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Neuronal specification in the spinal cord: Inductive signals and transcriptional codes
Abstract
Neural circuits are assembled with remarkable precision during embryonic
development, and the selectivity inherent in their formation helps to define
the behavioural repertoire of the mature organism. In the vertebrate central
nervous system, this developmental program begins with the differentiation
of distinct classes of neurons from progenitor cells located at defined positions
within the neural tube. The mechanisms that specify the identity of neural
cells have been examined in many regions of the nervous system and reveal
a high degree of conservation in the specification of cell fate by key signalling
molecules.
... Spinal cord (SC) development is orchestrated by coordinated actions of morphogens such as Sonic hedgehog (Shh), bone morphogenetic protein (BMP) and Wnt molecules that act by forming gradients in opposite directions along the dorsoventral axis of the developing SC [1][2][3][4][5]. Shh is a key molecule for the specification of neuronal and glial cell lineages during SC development. ...
... Shh is produced initially by the notochord (NC), which acts as an organizing center, later during neurogenesis by the medial floor plate (MFP), and finally, during gliogenesis, by the lateral floor plate (LFP) [3,[6][7][8][9]. Shh regulates the combinatorial expression of a set of transcription factors that are necessary and sufficient to specify each neuronal subtype, resulting in the formation of five discrete ventral progenitor domains (p3, pMN, p2, p1, and p0) that are arrayed along the dorso-ventral axis of SC [2,6]. In the chick SC, Shh controls cell cycle progression of neural progenitors and their survival by maintaining progenitor cell proliferation and preventing cell death during SC development [9][10][11]. ...
... Shh produced initially by the notochord (NC) and later by medial (MFP) and lateral floor plate (LFP), forms a gradient [3,[6][7][8][9][10] that patterns the ventral neural tube into distinct progenitor domains, by controlling the expression of specific homeodomain and basic helix-loop-helix (bHLH) transcriptions factors [2,3,5,6,71,72]. ...
Cross-talk between Mirk/Dyrk1B kinase and Sonic hedgehog (Shh)/Gli pathway affects physiology and pathology. Here, we reveal a novel role for Dyrk1B in regulating ventral progenitor and neuron subtypes in the embryonic chick spinal cord (SC) via the Shh pathway. Using in ovo gain-and-loss-of-function approaches at E2, we report that Dyrk1B affects the proliferation and differentiation of neuronal progenitors at E4 and impacts on apoptosis specifically in the motor neuron (MN) domain. Especially, Dyrk1B overexpression decreases the numbers of ventral progenitors, MNs, and V2a interneurons, while the pharmacological inhibition of endogenous Dyrk1B kinase activity by AZ191 administration increases the numbers of ventral progenitors and MNs. Mechanistically, Dyrk1B overexpression suppresses Shh, Gli2 and Gli3 mRNA levels, while conversely, Shh, Gli2 and Gli3 transcription is increased in the presence of Dyrk1B inhibitor AZ191 or Smoothened agonist SAG. Most importantly, in phenotype rescue experiments, SAG restores the Dyrk1B-mediated dysregulation of ventral progenitors. Further at E6, Dyrk1B affects selectively the medial lateral motor neuron column (LMCm), consistent with the expression of Shh in this region. Collectively, these observations reveal a novel regulatory function of Dyrk1B kinase in suppressing the Shh/Gli pathway and thus affecting ventral subtypes in the developing spinal cord. These data render Dyrk1B a possible therapeutic target for motor neuron diseases.
... This procedure identified all the major cell types of the spinal cord and allowed us to establish a comprehensive catalog of 175 more granular subpopulations. We organized these subpopulations into a clustering tree 85 that recapitulated the known cellular hierarchy of the spinal cord ( Fig. 1e and Supplementary Fig. 5) 2,9,[47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][86][87][88] . ...
... The spinal cord encompasses dozens of anatomically, functionally, and transcriptionally distinct neuronal subpopulations 2,9,[47][48][49][50][51]53,56,57,59,60,[86][87][88] . The scale of our single-cell atlas, which comprised 80,315 single-neuron transcriptomes, allowed us to identify 60 distinct subpopulations of neurons ( Fig. 2i and Supplementary Fig. 11). ...
... The scale of our single-cell atlas, which comprised 80,315 single-neuron transcriptomes, allowed us to identify 60 distinct subpopulations of neurons ( Fig. 2i and Supplementary Fig. 11). Neuronal subpopulations were parcellated into dorsal versus ventral, excitatory versus inhibitory, and local (Nfib) versus long-projecting (Zfhx3) populations 59 39,86,[116][117][118] that could be separated into local (Nfib) and long-projecting (Zfhx3) subpopulations 59 . ...
Here, we introduce the Tabulae Paralytica - a compilation of four atlases of spinal cord injury (SCI) comprising a single-nucleus transcriptome atlas of half a million cells; a multiome atlas pairing transcriptomic and epigenomic measurements within the same nuclei; and two spatial transcriptomic atlases of the injured spinal cord spanning four spatial and temporal dimensions. We integrated these atlases into a common framework to dissect the molecular logic that governs the responses to injury within the spinal cord. The Tabulae Paralytica exposed new biological principles that dictate the consequences of SCI, including conserved and divergent neuronal responses to injury; the priming of specific neuronal subpopulations to become circuit-reorganizing neurons after injury; an inherent trade-off between neuronal stress responses and the activation of circuit reorganization programs; the necessity of reestablishing a tripartite neuroprotective barrier between immune-privileged and extra-neural environments after SCI; and a catastrophic failure to form this barrier in old mice. We leveraged the Tabulae Paralytica to develop a rejuvenative gene therapy that reestablished this tripartite barrier, and restored the natural recovery of walking after paralysis in old mice. The Tabulae Paralytica provides an unprecedented window into the pathobiology of SCI, while establishing a framework for integrating multimodal, genome-scale measurements in four dimensions to study biology and medicine.
... /10.5772/intechopen.114298 and fibroblast growth factors (FGFs) from the caudal mesoderm and tail bud, that together contribute to the specification of molecular identities of neural progenitor cells or domains along the d-v and r-c axes of the neural tube [8,9]. Shh drives the specification of motor neuron progenitor (pMN) and interneuron populations (V0, V1, V2, V3) in the ventral spinal cord, while RA and FGFs also contribute to motor neuron progenitor identities [10][11][12]. ...
... Moreover, the expression levels of Olig2/Ngn2 must be balanced to ensure timely motor neuron specific gene expression, since high expression of Olig2 sustains the pMN state, while high levels of Ngn2 activates the conversion of pMN to post-mitotic motor neurons [19]. Post-mitotic neuron classes, like the progenitor classes, are specified by the combinatorial expression of transcription factors, and in addition, develop specific patterns of connectivity, a neurotransmitter system and electrophysiological properties [8,9]. Newly born post-mitotic motor neurons express a set of TFs like Lhx3, Isl1 and Mnx1 and send axons peripherally to muscles, use acetylcholine and glutamate as neurotransmitters [20,21]. ...
Motor neurons operate at the interface between nervous system and movement apparatus and play several roles in movement generation. During development, motor neurons emerge from progenitor cells in the ventral neural tube and eventually settle into stereotypic position that predict the identity of their target muscles. The specification of these ‘positional’ identities has been studied in detail and involves a coordinate grid of intersecting extrinsic signals that result in the activation of unique combinations of transcription factors acting as cell-autonomous determinants. Eventually, motor neurons diversify into ‘functional’ (e.g., fast/intermediate/slow alpha, beta, and gamma) subtypes essential for proper movement execution, a process linked to the acquisition of unique sets of functional properties. Recent progress has provided insights into the molecular composition and specification of motor neuron functional identities, but little is known about their relationship to the mechanisms underlying the specification of positional identities. In this chapter, we attempt to provide a framework for consolidating both aspects of motor neuron diversification, in addition to outlining the gaps in our knowledge to guide future research directions aiming at understanding the events on a motor neuron’s journey from specification to function.
... Respiratory MNs are generated from "dorsal-most" MNPs in response to a combination of SHH 18 and RA 3,4 signaling gradients, and are characterized by lower expression of the transcription factor TLE and higher expression of the transcription factor Pax6 compared to "ventral-most" MNPs 25 . We therefore hypothesized that controlled activation of SHH signaling to promote the specification of hiPSC-MNPs (MNPs hereafter) displaying a The combined signals from graded SHH and RA are integrated primarily by the HOX transcription factors to specify rostro-caudal MN subtype identity 15 . ...
... During spinal cord development, phMNs derive from progenitors located in the dorsal-most part of the pMN domain at cervical level 3,4,17,18,25 . We therefore aimed at developing in vitro culture conditions that would instruct MNPs to acquire molecular characteristics expected of dorsal pMN progenitors at cervical level, such as a HOXA5 + (cervical trait) and ...
The fatal motor neuron (MN) disease Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive MN degeneration. Phrenic MNs (phMNs) controlling the activity of the diaphragm are prone to degeneration in ALS, leading to death by respiratory failure. Understanding of the mechanisms of phMN degeneration in ALS is limited, mainly because human experimental models to study phMNs are lacking. Here we describe a method enabling the derivation of phrenic-like MNs from human iPSCs (hiPSC-phMNs) within 30 days. This protocol uses an optimized combination of small molecules followed by cell-sorting based on a cell-surface protein enriched in hiPSC-phMNs, and is highly reproducible using several hiPSC lines. We show further that hiPSC-phMNs harbouring ALS-associated amplification of the C9orf72 gene progressively lose their activity and undergo increased death compared to isogenic controls. These studies establish a previously unavailable protocol to generate human phMNs, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction.
... The dorsoventral axis is dependent on the activities of morphogens, including BMPs, Wnts, and Shh, which induce spatially restricted expression of transcription factors (TFs) in progenitor cells along the axis. [1][2][3][4][5] Combinatorial TF expression directs the differentiation of progenitor cells towards molecularly and physiologically distinct neuronal subtypes, opposing adjacent transcriptional programs and sharpening boundaries between progenitor zones. 2,[6][7][8][9] Spinal cord development is also organized along a medial-lateral axis, with dividing progenitor cells located medially and differentiating progeny migrating laterally. ...
... Previous research in mice and chicks has demonstrated that NPCs in the developing spinal cord can be divided into distinct subtypes along a DV axis, with each subtype giving rise to functionally distinct neuronal types. 1,[5][6][7]66 Building on these findings, we used combinatorial TF expression patterns observed in mice as a reference to identify and characterize molecularly distinct NPC subtypes in the developing human spinal cord. Using our TF-seqFISH dataset, we observed that the spatial position of NPC subtypes along the DV axis in the human spinal cord is similar to that reported in mice. ...
The spinal cord is a crucial component of the central nervous system that facilitates sensory processing and motor performance. Despite its importance, the spatiotemporal codes underlying human spinal cord development have remained elusive. In this study, we have introduced an image-based single-cell transcription factor (TF) expression decoding spatial transcriptome method (TF-seqFISH) to investigate the spatial expression and regulation of TFs during human spinal cord development. By combining spatial transcriptomic data from TF-seqFISH and single-cell RNA-sequencing data, we uncovered the spatial distribution of neural progenitor cells characterized by combinatorial TFs along the dorsoventral axis, as well as the molecular and spatial features governing neuronal generation, migration, and differentiation along the mediolateral axis. Notably, we observed a sandwich-like organization of excitatory and inhibitory interneurons transiently appearing in the dorsal horns of the developing human spinal cord. In addition, we integrated data from 10× Visium to identify early and late waves of neurogenesis in the dorsal horn, revealing the formation of laminas in the dorsal horns. Our study also illuminated the spatial differences and molecular cues underlying motor neuron (MN) diversification, and the enrichment of Amyotrophic Lateral Sclerosis (ALS) risk genes in MNs and microglia. Interestingly, we detected disease-associated microglia (DAM)-like microglia groups in the developing human spinal cord, which are predicted to be vulnerable to ALS and engaged in the TYROBP causal network and response to unfolded proteins. These findings provide spatiotemporal transcriptomic resources on the developing human spinal cord and potential strategies for spinal cord injury repair and ALS treatment.
... Markers of primary sensory neurons (DRGX, PIEZO) are also found distributed throughout this territory (SFig 3e, f, g). These results indicate that unlike vertebrate spinal cord 18 , there is not a clear spatial separation of sensory and motor neurons within the ANC. ...
The prehensile arms of the cephalopod are among these animals most remarkable features, but the neural circuitry governing arm and sucker movements remains largely unknown. We studied the neuronal organization of the adult axial nerve cord (ANC) of Octopus bimaculoides with molecular and cellular methods. The ANCs, which lie in the center of every arm, are the largest neuronal structures in the octopus, containing four times as many neurons as found in the central brain. In transverse cross section, the cell body layer (CBL) of the ANC wraps around its neuropil (NP) with little apparent segregation of sensory and motor neurons or nerve exits. Strikingly, when studied in longitudinal sections, the ANC is segmented. ANC neuronal cell bodies form columns separated by septa, with 15 segments overlying each pair of suckers. The segments underlie a modular organization to the ANC neuropil: neuronal cell bodies within each segment send the bulk of their processes directly into the adjoining neuropil, with some reaching the contralateral side. In addition, some nerve processes branch upon entering the NP, forming short-range projections to neighboring segments and mid-range projections to the ANC segments of adjoining suckers. The septa between the segments are employed as ANC nerve exits and as channels for ANC vasculature. Cellular analysis establishes that adjoining septa issue nerves with distinct fiber trajectories, which across two segments (or three septa) fully innervate the arm musculature. Sucker nerves also use the septa, setting up a nerve fiber “suckerotopy” in the sucker-side of the ANC. Comparative anatomy suggests a strong link between segmentation and flexible sucker-laden arms. In the squid Doryteuthis pealeii , the arms and the sucker- rich club of the tentacles have segments, but the sucker-poor stalk of the tentacles does not. The neural modules described here provide a new template for understanding the motor control of octopus soft tissues. In addition, this finding represents the first demonstration of nervous system segmentation in a mollusc.
... Although developmentally defined V2a neurons that express Vsx2 (previously known as Chx10) [42][43][44][45] have been implicated in the production of reaching [46][47][48] and walking 46,[49][50][51][52][53][54][55][56][57][58][59] , Vsx2 ON neuronal subpopulations have not been demonstrated to participate in the regulation of blood pressure. Nonetheless, the results of our comparative snRNA-seq experiments dictate that the activation of SC Hoxa7::Nfib::Vsx2 neurons and SC Hoxa10::Zfhx3::Vsx2 neurons triggers autonomic dysreflexia. ...
Autonomic dysreflexia is a life-threatening medical condition characterized by episodes of uncontrolled hypertension that occur in response to sensory stimuli after spinal cord injury (SCI) 1–7 . The fragmented understanding of the mechanisms underlying autonomic dysreflexia hampers the development of therapeutic strategies to manage this condition, leaving people with SCI at daily risk of heart attack and stroke 8–18 . Here, we expose the complete de novo neuronal architecture that develops after SCI and causes autonomic dysreflexia. In parallel, we uncover a competing, yet overlapping neuronal architecture activated by epidural electrical stimulation of the spinal cord that safely regulates blood pressure after SCI. The discovery that these adversarial neuronal architectures converge onto a single neuronal subpopulation provided a blueprint for the design of a mechanism-based intervention that reversed autonomic dysreflexia in mice, rats, and humans with SCI. These results establish a path for the effective treatment of autonomic dysreflexia in people with SCI.
... 2 These spinal circuits are derived from six major developmentally defined classes of dorsal neurons and five classes of ventral neurons, each of which comprise several transcriptionally defined subtypes with distinct structural and physiological properties. [3][4][5] The brain shapes body movement in large part through the connections made with this diverse pool of spinal neurons, enabling countless patterns of muscle activation and regulating the structure of sensory feedback. Among the neuronal populations that project to the spinal cord, corticospinal neurons (CSNs) play an important role in this transformation. ...
... Within each rhombomere, a particular overlapping expression pattern of the Hox family of homeodomain-containing transcription factors regulates the characteristic molecular environment for specific identity and developmental fates of the segment to produce distinct cell types and neuronal networks (Murphy et al., 1989;Prin et al., 2014). Besides their location along the longitudinal axis of the hindbrain, the fates of progenitors are also determined according to their dorsoventral positions provided by gradients of Sonigh hedgehog and Bone Morphogenic Protein secreted by the floor plate and roof plate, respectively (Briscoe et al., 1999;Jessell, 2000). It was supposed that the rhombomeres are transient structures necessary to the early development of the neural tube, but it seems that their Hox expression pattern is maintained until the perinatal and even in adult stages in the mouse (Farago et al., 2006). ...
Normal brain development requires continuous communication between developing neurons and their environment filled by a complex network referred to as extracellular matrix (ECM). The ECM is divided into distinct families of molecules including hyaluronic acid, proteoglycans, glycoproteins such as tenascins, and link proteins. In this study, we characterize the temporal and spatial distribution of the extracellular matrix molecules in the embryonic and postnatal mouse hindbrain by using antibodies and lectin histochemistry. In the embryo, hyaluronan and neurocan were found in high amounts until the time of birth whereas versican and tenascin-R were detected in lower intensities during the whole embryonic period. After birth, both hyaluronic acid and neurocan still produced intense staining in almost all areas of the hindbrain, while tenascin-R labeling showed a continuous increase during postnatal development. The reaction with WFA and aggrecan was revealed first 4th postnatal day (P4) with low staining intensities, while HAPLN was detected two weeks after birth (P14). The perineuronal net appeared first around the facial and vestibular neurons at P4 with hyaluronic acid cytochemistry. One week after birth aggrecan, neurocan, tenascin-R, and WFA were also accumulated around the neurons located in several hindbrain nuclei, but HAPLN1 was detected on the second postnatal week. Our results provide further evidence that many extracellular macromolecules that will be incorporated into the perineuronal net are already expressed at embryonic and early postnatal stages of development to control differentiation, migration, and synaptogenesis of neurons. In late postnatal period, the experience-driven neuronal activity induces formation of perineuronal net to stabilize synaptic connections.
... Sub-threshold SUP Supra-threshold TEP Transspinal evoked potential TS Terminal stance TSw Terminal swing α-DFA Fractal scaling exponent from the detrended fluctuation analysis τ Appropriate delay time Spinal neuronal networks can generate flexible and adaptable rhythmic motor activity in absence of descending control and movement mediated afferent inputs [1][2][3] . These spinal neuronal networks are known as central pattern generators (CPGs) and have been extensively studied in animals under different preparations including genetic transcription factors and computational modeling [4][5][6][7] . Seminal works on the existence of mammal CPGs were the rhythmic motor discharges in a decerebrate, spinalized and deafferented cat postulated by Thomas Graham Brown and his proposal of "half-centres" on each side of the spinal cord followed by Anders Lundberg (1920Lundberg ( -2009 and Elzbieta Jankowska (1930-) works on the physiology and function of the spinal interneuronal networks and their control on stepping [8][9][10] . ...
Human locomotion is controlled by spinal neuronal networks of similar properties, function, and organization to those described in animals. Transspinal stimulation affects the spinal locomotor networks and is used to improve standing and walking ability in paralyzed people. However, the function of locomotor centers during transspinal stimulation at different frequencies and intensities is not known. Here, we document the 3D joint kinematics and spatiotemporal gait characteristics during transspinal stimulation at 15, 30, and 50 Hz at sub-threshold and supra-threshold stimulation intensities. We document the temporal structure of gait patterns, dynamic stability of joint movements over stride-to-stride fluctuations, and limb coordination during walking at a self-selected speed in healthy subjects. We found that transspinal stimulation (1) affects the kinematics of the hip, knee, and ankle joints, (2) promotes a more stable coordination at the left ankle, (3) affects interlimb coordination of the thighs, and (4) intralimb coordination between thigh and foot, (5) promotes greater dynamic stability of the hips, (6) increases the persistence of fluctuations in step length variability, and lastly (7) affects mechanical walking stability. These results support that transspinal stimulation is an important neuromodulatory strategy that directly affects gait symmetry and dynamic stability. The conservation of main effects at different frequencies and intensities calls for systematic investigation of stimulation protocols for clinical applications.
... During spinal cord development, phMNs emerge from specific MN progenitors (MNPs) located in the 'dorsal-most' MN progenitor (pMN) domain at cervical level 7,8,23,24 . Specification of the pMN domain is under the control of a ventral to dorsal gradient of Sonic hedgehog (SHH) signaling emanating from the notochord and floor plate [25][26][27] . Cervical identity is controlled by a rostro-caudal gradient of Retinoic Acid (RA), which regulates HOXA5 gene expression in the cervical segment of the spinal cord, contributing to phMN identity specification 7,8,24 . ...
The fatal motor neuron (MN) disease Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive MN degeneration. Phrenic MNs (phMNs) controlling the activity of the diaphragm are prone to degeneration in ALS, leading to death by respiratory failure. Understanding of the mechanisms of phMN degeneration in ALS is limited, mainly because human experimental models to study phMNs are lacking. Here we describe a method enabling the derivation of phrenic-like MNs from human iPSCs (hiPSC-phMNs) within 30 days. This protocol uses an optimized combination of small molecules followed by cell-sorting based on a cell-surface protein enriched in hiPSC-phMNs, and is highly reproducible using several hiPSC lines. We show further that hiPSC-phMNs harbouring ALS-associated amplification of the C9orf72 gene progressively lose their electrophysiological activity and undergo increased death compared to isogenic controls. These studies establish a previously unavailable protocol to generate human phMNs offering a disease-relevant system to study mechanisms of respiratory MN dysfunction.
... 13,14 Within the ventral spinal cord, the morphogen sonic hedgehog operates as a pivotal determinant, instigating the activation of specific transcription factor (TF) genes via a gradient emanating from the notochord and the floor plate. [15][16][17] This nuanced gradient delineates five distinct progenitor domains (p0-2, pMN, and p3), with the pMN domain serving as the precursor of post-mitotic motor neurons (MNs), ultimately shaping their emergence from the developmental milieu. 17 MNs exert a central role as the primary neural entities extending beyond the confines of the spinal cord, orchestrating both respiratory and motor functions. ...
Long noncoding RNAs (lncRNAs) play pivotal roles in modulating gene expression during development and disease. Despite their high expression in the central nervous system (CNS), understanding the precise physiological functions of CNS-associated lncRNAs has been challenging, largely due to the in vitro-centric nature of studies in this field. Here, utilizing mouse embryonic stem cell (ESC)-derived motor neurons (MNs), we identified an unexplored MN-specific lncRNA, Litchi (Long Intergenic RNAs in Chat Intron). By employing an "exon-only" deletion strategy in ESCs and a mouse model, we reveal that Litchi deletion profoundly impacts MN dendritic complexity, axonal growth, and altered action potential patterns. Mechanistically, voltage-gated channels and neurite growth-related genes exhibited heightened sensitivity to Litchi deletion. Our Litchi-knockout mouse model displayed compromised motor behaviors and reduced muscle strength, highlighting Litchi’s critical role in motor function. This study unveils an underappreciated function of lncRNAs in orchestrating MN maturation and maintaining robust electrophysiological properties.
... Regarding its structural function, encodes for a transmembrane protein with a patched domain that was initially described in the Drosophila, which has the function of carrying sterols and lipids and works as a receptor for the sonic hedgehog (Hh) (6). Hh is one of the key signaling pathways involved in the formation of the neural tube and brain (7) and in the post-natal brain, it plays a key role in controlling neuronal precursor proliferation in the developing cerebellum and developing dentate granule cells and adult neural precursors. For this reason, while in the past it has been hypothesized as a possible contribution of the gene to the sonic hedgehog (Shh) signaling and synapse formation (8,9), recent studies have demonstrated that, unlike PTCH1, PTCHD1 ectodomains do not bind Shh but a series of RNA binding proteins involved in stress granule and ribonucleoprotein granule assembly (5). ...
Introduction
X-linked PTCHD1 gene has recently been pointed as one of the most interesting candidates for involvement in neurodevelopmental disorders (NDs), such as intellectual disability (ID) and autism spectrum disorder (ASD). PTCHD1 encodes the patched domain-containing protein 1 (PTCHD1), which is mainly expressed in the developing brain and adult brain tissues. To date, major studies have focused on the biological function of the PTCHD1 gene, while the mechanisms underlying neuronal alterations and the cognitive-behavioral phenotype associated with mutations still remain unclear.
Methods
With the aim of incorporating information on the clinical profile of affected individuals and enhancing the characterization of the genotype–phenotype correlation, in this study, we analyze the clinical features of four individuals (two children and two adults) in which array-CGH detected a PTCHD1 deletion or in which panel for screening non-syndromal XLID (X-linked ID) detected a PTCHD1 gene variant. We define the neuropsychological and psychopathological profiles, providing quantitative data from standardized evaluations. The assessment consisted of clinical observations, structured interviews, and parent/self-reported questionnaires.
Results
Our descriptive analysis align with previous findings on the involvement of the PTCHD1 gene in NDs. Specifically, our patients exhibited a clinical phenotype characterized by psychomotor developmental delay- ID of varying severity. Interestingly, while ID during early childhood was associated with autistic-like symptomatology, this interrelation was no longer observed in the adult subjects. Furthermore, our cohort did not display peculiar dysmorphic features, congenital abnormalities or comorbidity with epilepsy.
Discussion
Our analysis shows that the psychopathological and behavioral comorbidities along with cognitive impairment interfere with development, therefore contributing to the severity of disability associated with PTCHD1 gene mutation. Awareness of this profile by professionals and caregivers can promote prompt diagnosis as well as early cognitive and occupational enhancement interventions.
... Opposing morphogen gradients establish 11 progenitor domains that give rise to discrete postmitotic cardinal IN classes and motor neurons. 4 While neurons within each cardinal IN class share early developmental origins and molecular signatures, each class further displays functional 2,5,6 and molecular 7,8 subtype divergence by postnatal stages. In particular, recent transcriptomic studies have revealed immense transcription factor (TF) networks diversifying spinal neuron molecular clusters well beyond their 11 progenitor domains of origin. ...
... Recent studies are further exploiting the capacity of retinoic acid independently and in combination with other growth factors and morphogens, by mimicking embryogenesis in vitro. Indeed, as discussed before, retinoic acid, released from the somites and the SHH gradient produced by the floor plate and notochord [76][77][78], provides a rostral-caudal and ventral-dorsal gradient, respectively, influencing the emergence of the ventral progenitor interneuron domains (p0-p3) and a progenitor motor neuron domain (pMN) arranged in the ventral-dorsal axis, which ultimately mature into the ventral interneuron classes and motor neurons [76,79,80]. V2a interneurons, found in the spinal cord and the respiratory centers of the hindbrain, are synthesized from mouse embryonic stem cells that are treated with low concentrations of retinoic acid and high concentrations of SHH agonist (Pur) [81]. ...
Neuronal differentiation has been shown to be directed by retinoid action during embryo development and has been exploited in various in vitro cell differentiation systems. In this review, we summarize the role of retinoids through the activation of their specific retinoic acid nuclear receptors during embryo development and also in a variety of in vitro strategies for neuronal differentiation, including recent efforts in driving cell specialization towards a range of neuronal subtypes and glial cells. Finally, we highlight the role of retinoic acid in recent protocols recapitulating nervous tissue complexity (cerebral organoids). Overall, we expect that this effort might pave the way for exploring the usage of specific synthetic retinoids for directing complex nervous tissue differentiation.
... These circuits are likely fine-tuned during early postnatal development, at a time where the animal is moving independently (Blankenship and Feller, 2010;Inacio et al., 2016;Robinson et al., 2000). Early formation of neuronal circuits is specified by molecular mechanisms responsible for synaptic formation (Arber, 2012;Jessell, 2000; Lee and Pfaff, 2001). In sensory-motor circuits embedded within the spinal cord, information from the periphery is essential in the generation of smooth movement and key to counteract perturbations in locomotion ( In mature neuronal circuits, each motor neuron receives proprioceptive synapses originating from the homonymous muscle innervated by the motor neuron, as well as from proprioceptive fibers arising from synergistic muscles (Mears and Frank, 1997;Mendelsohn et al., 2015). ...
Overground movement in mammals requires the timely and appropriate assembly of spinal sensory-motor circuits. Within the spinal cord, sensory neurons, spinal interneurons, and motor neurons are key players forming intricate neuronal circuits to ensure proper motor control. The formation of neuronal circuits starts at the embryonic period and continues into postnatal development. During this process, supernumerary synapses are pruned, and refinement of immature circuits occurs, making way for the emergence of mature circuits. A major aspect of the sensory-motor circuit refinement involves the elimination of inappropriate synapses, the molecular mechanisms of which are relatively unknown. We have investigated the molecular mechanisms involved in the elimination of inappropriate proprioceptive synapses from motor neurons by focusing on the classical complement proteins C3 and C1q, as well as the integrin-associated protein CD47. Using mouse genetics, and viral-mediated neuronal map strategies together with physiological, morphological, and molecular biology assays, we found that inappropriate synaptic elimination occurs during early development utilizing both C3 and C1q proteins, but importantly and totally unexpectedly, the CD47 protein. Taken together, our study demonstrates that the refinement of immature sensory-motor circuits in the spinal cord is mediated by classical complement-dependent mechanisms as well as CD47-dependent mechanisms, uncovering a new role for CD47 as a major player in synapse elimination.
... Studies have shown that the multiple motoneurons involved in coordinating muscle contraction are arranged in the spinal cord in the form of motor columns (Tosolini and Morris, 2012;Tosolini et al., 2013;Mohan et al., 2015), and that mammalian limb muscles are innervated only by the lateral motor column (LMC) (Jessell, 2000;Kanning et al., 2010). In the LMC, motoneurons from the medial compartment project to the ventral muscles, and those from the lateral compartment project to the dorsal muscles (Landmesser, 1978). ...
JOURNAL/nrgr/04.03/01300535-202407000-00036/figure1/v/2023-11-20T171125Z/r/image-tiff
Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneurons. However, current research on the spatial location of the spinal motoneurons innervating different muscles is limited. In this study, we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs, which were innervated by the obturator nerve, femoral nerve, inferior gluteal nerve, deep peroneal nerve, and tibial nerve. Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared organs (3DISCO) and 3-D imaging technology based on lightsheet fluorescence microscopy (LSFM). Additionally, we propose the hypothesis that “messenger zones” exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups. We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons. Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse. Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles, but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.
... This method recapitulates the in vivo embryonic neural tube development, and neuroectoderm to the floor plate [39]. Our work supports the role of the FGF-8b and SHH signaling pathways in midbrain patterning, as previously observed [40,41]. It is notable that these organoids can achieve a diameter of over 2 mm, reflecting the capability of this in vitro system to generate MOs of considerable size and complexity. ...
Human pluripotent stem cell-derived midbrain organoids offer transformative potential for elucidating brain development, disease representation, and therapeutic innovations. We introduce a novel methodology to generate midbrain-specific organoids from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). By employing tailored differentiation cues, notably dual-SMAD inhibition combined with FGF-8b and Sonic hedgehog agonist purmorphamine, we direct pluripotent stem cells towards a midbrain lineage. These organoids, growing beyond 2mm in diameter, house diverse neuroepithelial cell populations. Their midbrain character is affirmed by the pronounced expression of midbrain-specific markers and the absence of forebrain and hindbrain indicators. Critically, these organoids differentiate into dopaminergic neurons characteristic of the midbrain, displaying both morphological sophistication and electrophysiological vigor. Additionally, our experiments with POLG iPSC-derived midbrain organoids revealed a marked loss of dopaminergic neurons and diminished expression of genes governing mitochondrial pathways. This evidence underscores the model's potential in simulating mitochondrial diseases and neurodegenerative conditions, notably Parkinson's disease. Our protocol thus emerges as a pivotal instrument for crafting functionally adept, midbrain-centric organoids, paving avenues for advanced studies in midbrain evolution, disorders like Parkinson's disease, and their interplay with mitochondrial dysfunction.
... Another challenge in the production of dorsal spinal neurons has been the lack of knowledge on the developmental pathways contributing to the formation of each subtype. While the ventral subpopulations may be consistently produced via concentration dependent application of sonic hedgehog pathway activators, only superficial dorsal progenitor layers exhibit a similar response to application of BMP (Jessell, 2000;Andrews et al., 2017;Hernandez-Miranda et al., 2017). No single pathway has yet been identified that can produce all six dorsal populations. ...
Spinal cord injury can attenuate both motor and sensory function with minimal potential for full recovery. Research utilizing human induced pluripotent stem cell (hiPSC) -derived spinal cell types for in vivo remodeling and neuromodulation after spinal cord injury has grown substantially in recent years. However, the majority of protocols for the differentiation of spinal neurons are lengthy, lack the appropriate dorsoventral or rostrocaudal specification, and are not typically replicated in more than one cell line. Furthermore, most researchers currently utilize hiPSC-derived motor neurons for cell transplantation after injury, with very little exploration of spinal sensory neuron transplantation. The lack of studies that utilize sensory populations may be due in part to the relative scarcity of dorsal horn differentiation protocols. Building upon our previously published work that demonstrated the rapid establishment of a primitive ectoderm population from hiPSCs, we describe here the production of a diverse population of both ventral spinal and dorsal horn progenitor cells. Our work creates a novel system allowing dorsal and ventral spinal neurons to be differentiated from the same intermediate ectoderm population, making it possible to construct the dorsal and ventral domains of the spinal cord while decreasing variability. This technology can be used in tandem with biomaterials and pharmacology to improve cell transplantation for spinal cord injury, increasing the potential for neuroregeneration.
... In order to examine whether an anatomical and functional propriospinal pathway exists to coordinate activity in locomotorrelated and autonomic sympathetic neural circuitry, we chose to focus on one of the 11 cardinal classes of genetically identified spinal neurons (Jessell, 2000). We focused on the V3 population of spinal neurons because the V3 population of interneurons (INs) are important for stabilizing the frequency of locomotion (Zhang et al., 2008), are excitatory, glutamatergic and project both ipsilaterally and contralaterally (Zhang et al., 2008;Blacklaws et al., 2015;Chopek et al., 2018). ...
Although sympathetic autonomic systems are activated in parallel with locomotion, the neural mechanisms mediating this coordination are incompletely understood. Sympathetic preganglionic neurons (SPNs), primarily located in the intermediate laminae of thoracic and upper lumbar segments (T1-L2), increase activation of tissues and organs that provide homeostatic and metabolic support during movement and exercise. Recent evidence suggests integration between locomotor and autonomic nuclei occurs within the brainstem, initiating both descending locomotor and sympathetic activation commands. However, both locomotor and sympathetic autonomic spinal systems can be activated independent of supraspinal input, in part due to a distributed network involving propriospinal neurons. Whether an intraspinal mechanism exists to coordinate activation of these systems is unknown. We hypothesized that ascending spinal neurons located in the lumbar region provide synaptic input to thoracic SPNs. Here, we demonstrate that synaptic contacts from locomotor-related V3 interneurons (INs) are present in all thoracic laminae. Injection of an anterograde tracer into lumbar segments demonstrated that 8–20% of glutamatergic input onto SPNs originated from lumbar V3 INs and displayed a somatotopographical organization of synaptic input. Whole cell patch clamp recording in SPNs demonstrated prolonged depolarizations or action potentials in response to optical activation of either lumbar V3 INs in spinal cord preparations or in response to optical activation of V3 terminals in thoracic slice preparations. This work demonstrates a direct intraspinal connection between lumbar locomotor and thoracic sympathetic networks and suggests communication between motor and autonomic systems may be a general function of the spinal cord.
... Spinal MNs (sMNs) are responsible for transmitting commands from the CNS to muscles to control motor activity appropriately. Signaling pathways and transcrip-tion factors contributing to the sMN development have been unveiled (Jessell, 2000;Muñoz-Sanjuán and Brivanlou, 2002;Shirasaki and Pfaff, 2002;Stifani, 2014). For example, neuroectoderm is activated in the fibroblast growth factor (FGF) and WNT pathways and/or inhibited by bone morphogenetic protein (BMP) and transforming growth factor-b (TGF-b) signalings (De Robertis, 2006;Nordström et al., 2002). ...
Sporadic amyotrophic lateral sclerosis (sALS) is the majority of ALS, and the lack of appropriate disease models has hindered its research. Induced pluripotent stem cell (iPSC) technology now permits derivation of iPSCs from somatic cells of sALS patients to investigate disease phenotypes and mechanisms. Most existing differentiation protocols are time-consuming or low efficient in generating motor neurons (MNs). Here we report a rapid and simple protocol to differentiate MNs in monolayer culture using small molecules, which led to nearly pure neural stem cells in 6 days, robust OLIG2 + pMNs (73%-91%) in 12 days, enriched CHAT + cervical spinal MNs (sMNs) (88%-97%) in 18 days, and functionally mature sMNs in 28 days. This simple and reproducible protocol permitted the identification of hyperexcitabil-ity phenotypes in our sALS iPSC-derived sMNs, and its application in neurodegenerative diseases should facilitate in vitro disease modeling, drug screening, and the development of cell therapy.
... Sciatic motor neurons of the hindlimb are derived from the LMC of the developing lumbar neural tube. The dorsal ( peroneal) branch of the sciatic nerve is derived from neurons of the lateral component of the LMC (lLMC), which are characterised by expression of genes encoding marker LIM homeodomain proteins Lim1 (also known as Lhx1) and Islet 2 (Isl2), and the ventral (tibial/sural) branch is derived from the medial LMC neurons (mLMC) expressing Islet 1 and 2 (reviewed by Jessell, 2000). Lim1-deficient lLMCs project illicitly along the ventral pathway (Kania et al., 2000). ...
... KEYWORDS neuroscience, spinal cord, sensorimotor system, dorsal root ganglion (DRG), genetic tools Introduction Lineage-tracing studies have revealed distinct molecularly defined clusters of neural progenitor cells that assemble into circuits responsible for various functions. In the spinal cord, this methodology has led to the identification and genetic targeting of key neural lineages that develop into populations responsible for somatosensory and locomotor behaviors in adult (Jessell, 2000;Lee and Pfaff, 2001;Butler and Bronner, 2015;Lu et al., 2015;Lai et al., 2016). Combining these lineage-defined Cre mouse lines with fluorophores and actuators has allowed key insights into the relationship between cell identity and function. ...
Improvements in the speed and cost of expression profiling of neuronal tissues offer an unprecedented opportunity to define ever finer subgroups of neurons for functional studies. In the spinal cord, single cell RNA sequencing studies support decades of work on spinal cord lineage studies, offering a unique opportunity to probe adult function based on developmental lineage. While Cre/Flp recombinase intersectional strategies remain a powerful tool to manipulate spinal neurons, the field lacks genetic tools and strategies to restrict manipulations to the adult mouse spinal cord at the speed at which new tools develop. This study establishes a new workflow for intersectional mouse-viral strategies to dissect adult spinal function based on developmental lineages in a modular fashion. To restrict manipulations to the spinal cord, we generate a brain-sparing Hoxb8 FlpO mouse line restricting Flp recombinase expression to caudal tissue. Recapitulating endogenous Hoxb8 gene expression, Flp-dependent reporter expression is present in the caudal embryo starting day 9.5. This expression restricts Flp activity in the adult to the caudal brainstem and below. Hoxb8 FlpO heterozygous and homozygous mice do not develop any of the sensory or locomotor phenotypes evident in Hoxb8 heterozygous or mutant animals, suggesting normal developmental function of the Hoxb8 gene and protein in Hoxb8 FlpO mice. Compared to the variability of brain recombination in available caudal Cre and Flp lines, Hoxb8 FlpO activity is not present in the brain above the caudal brainstem, independent of mouse genetic background. Lastly, we combine the Hoxb8 FlpO mouse line with dorsal horn developmental lineage Cre mouse lines to express GFP in developmentally determined dorsal horn populations. Using GFP-dependent Cre recombinase viruses and Cre recombinase-dependent inhibitory chemogenetics, we target developmentally defined lineages in the adult. We show how developmental knock-out versus transient adult silencing of the same ROR𝛃 lineage neurons affects adult sensorimotor behavior. In summary, this new mouse line and viral approach provides a blueprint to dissect adult somatosensory circuit function using Cre/Flp genetic tools to target spinal cord interneurons based on genetic lineage.
... To address how TFs play distinct roles in different cellular contexts, we examined the pancreas and central nervous system (CNS)-two tissues that share several common TFs, each of which is essential for unique cell specification decisions within the two systems (Ericson et al. 1997;Sussel et al. 1998;Briscoe et al. 1999Briscoe et al. , 2000Jessell 2000;Sander et al. 2000a,b;Jørgensen et al. 2007;Whyte et al. 2013). Specifically, we focused on the strategies by which NKX2.2 drives cell fate determination, as deletion of NKX2.2 in either the pancreas or CNS results in tissue-intrinsic cell fate conversions (Briscoe et al. 1999;Prado et al. 2004). ...
The consolidation of unambiguous cell fate commitment relies on the ability of transcription factors (TFs) to exert tissue-specific regulation of complex genetic networks. However, the mechanisms by which TFs establish such precise control over gene expression have remained elusive—especially in instances in which a single TF operates in two or more discrete cellular systems. In this study, we demonstrate that β cell-specific functions of NKX2.2 are driven by the highly conserved NK2-specific domain (SD). Mutation of the endogenous NKX2.2 SD prevents the developmental progression of β cell precursors into mature, insulin-expressing β cells, resulting in overt neonatal diabetes. Within the adult β cell, the SD stimulates β cell performance through the activation and repression of a subset of NKX2.2-regulated transcripts critical for β cell function. These irregularities in β cell gene expression may be mediated via SD-contingent interactions with components of chromatin remodelers and the nuclear pore complex. However, in stark contrast to these pancreatic phenotypes, the SD is entirely dispensable for the development of NKX2.2-dependent cell types within the CNS. Together, these results reveal a previously undetermined mechanism through which NKX2.2 directs disparate transcriptional programs in the pancreas versus neuroepithelium.
... Heparin-binding growth factor bFGF promotes stem cell proliferation at higher concentrations, while EGF promotes differentiation at lower values [170]. A crucial component of the hedgehog signaling system, sonic hedgehog (Shh), activates the homeodomain proteins NKx2.2 and Pax6, which bind to DNA sequence specifically to alter the destiny of NSCs in response to concentration gradients [82]. Growth factors and morphogens impact a wide range of cell surface receptors to coordinate signaling pathways for this process. ...
Over the past few decades, the application of mesenchymal stem cells has captured the attention of researchers
and practitioners worldwide. These cells can be obtained from practically every tissue in the body and are used to
treat a broad variety of conditions, most notably neurological diseases such as Parkinson’s disease, multiple
sclerosis, amyotrophic lateral sclerosis, and Alzheimer’s disease. Studies are still being conducted, and the results
of these studies have led to the identification of several different molecular pathways involved in the neuroglial
speciation process. These molecular systems are closely regulated and interconnected due to the coordinated
efforts of many components that make up the machinery responsible for cell signaling. Within the scope of this
study, we compared and contrasted the numerous mesenchymal cell sources and their cellular features. These
many sources of mesenchymal cells included adipocyte cells, fetal umbilical cord tissue, and bone marrow. In
addition, we investigated whether these cells can potentially treat and modify neurodegenerative illnesses.
At the end of the eighteenth century, William Smith, an engineer who was working on the canals of Britain, discovered an empirical rule for identifying the sedimentary rocks of different regions. Smith noticed that any sedimentary stratum contains fossils that are never found in higher or in lower strata. Hence the idea that if two rocks have the same fossils, they also have the same geological age, even if one is at the bottom of a valley and the other on the top of a mountain. The fossils became in this way the key for reconstructing the past movements of the Earth, and Smith used them to draw the first geological map of the United Kingdom.
The corticospinal tract exerts its influence on movement through spinal neurons, which can be divided into types that exhibit distinct functions. However, it remains unknown whether these functional distinctions are reflected in the corticospinal inputs that different types of spinal neurons receive. Using rabies monosynaptic tracing from individual neuron types in the cervical cord and 3D histological reconstruction in mice, we discovered that different types receive inputs distinctly distributed across cortex, and aligned with cell type function. This included a distinct, sparse distribution of direct inputs from cortex onto motor neurons. Coupling rabies tracing with activity measurement during motor behavior revealed different interneuron types receive different input activity patterns, primarily due to the topographical distribution of the corticospinal neurons contacting them. Our results establish that different spinal neuron types get distinct anatomical and functional inputs from the cortex, and reveal functionally relevant homology to primate corticospinal organization.
Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.
Those studying neural systems within the brain have historically assumed that lower-level processes in the spinal cord act in a mechanical manner, to relay afferent signals and execute motor commands. From this view, abstracting temporal and environmental relations is the province of the brain. Here we review work conducted over the last 50 years that challenges this perspective, demonstrating that mechanisms within the spinal cord can organize coordinated behavior (stepping), induce a lasting change in how pain (nociceptive) signals are processed, abstract stimulus–stimulus (Pavlovian) and response-outcome (instrumental) relations, and infer whether stimuli occur in a random or regular manner. The mechanisms that underlie these processes depend upon signal pathways (e.g., NMDA receptor mediated plasticity) analogous to those implicated in brain-dependent learning and memory. New data show that spinal cord injury (SCI) can enable plasticity within the spinal cord by reducing the inhibitory effect of GABA. It is suggested that the signals relayed to the brain may contain information about environmental relations and that spinal cord systems can coordinate action in response to descending signals from the brain. We further suggest that the study of stimulus processing, learning, memory, and cognitive-like processing in the spinal cord can inform our views of brain function, providing an attractive model system. Most importantly, the work has revealed new avenues of treatment for those that have suffered a SCI.
The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing³ to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4–8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.
The embryological development of the extracranial neuraxis represents a complex interplay between local processes occurring in a temporally organized manner, which reflects neural tube formation, proliferation of progenitor cells, and production of individual cell types. These processes reflect a highly coordinated chain of morphogenetic steps occurring in sequence with other parts of the developing nervous system, notably supraspinal structures, which are linked by spinofugal and petal projections, and the evolving peripheral target providing the target and source of afferent and efferent linkages with specific neuraxial substrates. This chapter reviews these linkages, the biology associated with the trophic targeting of these linkages, and the time course of the development process of these coordinated events.
Human locomotion is controlled by spinal neuronal networks of similar properties, function, and organization to those described in animals. Transspinal stimulation affects the spinal locomotor networks and is used to improve standing and walking ability in paralyzed people. However, the function of locomotor centers during transspinal stimulation at different frequencies and intensities is not known. Here, we document the 3D joint kinematics and spatiotemporal gait characteristics during transspinal stimulation at 15, 30, and 50 Hz at sub-threshold and supra-threshold stimulation intensities. We document the temporal structure of gait patterns, dynamic stability of joint movements over stride-to-stride uctuations, and limb coordination during walking at a self-selected speed in healthy subjects. We found that transspinal stimulation 1) affects the kinematics of the hip, knee, and ankle joints, 2) promotes a more stable coordination at the left ankle, 3) improves interlimb coordination of the thighs, 4) improves intralimb coordination between thigh and foot, 5) promotes greater dynamic stability of the hips, and lastly 6) affects the mechanical stability of the joints. These results support that transspinal stimulation is an important neuromodulatory strategy that directly affects gait symmetry and dynamic stability. The conservation of main effects at different frequencies and intensities calls for systematic investigation of stimulation protocols for clinical applications.
Developing tissues are patterned in space and time; this enables them to differentiate their cell types and form complex structures to support different body plans. Although space and time are two independent entities, there are many examples of spatial patterns that originate from temporal ones. The most prominent example is the expression of the genes hunchback, Krüppel, pdm, and castor, which are expressed temporally in the neural stem cells of the Drosophila ventral nerve cord and spatially along the anteroposterior axis of the blastoderm stage embryo. In this Viewpoint, we investigate the relationship between space and time in specific examples of spatial and temporal patterns with the aim of gaining insight into the evolutionary history of patterning.
Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context‐dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell‐ and tissue‐specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.
Axon regeneration can be induced across anatomically complete spinal cord injury (SCI), but robust functional restoration has been elusive. Whether restoring neurological functions requires directed regeneration of axons from specific neuronal subpopulations to their natural target regions remains unclear. To address this question, we applied projection-specific and comparative single-nucleus RNA sequencing to identify neuronal subpopulations that restore walking after incomplete SCI. We show that chemoattracting and guiding the transected axons of these neurons to their natural target region led to substantial recovery of walking after complete SCI in mice, whereas regeneration of axons simply across the lesion had no effect. Thus, reestablishing the natural projections of characterized neurons forms an essential part of axon regeneration strategies aimed at restoring lost neurological functions.
The brain stem is composed of the midbrain (the mesencephalon) and the hindbrain (the rhombencephalon), and is, at least during development, segmentally organized. The midbrain is composed of two temporarily present segments known as mesomeres, whereas the hindbrain is composed of eight, and more recently of 12, rhombomeres (r0–r12), counting the isthmic rhombomere as r0. The cerebellum arises from the first and second rhombomere (r0 and r1). The brain stem also contributes 10 of the 12 cranial nerves, III–XII. A great number of genes are involved in the proper development of the brain stem. The isthmus organizer regulates the early development of the mesencephalon and of the rostral part of the rhombencephalon. Each rhombomere is characterized by a unique combination of Hox genes, its Hox code. In mice, spontaneous and targeted (knockout) mutations in these genes result in specific, rhombomere-restricted disruptions in the development of motor nuclei of cranial nerves. Such a “rhombomeropathy” has been described for the HOXA1 gene.In this chapter, patterning of the brain stem and its segmentation are discussed in ► Sect. 7.2, followed by an overview of the development and developmental disorders of the cranial nerves (► Sect. 7.3). In ► Sect. 7.4, the development of the auditory system, its molecular basis, some of its disorders, and genes associated with deafness are discussed. Clinical cases illustrate some major malformations.KeywordsDevelopment brain stemMesomeresRhombomeresDevelopment cranial nervesCongenital cranial dysinnervation disordersRhombomeropathyDevelopment auditory systemMolecular basis development earHearing defectsGenes associated with deafness
Many of the mechanisms underlying neural development are basically similar in vertebrates and invertebrates. Among vertebrates, popular species for experimental studies are zebrafish, the South African clawed toad, the chick embryo and mice. In mice, many spontaneously occurring mutations affecting the cerebral cortex and the cerebellum have been described. Their molecular analysis, combined with transgenic technology to achieve ectopic gene expression and targeted gene ablation, has made the mouse the mammal of choice for molecular genetic studies of early development.In this chapter, mechanisms of development will be discussed with emphasis on neural induction (► Sect. 2.2), cell lineage studies and fate mapping (► Sect. 2.3), pattern formation of the forebrain and the hindbrain (► Sect. 2.4), specification of cell fate from the spinal cord to the telencephalon (► Sect. 2.5), neurogenesis, gliogenesis and migration, of the cerebral cortex in particular (► Sect. 2.6), axon outgrowth and guidance, focussing on the corpus callosum, the pyramidal tract, thalamocortical projections and the formation of topographic maps (► Sect. 2.7) and programmed cell death (► Sect. 2.8).KeywordsMechanisms of developmentGene expressionNeural inductionCell lineageFate mappingSpecification of cell fateNeurogenesisGliogenesisAxon outgrowthFormation topographic mapsProgrammed cell death
Even after its development is complete, the spinal cord remains a rather simple structure with a ventral motor horn, a dorsal sensory horn and an intermediate zone in between (► Sect. 6.2). Classic birthdating studies have demonstrated a ventral-to-dorsal gradient of histogenesis in the spinal cord with motoneurons appearing first, followed by neurons in the intermediate zone and, finally, neurons in the dorsal horn (► Sect. 6.3). More recent studies have unravelled many of the molecular mechanisms that specify cell fates in the spinal cord (► Sect. 6.4). A number of homeodomain and basic helix-loop-helix combining transcription factors have been identified that are expressed in the spinal ventricular zone in specific dorsoventral domains.In the spinal cord, the sequential production of motor relay and interneuron populations is paralleled by the appearance of descending supraspinal, propriospinal and ascending spinal projections around the same time with dorsal root fibres clearly lagging behind. In ► Sects. 6.5–6.7, the development of dorsal root, and spinal ascending and descending supraspinal projections is discussed. The most frequent developmental disorders of the spinal cord are due to neural tube defects (see ► Chap. 4). Other malformations may also result in developmental anomalies of the spinal cord, such as duplication of the cord, displacement of the cord by neurenteric cysts, syringomyelia and abnormal course or even absence of main fibre tracts such as the pyramidal tract (► Sect. 6.8). Several Clinical Cases illustrate these malformations.KeywordsDevelopment spinal cordHistogenesisBirthdating studiesMolecular mechanismsSpecification of cell fateDevelopment of fibre connectionsDuplication of spinal cordSyringomyeliaAbsence of pyramidal tract
Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.
The vagus nerve, with its myriad constituent axon branches and innervation targets, has long been a model of anatomical complexity in the nervous system. The branched architecture of the vagus nerve is now appreciated to be highly organized around the topographic and/or molecular identities of the neurons that innervate each target tissue. However, we are only just beginning to understand the developmental mechanisms by which heterogeneous vagus neuron identity is specified, patterned, and used to guide the axons of particular neurons to particular targets. Here, we summarize our current understanding of the complex topographic and molecular organization of the vagus nerve, the developmental basis of neuron specification and patterned axon guidance that supports this organization, and the regenerative mechanisms that promote, or inhibit, the restoration of vagus nerve organization after nerve damage. Finally, we highlight key unanswered questions in these areas and discuss potential strategies to address these questions.
The vertebrate nervous system is composed of a wide range of neurons and complex synaptic connections, raising the intriguing question of how neuronal diversity is generated. The spinal cord provides an excellent model for exploring the mechanisms governing neuronal diversity due to its simple neural network and the conserved molecular processes involved in neuron formation and specification during evolution. This review specifically examines two distinct progenitor domains present in the zebrafish ventral spinal cord: the lateral floor plate (LFP) and the p2 progenitor domain. The LFP is responsible for the production of GABAergic Kolmer–Agduhr neurons (KA″), glutamatergic V3 neurons, and intraspinal serotonergic neurons, while the p2 domain generates V2 precursors that subsequently differentiate into three unique subpopulations of V2 neurons, namely glutamatergic V2a, GABAergic V2b, and glycinergic V2s. Based on recent findings, we will examine the fundamental signaling pathways and transcription factors that play a key role in the specification of these diverse neurons and neuronal subtypes derived from the LFP and p2 progenitor domains.
A fundamental question in developmental biology is how a single genome gives rise to the diversity of cell fates. In essence, each cell fate in the human body is a unique but stable output state of the genome, maintained by positive and negative feedbacks from both inside and outside the cell (a stable cell state). Traditionally, defining a cell fate means identifying a unique combination of transcriptional factors expressed by the specific cell type. The hundreds of transcriptional factors in the genome, however, have complicated the task of simplifying cell fate representation and obtaining insights into its regulation. Moreover, results from this approach provides only a mostly static picture, with each cell fate/state disconnected from one another. An alternative approach instead defines cell fates by determining their relationship to each other, through identifying the signaling pathways that control each step of their lineage transition from a common progenitor during development. Decades of studies have shown only a handful of signaling pathways are sufficient to specify all cell fates in the body, simplifying the execution of such a strategy. In this review, I will argue this alternative approach is not only feasible but also has the potential of simplifying the cell fate landscape as well as facilitating the engineering of different cell fates for regenerative medicine.
Spinal cord injury (SCI) is a debilitating condition with significant personal, societal, and economic burden. The highest proportion of traumatic injuries occur at the cervical level, which results in severe sensorimotor and autonomic deficits. Following the initial physical damage associated with traumatic injuries, secondary pro-inflammatory, excitotoxic, and ischemic cascades are initiated further contributing to neuronal and glial cell death. Additionally, emerging evidence has begun to reveal that spinal interneurons undergo subtype specific neuroplastic circuit rearrangements in the weeks to months following SCI, contributing to or hindering functional recovery. The current therapeutic guidelines and standards of care for SCI patients include early surgery, hemodynamic regulation, and rehabilitation. Additionally, preclinical work and ongoing clinical trials have begun exploring neuroregenerative strategies utilizing endogenous neural stem/progenitor cells, stem cell transplantation, combinatorial approaches, and direct cell reprogramming. This review will focus on emerging cellular and noncellular regenerative therapies with an overview of the current available strategies, the role of interneurons in plasticity, and the exciting research avenues enhancing tissue repair following SCI.
Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show that Noggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog (Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.
Sonic Hedgehog (Shh) is a secreted protein that controls cell fate and mitogenesis in the developing nervous system. Here we show that a constitutively active form of Smoothened (Smo-M2) mimics concentration-dependent actions of Shh in the developing neural tube, including activation of ventral marker genes (HNF3beta, patched, Nkx2.2, netrin-1), suppression of dorsal markers (Pax-3, Gli-3, Ephrin A5) and induction of ventral neurons (dopaminergic, serotonergic) and ventrolateral motor neurons (Islet-1+, Islet-2+, HB9+) and interneurons (Engrailed-1+, CHX10+). Furthermore, Smo-M2's patterning activities were cell autonomous, occurring exclusively in cells expressing Smo-M2. These findings suggest that Smo is a key signaling component in the Hh receptor and that Shh patterns the vertebrate nervous system as a morphogen, rather than through secondary relay signals.
In this paper we report studies on the organization of the motor projections to chick hindlimbs lacking limb segments as a result of surgical manipulations during early embryonic development. The innervation of partial limbs, missing either a thigh or both a calf and a foot, was studied using both retrograde and orthograde horseradish peroxidase nerve-tracing techniques, as well as by serial reconstruction. In addition, [3H]thymidine autoradiography was used to characterize motoneuron production and loss. Motor organization was assessed both before and after the period of naturally occurring motoneuron death. Prior to the period of cell death, we verified by autoradiography that the organization of the motor column (i.e., motoneuron birthdates and settling patterns) was normal despite deletions of the periphery. It was also found that, initially, the entire motor column projected to the partial limbs, entering via normal crural and sciatic pathways. The proximal branching patterns of the nerves leaving the plexus were normal; however, the distal projections of the nerves which would normally serve the missing limb segment were truncated. After the period of cell death, the motor columns serving segment-deleted limbs were excessively and selectively depleted of target-deprived motoneurons. The remaining motor pools appropriately innervated their normal targets. Thus, when both thigh- and calf-serving motor axons invaded a given limb segment, only the axons which normally serve that segment successfully innervated it. Our findings on the innervation of partial limbs cannot be explained on the assumption that motor projection patterns are determined solely by the timing or spatial arrangement of axon arrival at the limb bud. They indicate that motor axons destined to innervate thigh and calf muscles differ from one another such that their initial growth trajectories are distinctive and their final termination sites are appropriate for the muscles available. These differences are evident both from the pattern of selective cell death and from the normal projections of the surviving motoneurons.
During development, the neural tube produces a large diversity of neuronal phenotypes from a morphologically homogeneous pool of precursor cells. In recent years, the cellular and molecular mechanisms by which specific types of neurons are generated have been explored, in the hope of discovering features common to development throughout the nervous system. This article focuses on three strategies employed by the CNS to generate distinct classes of neuronal phenotypes during development: dorsal-ventral polarization in the spinal cord, segmentation in the hindbrain, and a lamination in the cerebral cortex. The mechanisms for neurogenesis exemplified by these three strategies range from a relatively rigid, cell lineage-dependent specification with a high degree of subservance to early patterns of gene expression, to inductions and cell-cell interactions that determine cell fates more flexibly.
Intercellular signaling molecules of the vertebrate hedgehog family and transcription factors of the winged-helix family have been implicated in floor plate development. We have examined the consequences of misexpressing the vertebrate hedgehog gene vhh-1 (sonic hedgehog, shh) and the winged-helix gene HNF-3 beta in the neural plate and neural tube of frog embryos. Misexpression of either of these genes induces floor plate differentiation at ectopic locations. However, ectopic floor plate induction in response to both vhh-1 and HNF-3 beta was temporally and spatially restricted. At neural plate stages, ectopic floor plate differentiation was not detected. After neural tube closure, ectopic floor plate differentiation was detected but was restricted predominantly to the dorsal region of the neural tube. The ability of winged-helix and vertebrate hedgehog genes to induce floor plate differentiation in vivo may, therefore, be constrained by additional signals that specify the time and position of floor plate differentiation.
The differentiation of floor plate cells and motor neurons can be induced by Sonic hedgehog (SHH), a secreted signaling protein that undergoes autoproteolytic cleavage to generate amino- and carboxy-terminal products. We have found that both floor plate cells and motor neurons are induced by the amino-terminal cleavage product of SHH (SHH-N). The threshold concentration of SHH-N required for motor neuron induction is about 5-fold lower than that required for floor plate induction. Higher concentrations of SHH-N can induce floor plate cells at the expense of motor neuron differentiation. Our results suggest that the induction of floor plate cells and motor neurons by the notochord in vivo is mediated by exposure of neural plate cells to different concentrations of the amino-terminal product of SHH autoproteolytic cleavage.
The secreted protein products of the hedgehog (hh) gene family are associated with local and long-range signalling activities that are responsible for developmental patterning in multiple systems, including Drosophila embryonic and larval tissues and vertebrate neural tube, limbs and somites. In a process that is critical for full biological activity, the hedgehog protein (Hh) undergoes autoproteolysis to generate two biochemically distinct products, an 18K amino-terminal fragment, N, and a 25K carboxy-terminal fragment, C (ref. 16); mutations that block autoproteolysis impair Hh function. We have identified the site of autoproteolytic cleavage and find that it is broadly conserved throughout the hedgehog family. Knowing the site of cleavage, we were able to test the function of the N and C cleavage products in Drosophila assays. We show here that the N product is the active species in both local and long-range signalling. Consistent with this, all twelve mapped hedgehog mutations either affected the structure of the N product directly or otherwise blocked the release of N from the Hh precursor as a result of deletion or alteration of sequences in the C domain.
Motor neuron differentiation is accompanied by the expression of a LIM homeodomain transcription factor, Islet1 (ISL1). To assess the involvement of ISL1 in the generation of motor neurons, we analyzed cell differentiation in the neural tube of embryos in which ISL1 expression has been eliminated by gene targeting. Motor neurons are not generated without ISL1, although many other aspects of cell differentiation in the neural tube occur normally. A population of interneurons that express Engrailed1 (EN1), however, also fails to differentiate in Isl1 mutant embryos. The differentiation of EN1+ interneurons can be induced in both wild-type and mutant neural tissue by regions of the neural tube that contain motor neurons. These results show that ISL1 is required for the generation of motor neurons and suggest that motor neuron generation is required for the subsequent differentiation of certain interneurons.
In zebrafish, individual primary motoneurons can be uniquely identified by their characteristic cell body positions and axonal projection patterns. The fate of individual primary motoneurons remains plastic until just prior to axogenesis when they become committed to particular identities. We find that distinct primary motoneurons express particular combinations of LIM homeobox genes. Expression precedes axogenesis as well as commitment, suggesting that LIM homeobox genes may contribute to the specification of motoneuronal fates. By transplanting them to new spinal cord positions, we demonstrate that primary motoneurons can initiate a new program of LIM homeobox gene expression, as well as the morphological features appropriate for the new position. We conclude that the patterned distribution of different primary motoneuronal types within the zebrafish spinal cord follows the patterned expression of LIM homeobox genes, and that this reflects a highly resolved system of positional information controlling gene transcription.
The source of environmental cues determining the central connections of muscle sensory neurons was investigated by manipulating chick embryos so that sensory neurons supplied a duplicate set of dorsal thigh muscles. These neurons projected out ventral nerve pathways and along motor axons that normally project to ventral muscles but their ultimate target tissue was the duplicate set of dorsal muscles. The central connections of these sensory neurons to motoneurons supplying normal dorsal muscles were then determined with intracellular recordings in isolated spinal cord preparations. Sensory neurons supplying individual duplicate dorsal muscles made the same connections as those supplying the corresponding normal dorsal muscles; the pattern of these connections was different than that made by afferents supplying ventral muscles. Sensory neurons thus made synaptic connections appropriate for their target muscle rather than for their more proximal ventral environment. These findings suggest that the target muscle is the source of cues that determine the central connections of the sensory neurons projecting to it. Motoneurons forced to innervate novel muscle received many of the same sensory inputs they would normally receive, suggesting that motoneurons are less influenced by their target tissue than sensory neurons.
When 3-4 segments of the chick lumbosacral neural tube are reversed in the anterior-posterior axis at stage 15 (embryonic day 2.5), the spinal cord develops with a reversed organization of motoneurons projecting to individual muscles in the limb (C. Lance-Jones and L. Landmesser (1980) J. Physiol. 302, 581-602). This finding indicated that motoneuron precursors or components of their local environment were specified with respect to target by stage 15. To identify the timing of this event, we have now assessed motoneuron projections after equivalent neural tube reversals at earlier stages of development.
Lumbosacral neural tube segments 1-3 (± one segment cranial or caudal) were reversed in the anterior-posterior axis at stages 13 and 14 (embryonic day 2). The locations of motoneurons innervating two thigh muscles, the sartorius and femorotibialis, were mapped via retrograde horseradish peroxidase labeling at stages 35-36 (embryonic days 9-10). In a sample of embryos, counts were made of the total number of motoneurons in the lateral motor columns of reversed segments. The majority of motoneurons projecting to the sartorius and femorotibialis were in a normal position within the spinal cord. Segmental differences in motor column size were also similar to normal. These observations indicate that positional cues external to the LS neural tube can affect motoneuron commitment and number at stages 13-14. Since these observations stand in contrast to results following stage 15 reversals, we conclude that regional differences related to motoneuron target identity are normally specified or stabilized within the anterior LS neural tube between stages 14 and 15.
To examine the role of the notochord in this process, neural tube reversals were performed at stages 13-14 as described above, except that the underlying notochord was also reversed. Projections to the sartorius and femorotibialis muscles did not differ significantly from those in embryos with neural tube reversals alone, indicating that the notochord is not the source of cues for target identity at stages 13-14.
We thank Javier Sampedro for planning Figure 4Figure 4 and Dan Barbash, Jose Casal, Jim Smith, and Jean-Paul Vincent for help with the manuscript. The advice and support of Hugh Pelham and Jim Smith is much appreciated.
The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.
Antibodies that block Sonic Hedgehog (SHH) signaling have been used to show that SHH activity is required for the induction of floor plate differentiation by the notochord and independently for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: an early period during which naive neural plate cells are converted into ventralized progenitors and a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. The ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.
Thymocyte winged helix (TWH) is a putative transcription factor expressed in the developing neural tube. At midgestation, TWH expression identifies subsets of spinal cord motor neurons and interneurons. TWH-expressing motor neurons were restricted to specific spinal cord levels, distinguishing motor neurons at lumbar from those at cervical levels. To understand the developmental role of TWH, we replaced the TWH gene with the lacZ reporter gene and generated mice with a homozygous disruption of the TWH gene. TWH(-/-) mutant mice had increased perinatal mortality, retarded postnatal growth, and motor weakness. The TWH(-/-) mutation resulted in alterations in the sizes and position of different neuronal populations. Our results demonstrate that TWH plays a critical role in neuronal development and suggest that TWH regulates the early differentiation of neural progenitors.
Distinct classes of motor neurons and ventral interneurons are generated by the graded signaling activity of Sonic hedgehog (Shh). Shh controls neuronal fate by establishing different progenitor cell populations in the ventral neural tube that are defined by the expression of Pax6 and Nkx2.2. Pax6 establishes distinct ventral progenitor cell populations and controls the identity of motor neurons and ventral interneurons, mediating graded Shh signaling in the ventral spinal cord and hindbrain.
The vertebrate spinal cord has long served as a useful system for studying the pattern of cell differentiation along the dorsoventral (d/v) axis. In this paper, we have defined the expression of several classes of genes expressed in restricted d/v domains in the intermediate region (IR) of the mouse spinal cord, in which most interneurons are generated. From this analysis, we have found that spinal cord interneurons and their precursors express unique combinations of transcription factors and Notch ligands at the onset of their differentiation. The domains of expression of a number of different classes of genes share similar boundaries, indicating that there could be a basic subdivision of the ventral IR into four distinct regions. This differential gene expression suggests that spinal cord interneurons acquire unique identities early in their development and that Notch signaling mechanisms may participate in the determination of cell fate along the d/v axis. Gene expression studies in Engrailed-1 (En-1) mutants showed that En-1-expressing and other closely positioned classes of neurons do not require the homeodomain protein En-1 for their early pattern of differentiation. Rather, it is suggested that En-1 may function to distinguish a subset of interneurons during the later maturation of the spinal cord.
A Sonic hedgehog (Shh) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter that binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway.
Members of the PAX family of transcription factors are candidates for controlling cell identity in the spinal cord. We have morphologically analyzed cells that express one of these transcription factors, PAX2, demonstrating multiple interneuron cell types express PAX2. Two ventral populations of PAX2-expressing interneurons in the spinal cord are marked by coexpression of the transcription factors, EN1 and EVX1. Interestingly, the expression domains of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the regulation of PAX2, EN1 and EVX1. We show that one of these patterning genes, Pax6, is required for the correct specification of ventral PAX2+ interneurons that coexpress EN1. These results demonstrate that the early activity of patterning genes in the ventricular zone determines interneuron identity in the spinal cord.
Signals that induce neural cell fate in amniote embryos emanate from a unique cell population found at the anterior end of the primitive streak. Cells in this region express a number of fibroblast growth factors (FGFs), a group of secreted proteins implicated in the induction and patterning of neural tissue in the amphibian embryo. Here we exploit the large size and accessibility of the early chick embryo to analyse the function of FGF signalling specifically during neural induction. Our results demonstrate that extraembryonic epiblast cells previously shown to be responsive to endogenous neural-inducing signals express early posterior neural genes in response to local, physiological levels of FGF signal. This neural tissue does not express anterior neural markers or undergo neuronal differentiation and forms in the absence of axial mesoderm. Prospective mesodermal tissue is, however, induced and we present evidence for both the direct and indirect action of FGFs on prospective posterior neural tissue. These findings suggest that FGF signalling underlies a specific aspect of neural induction, the initiation of the programme that leads to the generation of the posterior central nervous system.
The generation of distinct classes of motor neurons is an early step in the control of vertebrate motor behavior. To study the interactions that control the generation of motor neuron subclasses in the developing avian spinal cord we performed in vivo grafting studies in which either the neural tube or flanking mesoderm were displaced between thoracic and brachial levels. The positional identity of neural tube cells and motor neuron subtype identity was assessed by Hox and LIM homeodomain protein expression. Our results show that the rostrocaudal identity of neural cells is plastic at the time of neural tube closure and is sensitive to positionally restricted signals from the paraxial mesoderm. Such paraxial mesodermal signals appear to control the rostrocaudal identity of neural tube cells and the columnar subtype identity of motor neurons. These results suggest that the generation of motor neuron subtypes in the developing spinal cord involves the integration of distinct rostrocaudal and dorsoventral patterning signals that derive, respectively, from paraxial and axial mesodermal cell groups.
We have studied the role of Bmp signaling in patterning neural tissue through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. We demonstrate that Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. Our analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. We found that a presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord.
A Sonic hedgehog (Shh) response element was identified in the chicken ovalbumin upstream promoter–transcription factor II (COUP-TFII) promoter that binds to a factor distinct fromGli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII–, Islet1-, and Gli response element–dependent gene expression. This finding identifies another step in the Shh-N signaling pathway.
There is growing evidence that sonic hedgehog (Shh) signaling regulates ventral neuronal fate in the vertebrate central nervous system through Nkx-class homeodomain proteins. We have examined the patterns of neurogenesis in mice carrying a targeted mutation in Nkx6.1. These mutants show a dorsal-to-ventral switch in the identity of progenitors and in the fate of postmitotic neurons. At many axial levels there is a complete block in the generation of V2 interneurons and motor neurons and a compensatory ventral expansion in the domain of generation of V1 neurons, demonstrating the essential functions of Nkx6.1 in regional patterning and neuronal fate determination.
Keywords
• Nkx
• spinal cord
• motor neurons
• neuronal specification
• interneurons
Regional diversity along the anterior–posterior axis of the central nervous system is established during gastrulation and is subsequently refined by local organizing centres that are located at genetically defined positions. The isthmic organizer possesses midbrain- and cerebellum-inducing properties, and its positioning at the midbrain–hindbrain boundary is a crucial event that controls midbrain and cerebellum development. Recent work has shown that two transcription factors, Otx2 and Gbx2, are instrumental in positioning the isthmic organizer at the midbrain–hindbrain boundary.
We recently identifiedneurogenin(ngn), aneuroD-related bHLH gene, whoseXenopushomolog functions as a neuronal determination factor and upstream activator ofXneuroD(Maet al. Cell87:43–52, 1996). Here we identify two additionalngn's,ngn2andngn3, which together define a novel subfamily ofatonal-related mouse genes. Comparative analysis ofngnexpression indicates that these three genes define distinct progenitor populations in the developing CNS and PNS, exhibiting nonoverlapping expression in some areas and partial overlap in others. The expression of thengn's spatially overlaps and often temporally precedes that ofneuroD,suggesting that (as inXenopus) thengn's andneuroDfunction in a cascade. Thus, as in myogenesis, different bHLH determination factors may activate a common bHLH differentiation factor in different sublineages. Thengn's therefore represent both a family of putative mammalian neuronal determination genes and useful markers of the origins of neuronal diversity.
Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. We performed reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons by panning to identify RTKs involved in the early development of motoneuron. In situ hybridization revealed that Cek8, a member of the eph family, was specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles, and disappeared after the naturally occurring cell death period (E6–E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons.
1. Retrograde transport of horseradish peroxidase was used to map the initial projection patterns of lumbosacral motoneurones to the embryonic chick hind limb. 2. The stage 28 segmental projection pattern to each of the four primary muscle masses was characteristic and indistinguishable from the stage 36 projection pattern to the sum of the muscles derived from that mass. In addition, the adductor motoneurone pool was found to be similar in position (both rostro-caudal and mediolateral) at stages 29, 30, 32, 33 1/2 and 36. 3. Therefore axons from lumbosacral motoneurones project for the most part only to appropriate regions from early times shortly after they grow into the limb bud. Furthermore, the attainment of the segmental projection pattern occurs prior to the normal time of, and therefore without the aid of, cell death. This conclusion was supported by electrophysiological recordings made from muscle nerves. 4. A regionalization of the projection patterns within a single muscle mass could be shown both anatomically and physiologically prior to the cleavage of the mass into individual muscles and the projections were in a general way appropriate for the muscles derived from those regions. 5. Therefore the process of muscle cleavage does not in itself create the specific projection patterns observed, and motoneurone axons appear to grow to and to ramify and make synapses only within regions which correspond to their adult muscles. 6. Finally, the termination site of each motoneurone axon in the early limb was found to be tightly correlated in a somatotopic fashion with the position occupied by its soma in the cord. This suggests that some feature of the motoneurone related to its position may be of importance in achieving the specific projection patterns observed.
1. The motor nuclei supplying many of the hind limb muscles were localized in late chick embryos (stage 36-37; 10-11 days) by utilizing the technique of retrograde transport of horseradish peroxidase. 2. Each nucleus was found to be localized in a characteristic position in both the rostro-caudal and transverse plane of the spinal cord with only slight individual variation. 3. Each motor nucleus consisted of an elongate, coherent cluster of labelled cells, with few cells occurring outside the cluster. Thus, there did not appear to be extensive overlap of nuclei nor extensive intermingling of motoneurones projecting to different muscles. 4. The position of a motor nucleus in the transverse plane was not correlated with whether its muscle was used as an extensor or flexor; nor were adjacent nuclei necessarily co-activated during normal unrestrained walking movements as deduced from e.m.g. recordings. The position of a motor nucleus also was not correlated in a topographical manner with the adult position in the limb of the muscle to which it projected. 5. Further, while no correlation was found between the rostrocaudal position of a motor nucleus and the embryonic muscle mass from which its muscle was derived, such a relationship existed for the medio-lateral position; all muscles arising from the dorsal muscle mass, regardless of their function or adult position, were innervated by laterally situated motoneurones, all muscles arising from the ventral muscle mass by medially situated motoneurones. 6. It is concluded that motoneurone position is most closely correlated with ontogenetic events presumaeriphery. It can also be inferred that the central connexions onto motoneurones, responsible for their proper activation, cannot be achieved by a simple mechanism based largely on the position of the motoneurone soma.
An autoradiographic determination of the time of origin of the lateral motor columns (LMC) of the chick embryo has been made. The first motor neurons of the brachial LMC are born at stage 15; the earliest birthdates of lumbar LMC neurons are at stage 17. At least 95% of the motor neurons of both brachial and lumbar columns are produced by stage 23 (4 days). The remaining 5% of the motor neurons are produced during the next two days. A clear rostrocaudal gradient of motor neuron production is seen beoth between the brachial and lumbar LMCs and within the LMCs themselves. The LMCs are assembled in a mediolateral sequence: the early-born motor neurons settle medially, the later-born motor neurons settle more laterally. Observations were made of other large early-born neurons which remain permanently in the dorsal gray of the spinal cord.
The developmental determination of primary motoneurons was investigated by transplanting identified motoneurons in embryonic
zebrafish to new spinal cord positions. Some cells moved from the new positions in which they were placed back to their original
positions, thus it was difficult to evaluate whether they were determined. Among cells that remained in their new positions,
those transplanted about 1 hour before axogenesis developed axonal trajectories that were appropriate for their original soma
positions, whereas those transplanted 2 to 3 hours before axogenesis developed morphologies appropriate for their new soma
positions. These results suggest that motoneuronal identity is determined before axogenesis.
Motor neurons located at different positions in the embryonic spinal cord innervate distinct targets in the periphery, establishing a topographic neural map. The topographic organization of motor projections depends on the generation of subclasses of motor neurons that select specific paths to their targets. We have cloned a family of LIM homeobox genes in chick and show here that the combinatorial expression of four of these genes, Islet-1, Islet-2, Lim-1, and Lim-3, defines subclasses of motor neurons that segregate into columns in the spinal cord and select distinct axonal pathways. These genes are expressed prior to the formation of distinct motor axon pathways and before motor columns appear. Our results suggest that LIM homeobox genes contribute to the generation of motor neuron diversity and may confer subclasses of motor neurons with the ability to select specific axon pathways, thereby initiating the topographic organization of motor projections.
Recent studies have uncovered new roles for the notochord and floor plate in patterning adjacent cells, elaborating their importance as essential organizers of neural and paraxial tissue. The identification of key molecules that mediate the ability of notochord and floor plate to induce cells to adopt distinct fates has provided a first step in elucidating the mechanisms underlying these events.
The identity and patterning of ventral cell types in the vertebrate central nervous system depends on cell interactions. For example, induction of a specialized population of ventral midline cells, the floor plate, appears to require contact-mediated signalling by the underlying notochord, whereas diffusible signals from the notochord and floor plate can induce ventrolaterally positioned motor neurons. Sonic hedgehog (Shh), a vertebrate hedgehog-family member, is processed to generate two peptides (M(r) 19K and 26/27K) which are secreted by both of these organizing centres. Moreover, experiments in a variety of vertebrate embryos, and in neural explants in vitro, indicate that Shh can mediate floor-plate induction. Here we have applied recombinant Shh peptides to neural explants in serum-free conditions. High concentrations of Shh bound to a matrix induce floor plate and motor neurons, and addition of Shh to the medium leads to dose-dependent induction of motor neurons. All inducing activity resides in a highly conserved amino-terminal peptide (M(r) 19K). Moreover, antibodies that specifically recognize this peptide block induction of motor neurons by the notochord. We propose that Shh acts as a morphogen to induce distinct ventral cell types in the vertebrate central nervous system.
The transcription factor gene HNF-3 beta is expressed in the ventral midline of the mouse embryonic neural tube, including the floor plate, a structure important for dorsoventral patterning and axonal guidance. To assess HNF-3 beta function, the gene has been ectopically expressed in the midbrain/hindbrain of transgenic embryos using an En-2 promoter/enhancer. By 18.5 days postcoitum, transgenic brains show a range of abnormalities, including absent inferior colliculus and reduced cerebellum. Earlier, several genes normally expressed in the floor plate (BMP-1, Steel factor, and HNF-3 alpha) are induced within the same ectopic dorsal domain as HNF-3 beta, and autoactivation of the endogenous HNF-3 beta is observed. Conversely, expression of the dorsal gene Pax-3 is suppressed. Ectopic dorsal neuronal differentiation and abnormal dorsal axonal projections are also seen. These results suggest that HNF-3 beta is an important regulator of floor plate development in vivo.
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The signaling protein Hedgehog (Hh) controls cell fate and polarizes tissues in both flies and vertebrates. In flies, Hh exerts its effects by opposing the function of a novel transmembrane protein, Patched, while also locally inducing patched (ptc) transcription. We have identified a mouse homolog of ptc which in many tissues is transcribed near cells making either Sonic or Indian hedgehog. In addition, ectopic Sonic hedgehog expression in the mouse central nervous system induces ptc transcription. As in flies, mouse ptc transcription appears to be indicative of hedgehog signal reception. The results support the existence of a conserved signaling pathway used for pattern formation in insects and mammals.
Ventral cell fates in the central nervous system are induced by Sonic hedgehog, a homolog of hedgehog, a secreted Drosophila protein. In the central nervous system, Sonic hedgehog has been identified as the signal inducing floor plate, motor neurons, and dopaminergic neurons. Sonic hedgehog is also involved in the induction of ventral cell type in the developing somites. ptc is a key gene in the Drosophila hedgehog signaling pathway where it is involved in transducing the hedgehog signal and is also a transcriptional target of the signal. PTC, a vertebrate homolog of this Drosophila gene, is genetically downstream of Sonic hedgehog (Shh) in the limb bud. We analyze PTC expression during chicken neural and somite development and find it expressed in all regions of these tissues known to be responsive to Sonic hedgehog signal. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. This conservation of regulation allows us to use PTC as a marker for Sonic hedgehog response. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than have been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling.
Evidence that region- and cell-type-specific transcription factors regulate morphogenesis and differentiation of the vertebrate nervous system comes from numerous studies, including descriptions of discrete patterns of expression during neural development and analysis of mutant phenotypes. Recently published works provide insights into the roles of vertebrate transcription factors in regulating the generation of neural precursors, regionalization of the nervous system, and subsequent differentiation of specific cell types within these regions. For instance, misexpression studies in Xenopus embryos show that the newly isolated basic helix-loop-helix protein NeuroD is able to promote neurogenesis, whereas analysis of mouse embryos mutant for the homeobox gene En-1 demonstrates that this transcription factor is required for proper development of the midbrain-hindbrain region. A recent study in chick shows that the combinatorial expression of Islet-1, Lim-1, and two other LIM homeobox genes, Islet-2 and Lim-3, defines subclasses of motor neurons in the spinal cord, supporting a model where combinatorial repertoires of transcription factors may act to generate diverse cell types.
Retinoic acid, a developmental signal implicated in the formation of the neural axis, is present at high levels in the early embryonic trunk region, where it is synthesized by a novel dehydrogenase. Here we show that the same enzyme is inducible by retinoic acid in P19 teratocarcinoma cells, and we report the cloning from P19 cells of a cDNA encoding a novel dehydrogenase, named retinaldehyde dehydrogenase-2 (RALDH-2). Expression in COS cells shows RALDH-2 to be highly effective in oxidation of retinaldehyde, with no detectable activity on any other aldehyde tested. In situ hybridization histochemistry on the embryonic trunk reveals RALDH-2 mRNA both in mesoderm and neuroectoderm, with highest neuroectodermal expression in the ventral horn of the spinal cord at two restricted locations along the anteroposterior axis, presumably the subpopulation of motoneurons that innervate the limbs.
Tyro4 is a member of the eph family of receptor protein-tyrosine kinases. We present sequence analysis that identifies Tyro4 as the rat homolog of mouse Mek4 and chick Cek4. We also present expression studies that demonstrate an evolutionarily conserved pattern of expression for Tyro4, Mek4, and Cek4. Most strikingly, we find this receptor to be specifically expressed, in all three species, in a subset of motor neurons in the medial motor column and in a subset of axial, but not limb, muscles. Mek4 has previously been ascribed a role in guiding retinal axons to their targets in the optic tectum. Our results extend the purported role of Mek4 in axon guidance to include motor neurons of the medial motor column.
Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.
Neuraxial patterning is a continuous process that extends over a protracted period of development. During gastrulation a crude anteroposterior pattern, detectable by molecular markers, is conferred on the neuroectoderm by signals from the endomesoderm that are largely inseparable from those of neural induction itself. This coarse-grained pattern is subsequently reinforced and refined by diverse, locally acting mechanisms. Segmentation and long-range signaling from organizing centers are prominent among the emerging principles governing regional pattern.
DSL (Delta, Serrate, Lag-2) ligands activate Notch signaling and thereby regulate the differentiation of many different cell types during development. We have isolated a novel Serrate-like gene, Jagged2, whose amino acid sequence and expression pattern during rat embryogenesis suggest that it functions as a ligand for Notch. In contrast to previously described DSL ligands for Notch, Jagged2 is not widely expressed in the developing central nervous system. However, Jagged2 and Notch1 are coexpressed in the apical ectodermal ridge (AER), suggesting a role for this ligand-receptor pair in limb development. Furthermore, unlike Jagged1, Jagged2 is coexpressed with Notch in the developing thymus, where it may induce Notch signaling to direct T-cell fate. Our data are consistent with the idea that the diversity of cell types regulated by Notch signaling is a consequence of activation of unique Notch isoforms by different DSL ligands.
Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early embryo. Here we report the expression domains of the RALDH-2 gene during mouse embryogenesis, which are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis.
Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria [1] is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein-GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system [2,3], the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90-120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 micron-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein-GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology [4], and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) [5].
Graded Sonic hedgehog signaling may generate neuronal diversity in the ventral spinal cord in two steps: the creation of a generic population of ventral progenitors, followed by the Pax6-dependent generation of distinct subpopulations within these ventral progenitors.