 deficient mice. Furthermore axonal regeneration occurs normally. Schwann cell death occurs developmentally and the effect of Krox-24 deficiency on this phenomenon has been investigated using TUNEL. 1 day after transection of newborn sciatic nerves cell death is elevated threefold in Krox-24-/- mice compared to wiltype littermates. In a low density culture assay where cell death is measured in the absence of autocrine Schwann cell survival factors, there is no difference between Krox-24 null cells and normal Schwann cells. Additionally cell death induced by TGFβ is unaltered.
... Pit-1, which binds a specific consensus sequence found w ithin the Prolactin promoter, plays a critical role in pituitary-specific gene regulation (Anderson and Rosenfeil, 1994). Additional members o f the POU domain group o f transcription factors have been isolated by RT-PCR by using degenerate primers based on the conserved amino acids o f the POU-domain (He et al., 1989). POU domain factors have been implicated in the patterning o f the developing brain (Rubenstein and Puelles, 1994) The LIM family represent a novel family o f transcription factors which possess a conserved putative metal-binding Zn-fmger m otif and a homeodomain (Freyd et al., 1990;Karlson et al., 1990). ...
To study the regulation of expression of the murine c-ret proto-oncogene, we have cloned and initiated the molecular characterisation of the c-ret locus. cDNA clones representing the long and short isoforms of the murine c-ret proto-oncogene have been cloned from a cDNA library synthesised from mRNA isolated from adult mouse salivary glands. Isolated cDNA clones have been used to screen genomic libraries to isolate cosmid clones representing the murine c-ret locus. Approximately 60kb of the mouse genome, representing the entire c-ret gene structure, have been cloned as five overlapping cosmid clones spanning 12 kb 5' of the gene locus. Primer extension, S1 nuclease protection and DNA sequences analysis have been used to identify the transcription initiation site and to characterise the promoter of the murine c-ret gene. Two different transcription initiation sites, 40 bps apart, which synthesises a major and a minor population of c-ret mRNA have been identified in NB2α cells. The major transcription initiation site of the locus have been localised to be 254 bps upstream of translation initiation site of the gene. DNA sequence analysis of the promoter of the gene show the murine c-ret locus, like the human c-ret gene, to lack the TATA initiation consensus motif and have high GC content. The G+C content (67%) and CG:GC ratio (0.95) over the promoter and 5'UTR of the murine c-ret locus (719 bps) implies the presence of a putative methylation-free CpG island within this segment of the genome. Functional significance (if any) for the presence of this putative CpG island within the c-ret promoter remains to be analyzed. Analysis of interspecies promoter DNA sequence conservation showed low (37%) overall sequence homology within the mouse and human c-ret promoters. Apart from the conservation of putative Sp1 binding GC box sequences at -40 and -110 bps, there were no other significant alignment and conservation of transcription factor binding consensus motifs within the two promoters. Promoter deletion analysis of 5' flanking sequences of the murine c-ret locus in transgenic mice suggest a locus organised as linked modules of independent regulatory domains. The distal 6kb of the 5' flanking sequences harbours the putative regulatory sequences of the locus which induce the early expression of the gene at the proximal end of the primitive streak and facial ganglion. The proximal 6 kb of 5' flanking sequences of the locus harbours the putative cis-regulatory elements which direct expression to the PNS, CNS and the urogenital system. The 5' flanking sequences of the locus recapitulate the complex and dynamic expression of the endogenous gene in the sensory branch of the PNS. In addition to these, the 5' flanking sequences of the locus can also direct expression to a specific subset of cells of the thyroid and parathyroid. However, the 5' flanking sequences of the locus do not possess the cis- elements which regulate the full spectrum of the endogenous gene expression: the elements which direct expression to the enteric and sympathetic nervous systems and the mesonephros seems to be present elsewhere in the locus. Further characterisation of the reminder of the locus has to be performed in order to identify and characterise the regulatory elements which regulate the full spatial expression profile of the endogenous gene. In addition to these studies, we have initiated genetic experiments using the established transgenic lines to study the ontogeny of the c-ret expressing cell-lineage in the null genetic background. Preliminary analysis indicate a reduction in Lac Z staining intensity in DRG isolated from mutant transgenic neonates compared to wild type transgenic neonates. Conversely, preliminary experiments indicate increased numbers of Lac Z staining c-cells in the thyroids of mutant transgenic neonates compared to the wild type transgenic thyroids. Further, more detailed quantitative analysis is required to substantiate these interesting preliminary findings. The detailed analysis of the possibility of plasticity, functional compensation or redundancy in c-ret function within the neural crest cell linage which establish the sensory nervous system and the c-cells of the thyroid, remains for further study.
... (He et al., 1989;Bulfone et al., 1995;Hevner, 2007;Molyneaux et al., 2007;Toma and Hanashima, 2015;Glatzle et al., 2017). This revealed that, in ...
... (He et al., 1989;Bulfone et al., 1995;Hevner, 2007;Molyneaux et al., 2007;Toma and Hanashima, 2015;Glatzle et al., 2017). This revealed that, in ...
Open spina bifida (OSB) is one of the most prevalent congenital malformations of the CNS that often leads to severe disabilities. Previous studies reported the volume and thickness of the neocortex to be altered in children and adolescents diagnosed with OSB. Until now, the onset and the underlying cause of the atypical neocortex organization in OSB patients remain largely unknown. To examine the effects of OSB on fetal neocortex development, we analyzed human fetuses of both sexes diagnosed with OSB between 11 and 15 weeks of gestation by immunofluorescence for established neuronal and neural progenitor marker proteins and compared the results with healthy controls of the same, or very similar, gestational age. Our data indicate that neocortex development in OSB fetuses is altered as early as 11 weeks of gestation. We observed a marked reduction in the radial thickness of the OSB neocortex, which appears to be attributable to a massive decrease in the number of deep- and upper-layer neurons per field, and found a marked reduction in the number of basal progenitors (BPs) per field in the OSB neocortex, consistent with an impairment of cortical neurogenesis underlying the neuronal decrease in OSB fetuses. Moreover, our data suggest that the decrease in BP number in the OSB neocortex may be associated with BPs spending a lesser proportion of their cell cycle in M-phase. Together, our findings expand our understanding of the pathophysiology of OSB and support the need for an early fetal therapy (i.e., in the first trimester of pregnancy).
... 6 " The Pou1f1 lineages comprise three cell types within the anterior pituitary that all require the expression of the pituitary-specific POU domain transcription factor, Pou1f1, for their development and for their continued function in the adult pituitary. These cell types, the thyrotropes, lactotropes, and somatotropes, produce thyroid stimulating hormone, prolactin, and growth hormone, respectively [7][8][9] . Both mice and humans with mutations in the Pou1f1 gene are characterized by a deficiency of growth hormone, prolactin, and thyroid stimulating hormone, highlighting that the differentiation and expansion of all three of these cell lineages share a common reliance on the actions of Pou1f1 10,11 . ...
The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the growth hormone (GH) producing somatotropes and the prolactin (PRL) producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors, that contribute to the somatotrope and lactotrope cell identities. Here, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of mRNAs that are selectively enriched in each of the two populations includes a set of transcription factors. A subset of these factors may have roles in pituitary lineage divergence, and/or in regulating expression of cell-type specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope-enriched and two lactotrope-enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark somatotrope or lactotrope genes in a pituitary-derived cell culture model system. Conditional inactivation of the genes encoding these factors in mice revealed that two of these transcription factors impact the expression of landmark hormone genes and alter cell phenotypes in primary murine pituitary. Subsequent mechanistic studies revealed that these two factors act on their target genes either through a direct binding at the target promoter and release of paused Pol II complexes, or through indirect mechanism(s). The functions of these two factors are further remarkable in how they maintain hormone expression in the lactotrope and somatotrope lineages; one serves to enhance landmark Prl gene expression in lactotropes while the second serves to reciprocally repress expression of the Prl gene in somatotropes. In conclusion, these studies identify novel regulators of the somatotropes and lactotropes, explore their mechanisms of action, and establish a useful database for further study of these lineages in the anterior pituitary.
... 1 The importance of POU3F3 for the developing brain is reflected in its original name Brain-1 (Brn1). 2 Best known as a marker of upper-layer projection neurons in the cortex, 3 POU3F3 is implicated in the regulation of many key processes in the development of the central nervous system; these processes include cortical neuronal migration, 4 upper-layer specification and production, and neurogenesis. [5][6][7] However, the phenotypic consequences of pathogenic germline variants in human POU3F3 are currently unknown. ...
POU3F3, also referred to as Brain-1, is a well-known transcription factor involved in the development of the central nervous system, but it has not previously been associated with a neurodevelopmental disorder. Here, we report the identification of 19 individuals with heterozygous POU3F3 disruptions, most of which are de novo variants. All individuals had developmental delays and/or intellectual disability and impairments in speech and language skills. Thirteen individuals had characteristic low-set, prominent, and/or cupped ears. Brain abnormalities were observed in seven of eleven MRI reports. POU3F3 is an intronless gene, insensitive to nonsense-mediated decay, and 13 individuals carried protein-truncating variants. All truncating variants that we tested in cellular models led to aberrant subcellular localization of the encoded protein. Luciferase assays demonstrated negative effects of these alleles on transcriptional activation of a reporter with a FOXP2-derived binding motif. In addition to the loss-of-function variants, five individuals had missense variants that clustered at specific positions within the functional domains, and one small in-frame deletion was identified. Two missense variants showed reduced transactivation capacity in our assays, whereas one variant displayed gain-of-function effects, suggesting a distinct pathophysiological mechanism. In bioluminescence resonance energy transfer (BRET) interaction assays, all the truncated POU3F3 versions that we tested had significantly impaired dimerization capacities, whereas all missense variants showed unaffected dimerization with wild-type POU3F3. Taken together, our identification and functional cell-based analyses of pathogenic variants in POU3F3, coupled with a clinical characterization, implicate disruptions of this gene in a characteristic neurodevelopmental disorder.
... Expression changes in POU3F2 have been observed in neurons derived from SCZ patient-specific induced pluripotent stem cells (38). POU3F2 was discovered on the basis of the sequence similarity of the POU domain and is also known as Brain-2 (Brn-2), because it is expressed in the central nervous system (39). The function of POU3F2 was initially studied in melanocytic cells. ...
Schizophrenia and bipolar disorder are complex psychiatric diseases with risks contributed by multiple genes. Dysregulation of gene expression has been implicated in these disorders, but little is known about such dysregulation in the human brain. We analyzed three transcriptome datasets from 394 postmortem brain tissue samples from patients with schizophrenia or bipolar disorder or from healthy control individuals without a known history of psychiatric disease. We built genome-wide coexpression networks that included microRNAs (miRNAs). We identified a coexpression network module that was differentially expressed in the brain tissue from patients compared to healthy control individuals. This module contained genes that were principally involved in glial and neural cell genesis and glial cell differentiation, and included schizophrenia risk genes carrying rare variants. This module included five miRNAs and 545 mRNAs, with six transcription factors serving as hub genes in this module. We found that the most connected transcription factor gene POU3F2, also identified on a genome-wide association study for bipolar disorder, could regulate the miRNA hsa-miR-320e and other putative target mRNAs. These regulatory relationships were replicated using PsychENCODE/BrainGVEX datasets and validated by knockdown and overexpression experiments in SH-SY5Y cells and human neural progenitor cells in vitro. Thus, we identified a brain gene expression module that was enriched for rare coding variants in genes associated with schizophrenia and that contained the putative bipolar disorder risk gene POU3F2. The transcription factor POU3F2 may be a key regulator of gene expression in this disease-associated gene coexpression module.
... Expression changes in POU3F2 have been observed in neurons derived from SCZ patient-specific induced pluripotent stem cells (38). POU3F2 was discovered based on the sequence similarity of the POU domain and is also known as Brain-2 (Brn-2) since it is expressed in the central nervous system (39). The function of POU3F2 was initially studied in melanocytic cells. ...
Schizophrenia and bipolar disorder are complex psychiatric diseases with risks contributed by multiple genes. Dysregulation of gene expression has been implicated in these disorders, but little is known about such dysregulation in the human brain. We analyzed three transcriptome datasets from 394 postmortem brain tissue samples from patients with schizophrenia or bipolar disorder or from healthy control individuals without a known history of psychiatric disease. We built genome-wide coexpression networks that included microRNAs (miRNAs). We identified a coexpression network module that was differentially expressed in the brain tissue from patients compared to healthy control individuals. This module contained genes that were principally involved in glial and neural cell genesis and glial cell differentiation, and included schizophrenia risk genes carrying rare variants. This module included five miRNAs and 545 mRNAs, with six transcription factors serving as hub genes in this module. We found that the most connected transcription factor gene POU3F2, also identified on a genome-wide association study for bipolar disorder, could regulate the miRNA hsa-miR-320e and other putative target mRNAs. These regulatory relationships were replicated using PsychENCODE/BrainGVEX datasets and validated by knockdown and overexpression experiments in SH-SY5Y cells and human neural progenitor cells in vitro. Thus, we identified a brain gene expression module that was enriched for rare coding variants in genes associated with schizophrenia and that contained the putative bipolar disorder risk gene POU3F2. The transcription factor POU3F2 may be a key regulator of gene expression in this disease-associated gene coexpression module.
... neural tube (Mazet et al., 2005), NA (Albuixech-Crespo et al., 2017;Matsunaga et al., 2001;Miyamoto and Wada, 2013) Neural/neural tube marker Zic-r.a (zinc finger TF) Macho-1 Ciona in CNS with 2 gaps (Gostling and Shimeld, 2003), NA (Gostling and Shimeld, 2003;Aruga, 2004) Halocynthia roretzi, not in CNS (Aruga, 2004), (Knecht et al., 1993) Notch (transmembrane receptor) Halocynthia roretzi preferentially in CNS (Akanuma et al., 2002;Hori et al., 1997) NA (Holland et al., 2001;Louvi and Artavanis-tsakonas, 2006) Pou4 (Pou class IV homeobox TF) Ciona PNS and posterior sensory vesicle (Candiani et al., 2005), Ciona endostyle (Mazet et al., 2005), (Candiani et al., 2006;He et al., 1989) The genus Ciona, refers to either Ciona robusta or Ciona intestinalis (Brunetti et al., 2015), if the reference dates before 2015 and no indication of the type A or B was given. NS: nervous system; SV: sensory vesicle; CF: ciliated funnel. ...
During metamorphosis of solitary ascidians, part of the larval tubular nervous system is recruited to form the adult central nervous system (CNS) through neural stem-like cells called ependymal cells. The anteroposterior (AP) gene expression patterning of the larval CNS regionalize the distribution of the ependymal cells, which contains the positional information of the neurons of the adult nervous system. In colonial ascidians, the CNS of asexually developed zooids has the same morphology of the one of the post-metamorphic zooids. However, its development follows a completely different organogenesis that lacks embryogenesis, a larval phase and metamorphosis. In order to describe neurogenesis during asexual development (blastogenesis), we followed the expression of six CNS AP patterning genes conserved in chordates and five neural-related genes to determine neural cell identity in Botryllus schlosseri. We observed that a neurogenesis occurs de novo on each blastogenic cycle starting from a neurogenic transitory structure, the dorsal tube. The dorsal tube partially co-opts the AP patterning of the larval CNS markers, and potentially combine the neurogenesis role and provider of positional clues for neuron patterning. This study shows how a larval developmental module is reused in a direct asexual development in order to generate the same structures.
... The NR4A2 protein was enriched in the nuclei of lactotrope (PRL + ) cells (Fig. 3D), consistent with transcriptomic data. POU4F1, a member of the POU family of transcription factors, was selected for study owing to its role in stimulating aggressive proliferation of pituitary adenomas (58), and on its multiple regulatory roles in development throughout the neuroendocrine system (59,60). We observed that the POU4F1 protein was enriched in the nuclei of lactotrope cells in mouse pituitary, suggesting a potential for regulating the lactotrope lineage (Fig. 3E). ...
The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the growth hormone (GH) producing somatotropes and prolactin (PRL) producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors, that contribute to the somatotrope and lactotrope cell identities. Here, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of divergent mRNAs between the two populations includes a set of transcription factors that may have roles in pituitary lineage divergence, and/or in regulating expression of cell-type specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope-enriched and two lactotrope-enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark genes in a cell culture model system. These studies identify novel regulators of the somatotropes and lactotropes, and establish a useful database for further study of these lineages in the anterior pituitary.
... In contrast with these results, Pit1 expression has been demonstrated in other extra-pituitary locations in the reproductive system, in the chicken testes and vas deferens, and in the mammalian placental tissues (Harvey et al., 2004a). Pit1 protein and/or Pit1 mRNA have also been described in the developing nervous system, particularly in the brain and neural retina, but then disappeared during adulthood (Harvey et al., 2000;He et al., 1989). Thus, it is possible that the expression of Pit1 in extra-pituitary tissues and its influence upon GH expression may be dependent on developmental-stage or in the particular physiological status of the tissue, and this should be further investigated. ...
The human growth hormone (GH) locus is comprised by two GH (GH1 and GH2) genes and three chorionic somatomammotropin (CSH1, CSH2 and CSH-L) genes. While GH1 is expressed in the pituitary gland, the rest are expressed in the placenta. However, GH1 is also expressed in several extrapituitary tissues, including the eye. So to understand the role of this hormone in the eye we used the baboon (Papio hamadryas), that like humans has a multigenic GH locus; we set up to investigate the expression and regulation of GH locus in adult and fetal baboon ocular tissues.
We searched in baboon ocular tissues the expression of GH1, GH2, CSH1/2, Pit1 (pituitary transcription factor 1), GHR (growth hormone receptor), GHRH (growth hormone releasing hormone), GHRHR (growth hormone releasing hormone receptor), SST (somatostatin), SSTR1 (somatostatin receptor 1), SSTR2 (somatostatin receptor 2), SSTR3 (somatostatin receptor 3), SSTR4 (somatostatin receptor 4), and SSTR5 (somatostatin receptor 5) mRNA transcripts and derived proteins, by qPCR and immunofluorescence assays, respectively. The transcripts found were characterized by cDNA cloning and sequencing, having found only the one belonging to GH1 gene, mainly in the retina/choroid tissues. Through immunofluorescence assays the presence of GH1 and GHR proteins was confirmed in several retinal cell layers. Among the possible neuroendocrine regulators that may control local GH1 expression are GHRH and SST, since their mRNAs and proteins were found mainly in the retina/choroid tissues, as well as their corresponding receptors (GHRH and SSTR1-SSTR5). None of the ocular tissues express Pit1, so gene expression of GH1 in baboon eye could be independent of Pit1. We conclude that to understand the regulation of GH in the human eye, the baboon offers a very good experimental model.
... To determine the main period of neurogenesis, specifically the timing of deep and upper layer generation, in the domestic cat, pig and sheep neocortex, we analysed cortical sections from a broad range of developmental stages by immunohistochemistry for specific neuronal markers, that is Tbr1 characteristically expressed by deep layer neurons and Brn2 characteristically expressed by upper layer neurons (He et al., 1989;McEvilly et al., 2002;Hevner, 2007;Molyneaux et al., 2007;Toma and Hanashima, 2015;Sauerland et al., 2016). In all three species analysed, Tbr1+ neurons were born at early stages during development and predominantly accumulated in the deep layers of the CP whereas Brn2+ neurons were born at later stages during development and predominantly accumulated in the upper layers of the CP (Figs 1-3). ...
The neocortex plays a key role in cognition, volitional motor control and sensory perception and has undergone tremendous expansion during evolution. The mature neocortex consists of radially aligned neurons that are arranged in six layers. Layers II-VI are often split into two groups: deep and upper layers, both building up the so-called cortical plate during embryonic and foetal development. So far cortical neurogenesis, including the generation of deep and upper layers, has mostly been studied in laboratory rodents and primates. However, precise data for most companion animals are lacking. This study determined the main period of neurogenesis, specifically the timing of deep and upper layer generation, in the developing domestic cat, pig and sheep neocortex using immunohistochemistry for specific neuronal markers, that is Tbr1 and Brn2. We found that the general sequence of neural events is preserved among cat, pig, sheep and other mammalian species. However, we observed differences in the timing of the overall cortical neurogenic period and occurrence of distinct neural events when these three species were compared. Moreover, our data provide further evidence that the cortical neurogenic period and gestation length might be tightly related. Together, these data expand our current understanding of neocortex development and are important for future studies investigating neocortex development and expansion especially in companion animals.
... Due to crosscoupling between regulatory pathways (Schiile and Evans 1991), the number of regulatory proteins (transcriptional regulators and others) involved in most intracellular regulatory circuits is probably large. However, those circuits presumably relying to a large extent on transcriptional regulation may be quite small, involving 10 to 100 or fewer genes, according to the available circumstantial evidence (Ingham 1988;He et al. 1989;McGinnis and Krumlauf 1992). ...
During the development of a multicellular organism from a zygote, a large number of epigenetic interactions take place on every level of suborganismal organization. This raises the possibility that the system of epigenetic interactions may compensate or "buffer" some of the changes that occur as mutations on its lowest levels, and thus stabilize the phenotype with respect to mutations. This hypothetical phenomenon will be called "epigenetic stability." Its potential importance stems from the fact that phenotypic variation with a genetic basis is an essential prerequisite for evolution. Thus, variation in epigenetic stability might profoundly affect attainable rates of evolution. While representing a systemic property of a developmental system, epigenetic stability might itself be genetically determined and thus be subject to evolutionary change. Whether or not this is the case should ideally be answered directly, that is, by experimentation. The time scale involved and our insufficient quantitative understanding of developmental pathways will probably preclude such an approach in the foreseeable future. Preliminary answers are sought here by using a biochemically motivated model of a small but central part of a developmental pathway. Modeled are sets of transcriptional regulators that mutually regulate each other's expression and thereby form stable gene expression patterns. Such gene-expression patterns, crucially involved in determining developmental pattern formation events, are most likely subject to strong stabilizing natural selection. After long periods of stabilizing selection, the fraction of mutations causing changes in gene-expression patterns is substantially reduced in the model. Epigenetic stability has increased. This phenomenon is found for widely varying regulatory scenarios among transcription factor genes. It is discussed that only epistatic (nonlinear) gene interactions can cause such change in epigenetic stability. Evidence from paleontology, molecular evolution, development, and genetics, consistent with the existence of variation in epigenetic stability, is discussed. The relation of epigenetic stability to developmental canalization is outlined. Experimental scenarios are suggested that may provide further evidence.
... The putative promoter region of zPOMGnT1 gene contained putative binding sites for MyoD and E47 with flanking ( Figure S1). MyoD is a master regulator of skeletal muscle development belonging to the family of basic helix-loop-helix (bHLH) myogenic transcriptional factors which induce muscle-specific transcription upon binding to E-box consensus sequences, and acquires high affinity for the muscle E-box upon heterodimerization through the HLH domain with ubiquitous bHLH protein such as E47 so-called E proteins [44]. These results suggest that the function of zPOMGnT1 might be more essential to not only muscle but also female tissues during adulthood. ...
Muscular dystrophies are genetic diseases characterized by progressive muscle degeneration and muscular weakening. Defects in glycosylation of α-dystroglycan are responsible for certain congenital muscular dystrophies to be called α-dystroglycanopathies. The structure of glycans in α-dystroglycan is Siaα2-3Galβ1-4GlcNAcβ1- 2Manα1-Ser/Thr and required for binding basal lamina proteins. The first step of O-mannosyl glycan synthesis on α-dystroglycan is catalyzed by protein O-mannosyltransferases (POMT1 and POMT2), and detect in POMT1 or POMT2 result in Walker-Warburg syndrome one of the α-dystroglycanopathies. Next step is catalyzed by O-mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) and it is responsible for muscle-eye-brain disease. We have previously reported that protein O-mannosylation is necessary for normal embryonic development in zebrafish and revealed that zebrafish is a useful model for α-dystroglycanopathies. In this study, we focused on zebrafish POMGnT1. Zebrafish POMGnT1 revealed high level of expression in ovary and ubiquitously throughout early developmental stage as well as zebrafish POMT1 and POMT2. Morpholino experiments of zebrafish POMGnT1 in juvenile zebrafish showed several phenotypes of bended body, small eyes and edematous pericardium. More importantly, morpholino-injected zebrafish had reduction of the reactivity to the monoclonal antibody IIH6 that recognizes a glycosylated α-dystroglycan. Furthermore, phenotypes observed by knockdown of zebrafish POMGnT1 were similar to zebrafish POMT2 rather than zebrafish POMT1. Finally, in order to measure POMGnT1 activity, we cloned and expressed zebrafish POMGnT1 in human embryonic kidney 293T cells. As a result, zebrafish POMGnT1 had the enzymatic activity to transfer GlcNAc from UDP-GlcNAc to O-mannosyl peptide, indicating that O-mannosylation pathway of α-dystroglycan is conserved in zebrafish.
... POU3F1 (also known as OCT6, TST-1) is a POU-subclass homeobox transcription factor, and it is expressed in testis as well as in brain. In previous studies, POU3F1 has been examined as a regulator of neural cell development (13)(14)(15). Targeted disruption of POU3F1 expression in mice caused neonatal lethality in which pups displayed abnormal myelination of the axon sheath (13). POU3F1 was then first recognized as a candidate involved in SSCs when it was shown to be highly expressed in rat germ cells with enriched SSC activity (16). ...
As is similar to glial cell line-derived neurotrophic factor (GDNF), the Yangjing Capsule (YC) extract could also lead to proliferation of spermatogonial stem cells (SSCs) by stimulating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway; however, the regulatory effect of YC extract on the expression of POU3F1 still remains unknown. The objective of this study is to determine whether the transcription factor POU3F1 is up-regulated by YC extract through the PI3K/AKT signaling pathway to regulate SSCs survival and proliferation. Cultured GC-1 spermatogonial (spg) cells were treated with 0.01, 0.1, and 1 mg/mL YC extract for 48 h. Cell viability was analyzed using MTT assay, while POU3F1 expression was quantitatively detected using real time-polymerase chain reaction and Western blot analysis. POU3F1, GDNF family receptor alpha1 (GFRα1) short interfering ribonucleic acid (siRNA), and LY294002 (PI3K inhibitor) were applied as blockers to explore the underlying pathway. After 48 h treatment with YC extract, GC-1 spg cells proliferated and POU3F1 expression was significantly increased in a dose-dependent manner. POU3F1 siRNA partially blocked those effects of YC extract. Both GFRα1 siRNA and LY294002, as upstream blockers, reduced POU3F1 expression induced by YC extract. The conclusion is that YC extract promotes proliferation of GC-1 spg cells via up-regulation of POU3F1. The potential mechanism is that YC extract triggers the activation of the PI3K/AKT pathway and then up-regulates POU3F1 expression.
... Alternatively, the control of genes specifically expressed in the brain has been investigated by introduction of expression vectors into transgenic mice to map the sequences required for directed expression. For example, the 5'-flanking region of the glial-specific gene for myelin basic protein has been shown to direct appropriate expression in transgenic mice (6), as have the cloned nerve growth factor (NGF) receptor gene (7), the L7 purkinje cell gene (8), a rat brain type II sodium channel gene (9), the SCGlO gene (lo), and the amyloid precursor protein gene (1 l), among others. However, this approach does not provide access to the specific regulatory proteins important for targeting expression of these genes. ...
The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dram...
... POU is an acronym for Pit-1 expressed in the anterior pituitary, Oct-1 widely expressed, Oct-2 expressed in the immune and nervous systems, and Unc-86 involved in the neuronal cell development of the nematode Caenorhabditis elegans. With the occurrence of several proteins of the POU family in mammals (He et al., 1988), Drosophila (Johnson and Hirsch, 1990), and C. elegans (Burglin et al., 1989), it seems that the POU domains are highly conserved in eukaryotes. The POU domain is characterized by two regions, the POUspecific (POUs) domain (67-70 amino acids) and the POU homeodomain (POUhd) (60 amino acids), which are separated by a nonconserved sequence 14-25 amino acids long (Herr et al., 1988). ...
... Electrophoretic mobility shift assays (EMSAs) of nuclear extracts purified from mouse cell lines and tissues at different developmental stages with an octamer motif-containing DNA probe revealed 11 distinct band shifts, indicating the existence of 11 Oct factors (Oct-1 to -11) [3,5,6]. However, only eight Oct factors thus far, Oct-1 (POU2F1) [7], Oct-2 (POU2F2) [8], Oct-3/4 (POU5F1) [9], Oct-6 (POU3F1) [10], Oct-7 (POU3F2) [11,12], Oct-8 (POU3F3) [13], Oct-9 (POU3F4) [14], and Oct-11 (POU2F3) [5], have been cloned and characterized. At least some of the remaining band might represent degraded products or the splicing variants of these genes. ...
The expression of Oct-1 and -2 and their binding to the octamer motif in the mammary gland are developmentally and hormonally regulated, consistent with the expression of milk proteins. Both of these transcription factors constitutively bind to the proximal promoter of the milk protein gene β-casein and might be involved in the inhibition or activation of promoter activity via interactions with other transcription factors or cofactors at different developmental stages. In particular, the lactogenic hormone prolactin and glucocorticoids induce Oct-1 and Oct-2 binding and interaction with both the signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor on the β-casein promoter to activate β-casein expression. In addition, increasing evidence has shown the involvement of another Oct factor, Oct-3/4, in mammary tumorigenesis, making Oct-3/4 an emerging prognostic marker of breast cancer and a molecular target for the gene-directed therapeutic intervention, prevention and treatment of breast cancer. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin.
... For instance, the murine oncogene int-l, which is related to the Drosophila gene wingless, is expressed in a subset of cells at the lateral edge of the neural plate (Shackelford & Varmus, 1987;Wilkinson, Bailes, & McMahon, 1987). Also, the POU-domain genes encode a family of proteins that are expressed in various subsets of cells in the developing and the adult mammalian nervous system (He, Treacy, Simmons, Ingraham, Swanson, & Rosenfeld, 1989). Although it has not been proved, it is likely that the development of the human nervous system is controlled by such genes. ...
Developmental disorders affecting intelligence and behavior are among the most mystifying diseases of humankind. This is because they are primarily caused by abnormalities in the development of the most complex human organ, the brain. Perhaps the reason so little is understood about mental retardation, autism, childhood schizophrenia, attention deficit disorder, specific learning disorders, and Tourette disorder is that very little is still understood about how a normal brain develops. The premise of this chapter is that the key to understand these illnesses is an understanding of the processes involved in neurodevelopment.
... POUh et POUs sont séparés par un fragment peptidique appelé linker, non structuré, de longueur variable selon les facteurs de la famille POU (voir Sturm and Herr, 1988). Les propriétés du peptide linker ont été la base de la répartition des facteurs POU en 7 sous-familles (He et al., 1989;Ryan and Rosenfeld, 1997;Wegner et al., 1993). Au sein du peptide linker, on trouve notamment le signal de localisation nucléaire (KKMRRNRFK) permettant l'importation de HNF1alpha et de HNF1beta à travers l'enveloppe nucléaire (Chi et al., 2002;Lu et al., 2007). ...
HNF1beta est un facteur de transcription homeobox, dont les mutations sont fréquemment rencontrées chez des patients atteints d’anomalies congénitales du rein et du tractus urinaire (Congenital Abnomalities of the Kidney and the Urogenital Tract, CAKUT). HNF1beta est également impliqué dans le diabète de type Maturity Onset Diabetes of the Young 5 (MODY5). Le laboratoire d’accueil a démontré que HNF1beta était impliqué dans un mécanisme épigénétique, le Bookmarking, nécessaire à la réexpression post-mitotique de ses gènes cibles. En particulier, des expériences de vidéo-microscopie ont montré que la partie N-terminale de HNF1beta, contenant le domaine de liaison à l’ADN, en fusion avec la GFP (HNF1beta -GFP) est liée à la chromatine pendant la mitose. L’objectif de ma thèse était de caractériser les modalités biochimiques d’interaction de HNF1beta avec la chromatine mitotique. Nous avons mis en évidence le fait que la capacité de liaison à l’ADN est indispensable à la localisation mitotique de HNF1beta. En effet, la délétion de la troisième hélice alpha de l’homéo-domaine, responsable de l’interaction avec le grand sillon de l’ADN, entraîne la dissociation de la chromatine de HNF1beta pendant la mitose. Nous avons ensuite étudié l’effet de plusieurs mutations identifiées chez des patients MODY sur la localisation mitotique de HNF1beta. Nos résultats ont montré que certaines mutations faux-sens sont capables d’empêcher la fixation de la chromatine mitotique. Parmi ces mutations, certaines manifestent un phénotype dépendant de la température. Par exemple, à une température permissive, inférieure à 30°C, les mutations P256S et C273Y présentent une localisation mitotique normale. En revanche, à 37°C pour P256S et à 39°C pour le mutant C273Y, les protéines sont complètement dissociées, alors que dans toutes ces conditions de température, l’association de la protéine sauvage avec la chromatine mitotique n’est pas affectée. A température permissive (4°C), nous avons montré par retard sur gel (Electophoresis Mobility Shift Assay EMSA) que les mutants lient l’ADN avec un Kd apparent similaire à celui de la protéine sauvage. Par contre, à température restrictive, les mutants présentent des comportements différents. En effet, P256S perd sa capacité de liaison à l’ADN (de façon réversible), tandis que C273Y continue à lier l’ADN avec une affinité similaire à celui de la protéine sauvage. Le caractère thermosensible des mutants de HNF1beta nous a permis d’étudier les modalités de son recrutement sur la chromatine mitotique. Nos résultats ont montré que l’association des protéines à la chromatine mitotique présente une nature très dynamique. En effet, nous avons observé qu’une diminution rapide de température détermine la relocalisation mitotique réversible de la protéine, dans un délai de quelques secondes. Nous avons pu montrer que la relocalisation mitotique de HNF1beta induit par la température était affectée par une déplétion d’énergie, ainsi que par l’action d’un inhibiteur spécifique de l’importine-β (importazole). Nous avons enfin mis en évidence par immuno-précipitation de chromatine (ChIP) que la liaison de HNF1beta à la chromatine mitotique est séquence-spécifique. Nos résultats suggèrent que le recrutement de HNF1beta à la chromatine mitotique est énergie-dépendante, et nécessite le bon fonctionnement du système de transport lié à l’importine-beta. Mes résultats suggèrent que des mutations trouvées chez des patients MODY3 et MODY5 inactivent ou affaiblissent la capacité de HNF1beta de remplir son activité de Bookmarking.
... L'analyse réalisée au laboratoire des séquences régulatrices en cis dirigeant l'expression de Krox20 dans les cellules de Schwann a permis de caractériser deux éléments distincts (Ghislain et al., 2002). Le (He et al., 1989;Wegner et al., 1993). Bien que ces facteurs soient exprimés dans des domaines se recouvrant largement, ils ne sont pas reliés génétiquement et ne partagent aucune séquence régulatrice au niveau de leur locus génique (Alvarez-Bolado et al., 1995;Suzuki et al., 1990;Zwart et al., 1996). ...
During embryonic development, the posterior part of vertebrates brain, or hindbrain, goes through a transient segmentation process along the anterio-posterior axis, leading to the definition of metameric units called rhombomeres. They correspond to neuronal differenciation units and allow the organization of cranial nerves. They also play a major role in cranio-facial structures development through regionalized production of neural crest. The Krox20 gene encodes a transcription factor which is expressed in rhombomeres r3 and r5. It has been shown that its expression in these domains is necessary to define and maintain them, as well as to generate the corresponding cranial nerves properly. Krox20 controls indeed the transcriptional expression of various genes implicated in particular in rhombomeres identity and intactness. Some cofactors of Krox20, as Nab or HCF-1, had been previously described but their role in vivo had not been addressed, in particular during hindbrain development. The first aim of my PhD was to better understand the mechanisms on which Krox20 activity relies in vivo and in particular the regulating roles of some of its cofactors. To address these questions, I performed a structure-function study in vivo using chick embryo as a model. I showed that Krox20 activity during hindbrain development relies on different domains of the protein depending on the transcriptional target considered. I also showed that Nab proteins are implicated in a negative feedback loop regulating Krox20 activation function, whereas HCF-1 is a positive cofactor of Krox20 for the activation of two of its transcriptional targets. Furthermore, this study allowed us to characterize new interaction sites between HCF-1 and Krox20. Krox20 is also a key factor of the myelination process in the peripheral nervous system (PNS), on which the rapid saltatory conduction along nerves relies. Various human pathologies are due to hypomyelination or demyelinating events. A Krox20 knock-out mouse model previously generated in the laboratory had shown a lack of myelination of the PNS in the absence of Krox20. This was due to a defect of differenciation of the myelinating Schwann cells in which Krox20 is normally expressed. Following this study, various mutations in Krox20 had been characterized in hereditary myelinopathies of the PNS, such as Charcot-Marie-Tooth diseases (CMT). In particular, one of these mutations, that leads to a severe early-onset subtype of recessive CMT, appears to abolish the interaction between Krox20 and its cofactors Nab. The second aim of my PhD was to characterize the role of Nab interaction with Krox20 during the myelination process and the hindbrain segmentation in vivo. For this purpose, I generated a mouse model of CMT carrying the mutation abolishing Krox20/Nab interaction. Homozygous mutant mice show a phenotype similar to what is observed in human patients. These mice have locomotion defects evolving progressively to limbs paralysis and are affected by a severe defect in PNS myelination. My studies showed that the myelination process is first delayed, leading subsequently to an hypomyelination of the PNS, followed by demyelination. These defects appear to be linked to a delay in proliferation arrest of Schwann cells and a disregulation of gene expression in these cells. Surprisingly, Nab proteins appear to be positive cofactors of Krox20 in Schwann cells. Furthermore, I showed that these mutant mice present cranial nerves abnormalities which are compatible with functional impairments observed in human patients carrying this mutation. In summary, the studies made during my PhD allowed to show the complexity of Krox20 transcriptional activity, relying on different target-specific domains of the protein, as well as to better characterize the role of two of its cofactors during hindbrain development. This work also lead to the generation of a mouse model for a severe form of CMT, allowing a better understanding of its complex pathomechanisms.
The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of CN processing and integration. Nevertheless our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a genetic inducible fate mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. They are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral to medial progression. While some markers label a heterogeneous population of neurons sharing a common location (Brn2), others appear to be lineage specific (Tbr1, Lmx1a, Meis2). A comparative analysis of Tbr1 and Lmx1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings lay the groundwork for focused functional analyses of individual subpopulations of nuclear neurons.
Cancer stem-like cells (CSCs) have self-renewal abilities responsible for cancer progression, therapy resistance, and metastatic growth. The glioblastoma stem-like cells are the most studied among CSC populations. A recent study identified four transcription factors (SOX2, SALL2, OLIG2, and POU3F2) as the minimal core sufficient to reprogram differentiated glioblastoma (GBM) cells into stem-like cells. Transcriptomic data of GBM tissues and cell lines from two different datasets were then analyzed by the SWItch Miner (SWIM), a network-based software, and FOSL1 was identified as a putative regulator of the previously identified minimal core. Herein, we selected NTERA-2 and HEK293T cells to perform an in vitro study to investigate the role of FOSL1 in the reprogramming mechanisms. We transfected the two cell lines with a constitutive FOSL1 cDNA plasmid. We demonstrated that FOSL1 directly regulates the four transcription factors binding their promoter regions, is involved in the deregulation of several stemness markers, and reduces the cells’ ability to generate aggregates increasing the extracellular matrix component FN1. Although further experiments are necessary, our data suggest that FOSL1 reprograms the stemness by regulating the core of the four transcription factors.
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two devastating and fatal neurodegenerative conditions. While distinct, they share many clinical, genetic, and pathological characteristics, and both show selective vulnerability of layer 5b extratelencephalic-projecting cortical populations, including Betz cells in ALS and von Economo neurons (VENs) in FTLD. Here, we report the first high resolution single-cell atlas of the human primary motor cortex (MCX) and its transcriptional alterations in ALS and FTLD across ~380,000 nuclei from 64 individuals, including 17 control samples and 47 sporadic and C9orf72 -associated ALS and FTLD patient samples. We identify 46 transcriptionally distinct cellular subtypes including two Betz-cell subtypes, and we observe a previously unappreciated molecular similarity between Betz cells and VENs of the prefrontal cortex (PFC) and frontal insula. Many of the dysregulated genes and pathways are shared across excitatory neurons, including stress response, ribosome function, oxidative phosphorylation, synaptic vesicle cycle, endoplasmic reticulum protein processing, and autophagy. Betz cells and SCN4B + long-range projecting L3/L5 cells are the most transcriptionally affected in both ALS and FTLD. Lastly, we find that the VEN/Betz cell-enriched transcription factor, POU3F1, has altered subcellular localization, co-localizes with TDP-43 aggregates, and may represent a cell type-specific vulnerability factor in the Betz cells of ALS and FTLD patient tissues.
The Brn-3a and Brn-3b POU factors were originally identified in neuronal cells and neuroblastomas, but their expression in normal cervical and mammary cells has also been reported. As these factors may regulate the expression of genes involved in gynaecological cancers, their pattern of expression and potential role in tumourgenesis was investigated in both breast and cervical neoplastic tissues. The Brn-3 factors have been shown to have antagonistic effects on the transcription of the human papilloma virus types 16 and 18 (HPV-16/18) E6 and E7 oncogenes. Thus, while Brn-3a activates the expression of E6 and E7, Brn-3b represses it. The levels of Brn-3a and Brn-3b were therefore quantified in neoplastic and normal cervical biopsies. Brn-3a was seen to be over-expressed in high grade cervical intraepithelial neoplasia (CIN3) associated with HPV, as compared to normal cervical tissues lacking HPV. This effect was not observed for Brn-3b. To appreciate the role of Brn-3a in cervical neoplasia, its expression in cervical carcinoma-derived cell lines with or without endogenous HPV genes was manipulated. The artificial reduction of Brn-3a, reduces expression of the viral E6 and E7 products. Moreover, morphology, growth rate, saturation density and the anchorage independent proliferation of the cells were altered in the HPV-containing cervical carcinoma cells, but not in similar cells lacking HPV. This indicates that, Brn-3a may play a significant role in HPV-associated cervical cancer. It has also been shown that Brn-3b can repress the promoter of the BRCA-1 tumour suppressor gene. Thus, to Study the role played by Brn-3a and Brn-3b in breast tumourigenesis, the mRNA and protein expression levels of Brn-3a and Brn-3b and that of BRCA-1, in normal, benign and malignant breast tissues were compared. It was found that Brn-3b was over-expressed in a number of breast cancer samples and that these samples also showed reduced expression of BRCA- 1. No correlation was observed between Brn-3a and BRCA-1 expression. The data taken together with the results obtained from recent studies carried out in our laboratory, indicate for the first time that, the Brn-3b transcription factor regulates the expression of BRCA-1 in sporadic breast cancer.
ATP has been shown to directly activate sensory neurones in culture and evoke a sensation of pain on human blister bases. Molecular cloning of P2X3 showed that it is a sensory neurone-specific ATP-gated channel. The recombinant P2X3 thus provides a possible connection between ATP and nociception. The aim of this thesis is to characterise the expression pattern and function of the cloned P2X3 receptor. The full length of the P2X3 transcript is 3.8 kb, with a 397 amino acid coding region. The primary sequence of P2X3 shows a 36-48% homology to other P2X receptors and comprises a similar structure of two transmembrane domains, short intracellular N- and C-terminals, a large extracellular loop, and ten conserved cysteine residues. Northern analysis and in situ hybridisation showed that the P2X3 transcript is exclusively expressed in a subset of sensory neurones which are mainly peripherin positive and capsaicin sensitive small diameter neurones. Such neurones contain thin or nonmyelinated axons which are nociceptive fibres. The electrophysiological properties of the P2X3 receptor was examined using an oocyte expression system. By using a two electrode voltage clamp, an ATP evoked inward current with fast desensitisation can be detected in oocytes injected with P2X3 complementary RNA. In oocytes, the mechanism of P2X3 desensitisation was found to be calcium-dependent and regulated by calcineurin. Developmental studies showed that the expression of P2X3 transcript is as early as the period of sensory neurogenesis at rat E11.5 embryos. For further investigation of P2X3 expression and function, monoclonal antibodies against the P2X3 receptor were generated and characterised. In addition, a DRG-specific proton-gated channel ASIC β was also cloned. It belongs to the DEG/ENaC family whose structure is similar to P2X3 with two transmembrane spanning but with a larger extracellular loop and different conserved cysteine residues. Low pH (pH < 6.5) was found to evoke a non-selective inward cation flux in ASIC β transfected COS cells. As both ATP and protons are important chemical mediators in inflamed tissues, the corresponding ligand-gated channels P2X3 and ASIC β may be good targets for analgesic drug development.
The morphogenetic process of cranial neural tube closure was studied in normal and mutant splotch mouse embryos to identify factors contributing to the development of the neural tube defect (NTD), exencephaly. The study noted sex differences in the timing of the neurulation process and strain differences in the initiation of cranial neural tube closure, at the site termed closure 2. Splotch embryos exhibit closure 2 at a rostral level, within the forebrain region, and exencephaly results from failure of elevation of the midbrain neural folds in a proportion of homozygotes. Backcrossing the splotch mutation onto the DBA/2 background caused a caudal shift in the position of closure 2, to the midbrain region, and a reduction in the incidence of exencephaly, suggesting that altering the position of closure 2 may aid midbrain fold elevation and prevent the development of exencephaly. Whole-mount in situ hybridisation revealed that expression patterns of Pax2, Pax5 and Fgf8 remained constant within the forebrain- midbrain region, regardless of variation in the position of closure 2 in different strains. NTD in humans can be prevented by periconceptional supplementation with folic acid, although the mechanism of action of folate is unknown. The deoxyuridine (dU) suppression test was adapted for use in whole embryo culture and several genetic models of NTD were screened for defects of folate metabolism. The splotch mutant showed an abnormal dU suppression test, with excessive incorporation of 3H-thymidine. Administration of thymidine or folic acid prevented the NTD in splotch homozygotes, both in vitro and in vivo. Folic acid also ameliorated the excessive incorporation of 3H-thymidine and prevented the neural crest defects in homozygous embryos. The splotch mutant therefore appears to provide a model for folate-preventable NTD in humans.
One approach to understand the construction of complex systems is to investigate whether there are simple design principles that are commonly used in building such a system. In the context of nervous system development, one may ask whether the generation of its highly diverse sets of constituents, that is, distinct neuronal cell types, relies on genetic mechanisms that share specific common features. Specifically, are there common patterns in the function of regulatory genes across different neuron types and are those regulatory mechanisms not only used in different parts of one nervous system, but are they conserved across animal phylogeny? We address these questions here by focusing on one specific, highly conserved and well‐studied regulatory factor, the POU homeodomain transcription factor UNC‐86. Work over the last 30 years has revealed a common and paradigmatic theme of unc‐86 function throughout most of the neuron types in which Caenorhabditis elegans unc‐86 is expressed. Apart from its role in preventing lineage reiterations during development, UNC‐86 operates in combination with distinct partner proteins to initiate and maintain terminal differentiation programs, by coregulating a vast array of functionally distinct identity determinants of specific neuron types. Mouse orthologs of unc‐86 , the Brn3 genes, have been shown to fulfill a similar function in initiating and maintaining neuronal identity in specific parts of the mouse brain and similar functions appear to be carried out by the sole Drosophila ortholog, Acj6. The terminal selector function of UNC‐86 in many different neuron types provides a paradigm for neuronal identity regulation across phylogeny.
This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms
Invertebrate Organogenesis > Worms
Nervous System Development > Vertebrates: Regional Development
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