Article

A role for FGF8 in the initiation and maintenance of vertebrate limb bud outgrowth

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Abstract

The outgrowth of the vertebrate limb bud is the result of a reciprocal interaction between the mesenchyme and a specialized region of the ectoderm, the apical ectodermal ridge (AER), which overlies it. Signals emanating from the AER act to maintain the underlying mesenchyme, called the progress zone, in a highly proliferative and undifferentiated state. Removal of the AER results in the cessation of limb bud growth, thus causing limb truncation. The best candidates for this AER-derived signal are members of the fibroblast growth factor (FGF) family, in particular FGF-4, which can maintain limb bud outgrowth following removal of the AER. However, FGF-4 is only expressed after considerable outgrowth has occurred and a well-developed limb bud has formed, and then only in the posterior part of the AER. Likewise, the other FGFs studied to date are not candidates for this activity. We report evidence that a recently identified member of this family, FGF-8, is expressed in the ectoderm of the prospective limb territory prior to morphological outgrowth of the limb bud in both mouse and chick. Thereafter, expression is maintained throughout the AER during limb development. We have produced and purified the FGF-8 protein, and shown that it will substitute for the AER in maintaining limb bud outgrowth in mouse embryos from which the AER has been surgically removed. FGF-8 does not, however, maintain expression of the sonic hedgehog gene. These results indicate that FGF-8 is an AER-derived mitogen that stimulates limb bud outgrowth. Moreover, our data suggest that FGF-8 may also be an ectodermally derived mitogen that stimulates the onset of limb bud outgrowth (budding) in the absence of a morphological AER, and indicate the possible involvement of FGF-8 in the establishment of the limb field.

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... They are known to be involved in angiogenesis, otic, and limb development, as well as wound healing (Christen & Slack, 1997;Christensen, et al., 2002;Crossley, et al., 1996a;Crossley, et al., 1996b;P. H. Crossley & Martin, 1995;Grieshammer, et al., 2005;Mahmood, et al., 1997;Schimmang, 2007;Zeller, et al., 2009). The expression of FGF-8 was observed during limb regeneration in axolotl, where its activities were detected in the basal layer of the AEC and the underlying mesenchyme during blastema formation, suggesting that the signalling factor is involved in blastema formation as well as outgrowth of the regenerating limbs (Han, et al., 2001). ...
... Starting at the onset of the 20th century, a series of studies demonstrated that the signal-interplay between the ZPA and the AER that governs early limb development in chick and mouse involves the BMP-Grm signalling system in the limb bud mesenchyme. Normal development of vertebrate limb buds in early stages of embryogenesis is directed by a string of feedback loops formed by distant cis-regulatory elements including SHH in the ZPA and the FGFs in the overlying AER (Crossley, et al., 1996b;Drossopoulou, et al., 2000;Lewandoski, et al., 2000;Mahmood, et al., 1997;Niswander, 2002Niswander, , 2003. In 1999, the involvement of Grm activities in AER maintenance was indicated by the spatiotemporal expression pattern of the gene during outgrowth of chick limb bud and that ectopic overexpression of Grm in the wing bud resulted in persistence of the AER and mesenchymal FGF-4 expression, repression of apoptosis in the anterior necrotic zone and in the interdigital mesenchyme of the limb bud, leading to soft tissue overgrowths and truncation of distal cartilage elements (Capdevila, et al., 1999). ...
... It is involved in the patterning of developing mouse embryos, induction of midbrain development in chick embryos, the regulation of chick limb development, as well as many others (Crossley, et al., 1996a;P. Crossley, et al., 1996b;Crossley & Martin, 1995;Fletcher, et al., 2006;Fürthauer, et al., 2004;Mahmood, et al., 1997). Embryos of compound heterozygote FGF-8 mutant mice generated through Cre-and Flp-mediated recombination display a range of phenotypes including smaller size and/or delayed development compared to their WT or heterozygous litter mates, severely affected brain development, evidence of cardiac defects, as well as failure of posterior development (see detailed discussion in Meyers, et al., 1998). ...
... One common family of genetic regulators implicated in vertebrate limb regeneration are fibroblast growth factor (FGF) proteins, having functional roles in limb outgrowth and morphogenesis with signaling functions in the apical ectodermal ridge (AER) of the developing limb bud (Martin 1998). In chick embryos, FGF8 and FGF10 have been shown to be endogenous inducers of limb bud formation (an apical ectodermal factor; Mahmood et al. 1995;Ohuchi et al. 1997), while ectopic treatment with FGF1, FGF2, or FGF4 similarly induces the formation of supernumerary limbs during development (Cohn et al. 1995). Interestingly, during limb regeneration in developing frogs (i.e., limb amputation prior to metamorphosis), FGF8 is promptly reexpressed in the newly formed AEC (Christen and Slack 1997). ...
... In addition, inhibition of FGF signaling within regenerative systems effectively blocks the formation of blastema and subsequent regeneration (Poss et al. 2000;Lin and Slack 2008). FGF proteins are such potent stimulators of limb growth that forced expression can rescue limb development following AER removal (Niswander et al. 1993;Fallon et al. 1994;Mahmood et al. 1995), or can even lead to the formation of ectopic limbs during development (Crossley et al. 1996;Ohuchi et al. 1997, an apical ectodermal factor). The importance of FGF signaling is underscored by regenerative neuronal dependence. ...
Article
Limb loss due to traumatic injury or amputation is a major biomedical burden. Many vertebrates exhibit the ability to form and pattern normal limbs during embryogenesis from amorphous clusters of precursor cells, hinting that this process could perhaps be activated later in life to rebuild missing or damaged limbs. Indeed, some animals, such as salamanders, are proficient regenerators of limbs throughout their life span. Thus, research over the last century has sought to stimulate regeneration in species that do not normally regenerate their appendages. Importantly, these efforts are not only a vital aspect of regenerative medicine, but also have fundamental implications for understanding evolution and the cellular control of growth and form throughout the body. Here we review major recent advances in augmenting limb regeneration, summarizing the degree of success that has been achieved to date in frog and mammalian models using genetic, biochemical, and bioelectrical interventions. While the degree of whole limb repair in rodent models has been modest to date, a number of new technologies and approaches comprise an exciting near-term road map for basic and clinical progress in regeneration.
... Epithelial signals have also been shown to be required for the expression of homeobox-containing genes such as Msx-1 (Takahashi et al., 1991). The strong colocalization of epithelial Fgf-8 expression and underlying Barx-1 expression in the maxillary primordia, combined with the ability of FGF-8 to maintain/induce Barx-1 suggests that FGF-8 is an endogeneous factor controlling mesenchymal expression of Barx-1 (Mahmood et al., 1995;Wall and Hogan, 1995;Richman et al., 1997). While we propose that FGF-8 can activate mesenchymal expression of Barx-1, we note that Fgf-8 is only expressed on the medial side of the maxillary primordia, whereas Barx-1 expression extends across the mediolateral axis. ...
... The Barx-1-specific sense control riboprobes were made with T3 RNA polymerase using Xho1 linearized plasmid and the antisense riboprobes with T7 RNA polymerase using EcoR1 linearized plasmid. The Fgf-8 probe was obtained by PCR from stage-20 face cDNA using specific primers (5Ј end: GACTGCGTCTTCACC-GAG; 3Ј end: CATTACCAACAACTAGAG) (Mahmood et al., 1995). The 350 bp PCR product was subcloned into PCRScript (Stratagene) and linearized by EcoR1 digestion before antisense riboprobe synthesis with T3 RNA polymerase. ...
Article
The vertebrate face develops from a series of primordia surrounding the primitive mouth and is thought to be patterned by the differential expression of homeobox-containing genes. Here we describe the isolation of the chick homologue of the homeobox-containing gene, Barx-1, and show its expression in the developing facial primordia, stomach, and appendicular skeleton. In the maxillary primordia, mesenchymal expression of Barx-1 is complementary to that of Msx-1, which correlate with overlying epithelial expression of Fgf-8 and Bmp-4, respectively. We show that epithelial signals are required to maintain Barx-1 expression and that FGF-8 can substitute for the epithelium. By contrast, BMPs reduce Barx-1 expression and can antagonize FGF-8 sig-naling. This suggests that in vivo, FGF-8/BMP signaling may regulate Barx-1 gene expression. This provides evidence that the differential expression of FGF-8 and BMPs may determine ho-meobox-containing gene expression and hence patterning of the facial primordia. Dev Dyn 1999;214:291–302.
... This early expression of Fgf-8 suggests that it is the primary signal for inducing the expression of Lhx-7 and setting up the A-P axis of the first branchial arch, which is in agreement with previous results (Grigoriou et al., 1998). Fgf-8 is also expressed at the extreme lateral regions of each pharyngeal pouch and the overlying surface ectoderm of the pharyngeal groove (Mahmood et al., 1995; Crossley and Martin, 1995). Lhx-7 is expressed in restricted regions surrounding these areas at the base of the branchial arches, further linking the expression of Fgf-8 with Lhx-7 (Fig. 1F,G). ...
... In addition, FGF8 is considered to have an important biological role during embryonic development, especially in brain development, gastrulation, and limb morphogenesis (Heikinheimo et al. 1994;Ohuchi et al. 1994). In particular, with regard to mesencephalic development, FGF8 is expressed in cardiac mesoderm underlying the mesencephalic/metencephalic region (Crossley and Martin 1995;Heikinheimo et al. 1994;Mahmood et al. 1995). As such, it is necessary to develop methods that allow for abundant production of FGF8 with high bioactivity for future specific treatment of FGF8-related diseases. ...
Article
Full-text available
Human fibroblast growth factor 8b (FGF8b) was expressed based on a baculovirus expression vector system (BEVS) and identified as having a protective effect on Parkinson's disease. Immunoblotting demonstrated that rhFGF8b proteins were recognized by a human anti-FGF8b antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield and protein quality. Our results indicated that the rhFGF8b was first detectable at 36 h postinfection and reached a maximum at 60 h. A multiplicity of infection (MOI) of 8 pfu/mL was suitable for harvest. The target protein was purified by heparin-affinity chromatography. In vitro methylthiazol tetrazolium (MTT) assays demonstrated that the purified rhFGF8b could significantly stimulate proliferation of NIH3T3 cells. Furthermore, to elucidate the effect of rhFGF8b on Parkinson's disease, we used FGF8b pretreatment on a cell model of Parkinson's disease. The results indicated that rhFGF8b prevented necrosis and apoptosis of 1-METHYL-4-phenyl pyridine (MPP(+)) treated PC12 cells. Moreover, the effect of FGF8b on messenger RNA (mRNA) levels of apoptosis and ERS genes was investigated to clarify the molecular mechanisms of FGF8b. The results suggest that FGF8b exerts neuroprotective effects by alleviating endoplasmic reticulum (ER) stress during PD. These results suggest that FGF8b may be a promising candidate therapeutic drug for neurodegenerative diseases related to ER stress.
... A minimum of 4 mutant embryos were analysed for each genotype at each stage. mFgf10 [48] and mFgf8 [49] probes were reported previously. We used a full-length mouse Sall4 clone as a probe template. ...
Article
Full-text available
Author Summary Externally, the human form appears bilaterally symmetric. For example, each of our pairs of arms and legs are the same length. This external symmetry masks many asymmetries found in internal organs. In most people the heart is found on the left side of the chest. The stomach, liver and spleen are also positioned asymmetrically. The authors of this study demonstrate, using a mouse model, that bilateral symmetry of the arms is not a default, passive state but that mechanisms are in place that ensure symmetrical formation of the left and right limbs. Bilateral symmetry of the arms is achieved by the action of a gene Tbx5 that masks the effects of signals that acted earlier during embryogenesis, many days before limb formation, and imposed asymmetries on the forming internal organs. Maintaining bilateral symmetry of the arms is important for them to carry out their normal functions but this process can go wrong. Holt-Oram syndrome patients have upper limb defects, including shortened arms. Consistently the defects are more severe in their left arm than right. This birth defect is caused by disruption of the TBX5 gene. By linking the action of Tbx5 to symmetrical limb formation, the authors provide an explanation for why Holt-Oram syndrome patients have more severe defects in the left arms than right.
... Whole mount and section in situ hybridisation protocol and Tbx4, Pitx1, Hoxa13 probes have been described previously (DeLaurier et al. 2006, Riddle et al. 1993, Fgf10 probe (Bellusci et al. 1997), Col2a1 probe (Metsaranta et al. 1991), Sox9 probe (Kent et al. 1996), Fgf8 (Mahmood et al. 1995 (Capdevila et al. 1999, Mercader et al. 2000. Numbers of embryos processed with each probe is described in the respective figure legends. ...
Article
Full-text available
We dissect genetically a gene regulatory network, including the transcription factors Tbx4, Pitx1 and Isl1 that act cooperatively to establish the hindlimb bud and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects in the Tbx4 mutant result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing a novel explanation for the origins of proximally-biased limb defects.
... (F: 5′-CCGGAATTCAAGGGAGAGGTGCTGCGTGTGC-3′; R: 5′-CCGCTCGAGGAGGCAGGCACGGAGACATTGC-3′), Fgf10 (F: 5′-TGCTCTTTTTGGTGTCTTCGT-3′, R: 5′-GGGAGCTCCTTTTCCATTCAA-3′; 490 bp), RxLong (F: 5′-GGGGCTAGCATGCACCTGCCGGGCTGCGCG-3′, R: 5′-GGGGAATTCCTAGAGGGCTTGCCAGGGCTT-3′; 1029 bp), RxEx2 (F: 5′-AGCTACTAGGCCCTGCTAC-3′, R: 5′-GTCGGTTCTGGAACCATAC-3′; 275 bp)contained a 600-bp fragment of Pomc exon 3, a 730-bp fragment of Fgf855 and a full-length Shh cDNA (1300 bp). Antisense riboprobes were synthesised with a DIG labelling mix (Roche) using the digested vector as a template and SP6 (Roche) or T7 (Promega) RNA polymerases. ...
Article
The hypothalamus is a region of the anterior forebrain that controls basic aspects of vertebrate physiology, but the genes involved in its development are still poorly understood. Here, we investigate the function of the homeobox gene Rax/Rx in early hypothalamic development using a conditional targeted inactivation strategy in the mouse. We found that lack of Rax expression prior to embryonic day 8.5 (E8.5) caused a general underdevelopment of the hypothalamic neuroepithelium, while inactivation at later timepoints had little effect. The early absence of Rax impaired neurogenesis and prevented the expression of molecular markers of the dorsomedial hypothalamus, including neuropeptides Proopiomelanocortin and Somatostatin. Interestingly, the expression domains of genes expressed in the ventromedial hypothalamus and infundibulum invaded dorsal hypothalamic territory, showing that Rax is needed for the proper dorsoventral patterning of the developing medial hypothalamus. The phenotypes caused by the early loss of Rax are similar to those of eliminating the expression of the morphogen Sonic hedgehog (Shh) specifically from the hypothalamus. Consistent with this similarity in phenotypes, we observed that Shh and Rax are coexpressed in the rostral forebrain at late head fold stages and that loss of Rax caused a downregulation of Shh expression in the dorsomedial portion of the hypothalamus.
... Fgf8, for example, is expressed throughout the apical ectodermal ridge (AER) and mediates AER activity (Fernandez-Teran and Ros 2008; Lewandoski et al. 2000;Sun et al. 2002). A continuous feedback loop between Fgfs in the mesenchyme and AER helps to maintain cell proliferation and proximal-distal limb outgrowth (Crossley et al. 1996;Lewandoski et al. 2000;Mahmood et al. 1995;Mariani et al. 2008;Moon and Capecchi 2000;Yu and Ornitz 2008). Therefore, it is possible that differences in Fgf/ MAPK signaling are driving differences in cellular proliferation in opossum fore-and hind limbs, and this contributes to their differential growth rate. ...
Article
Full-text available
A fundamental question in biology is “how is growth differentially regulated during development to produce organs of particular sizes?” We used a new model system for the study of differential organ growth, the limbs of the opossum (Monodelphis domestica), to investigate the cellular and molecular basis of differential organ growth in mammals. Opossum forelimbs grow much faster than hindlimbs, making opossum limbs an exceptional system with which to study differential growth. We first used the great differences in opossum forelimb and hindlimb growth to identify cellular processes and molecular signals that underlie differential limb growth. We then used organ culture and pharmacological addition of FGF ligands and inhibitors to test the role of the Fgf/Mitogen-activated protein kinases (MAPK) signaling pathway in driving these cellular processes. We found that molecular signals from within the limb drive differences in cell proliferation that contribute to the differential growth of the forelimb and hindlimbs of opossums. We also found that alterations in the Fgf/MAPK pathway can generate differences in cell proliferation that mirror those observed between wild-type forelimb and hindlimbs of opossums and that manipulation of Fgf/MAPK signaling affects downstream focal adhesion-extracellular matrix (FA-ECM) and Wnt signaling in opossum limbs. Taken together, these findings suggest that evolutionary changes in the Fgf/MAPK pathway could help drive the observed differences in cell behaviors and growth in opossum forelimb and hindlimbs.
... They function in promoting cell survival and proliferation of undifferentiated mesenchymal cells (Niswander et al., 1994a(Niswander et al., , 1994bHara et al., 1998;Ngo-Muller and Muneoka, 2000;Han et al., 2001;Niswander, 2002;Weatherbee et al., 2006) as well as specifying cell fate during digit formation (Mariani et al., 2008;Lu et al., 2008). Fgf4 (genes indicated by italicized text, whereas proteins are indicated by Roman font), Fgf8, Fgf9, and Fgf17 are some of the many genes expressed in the apical ectoderm of developing limbs; but Fgf8 is the most important for normal limb outgrowth (Niswander et al., 1994a(Niswander et al., , 1994bMahmood et al., 1995;Vogel et al., 1996;Hara et al., 1998;Moon and Capecchi, 2000;Ngo-Muller and Muneoka, 2000;Sun et al., 2002;Talamillo et al., 2005;Verheyden and Sun, 2008). It is expressed earlier and at higher concentrations compared to other FGFs (Fernandez-Teran and Ros, 2008;Mariani et al., 2008). ...
... During late gastrulation and early neurulation stages Otx2 is expressed from the anterior limit of the neural plate to a posterior border at the presumptive MHB and Gbx2 is expressed in a complementary fashion in the posterior embryo (Wassarman, Lewandoski et al. 1997). Subsequently Pax2 is activated, followed by Eng1, Wnt1 and FGF8 (Crossley and Martin 1995;Mahmood, Bresnick et al. 1995;Rowitch and McMahon 1995;Reifers, Bohli et al. 1998). These genes are activated around the Otx2-Gbx2 interface consistent with the notion, that the region where Gbx2 and Otx2 abut demarcates the primordium of the MHB. ...
Thesis
A hindbrain derived neural stem cell line (c17.2) overexpressing Nurr1, an orphan nuclear receptor transcription factor, was examined to differentiate into dopaminergic neurons and to survive after transplantation. Nurr1 plays an important role in final differentiation of midbrain neural precursor cells into dopaminergic neurons. c17.2-cells overexpressing Pax2, a transcription factor crucial for development of midbrain-hindbrain boundary (MHB), were analyzed regarding the potential of Pax2 to induct neural differentiation and expression of genes more downstream on the way of dopaminergic differentiation. Nurr1-overexpressing c17.2 cells were transplanted together with radial glia cells into a neonatal rat model of Parkinson‟s disease (PD). Survival and differentiation of the cells were examined using immunohistochemistry. Moreover, those cells were pre-differentiated in vitro prior to transplantation using a co-culture together with FGF20-overexpressing Schwann cells. Pre-differentiated cells were transplanted into a neonatal and adult rat model of PD and analyzed by immunohistochemistry. Pax2-overexpressing c17.2 cells were differentiated in vitro, expression of downstream genes Pax5, FGF8 and Eng1 was analyzed using realtime-PCR. After transplantation together with radial glia cells Nurr1-c17.2 cells did not show transplant survival, whereas they demonstrated good survival after pre-differentiation in co-culture with FGF20-Schwann cells. Moreover, cells revealed tyrosine hydroxylase positive immunoreactivity and process growing in the neonatal rats. Pax2-c17.2 cells were kept in cell cycle and did not differentiate into neurons or glia cells. Pax5 was up-regulated, FGF8 unchanged and Eng1 displayed a trend to down-regulation after differentiation. Results call for further studies of FGF20 and Pax2 to induce dopaminergic differentiation and to increase the proportion of dopaminergic neurons. Moreover, transplantation protocols must be developed to enhance survival and functional integration of dopaminergic neurons within the adult host environment.
... The embryo then undergoes lateral folding, with the somites coming to lie dorsally and the edges of the lateral plate mesoderm eventually fusing in the ventral midline of the body; this folding brings the thickened mesoderm, which will form the wing buds to the sides of the body (Fig. 2, stage 16). By stage 16, Fgf8 is expressed in ectoderm cells of the wing bud which will form the apical ectodermal ridge (Mahmood et al. 1995;Crossley et al. 1996). Shh expression in the mesoderm at the posterior margin is detected a few hours later at stage 17 when the wing bud first becomes clearly visible ( Fig. 1; Riddle et al. 1993). ...
Article
The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors (FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein (BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis. © 2015 Anatomical Society.
... Bhlha9 regulates TP63 (Trp63) and Fgf8 expression TP63 (Trp63) and Fgf8 are expressed in the AER, and serve as key molecular modulators of limb development [5,6,19,20]. On the basis of gene expression analysis, abnormal expression of these genes was confirmed in E10.5 limb buds of Bhlha9-knockout mice. ...
Article
Split hand/foot malformation (SHFM) and SHFM combined with long-bone deficiency (SHFLD) are congenital dysgeneses of the limb. Although six different loci/mutations (SHFM1–SHFM6) have been found from studies on families with SHFM, the causes and associated pathogenic mechanisms for a large number of patients remain unidentified. On the basis of the identification of a duplicated gene region involving BHLHA9 in some affected families, BHLHA9 was identified as a novel SHFM/SHFLD-related gene. Although Bhlha9 is predicted to participate in limb development as a transcription factor, its precise function is unclear. Therefore, to study its physiological function, we generated a Bhlha9-knockout mouse and investigated gene expression and the associated phenotype in the limb bud. Bhlha9-knockout mice showed syndactyly and poliosis in the limb. Moreover, some apical ectodermal ridge (AER) formation related genes, including Trp63, exhibited an aberrant expression pattern in the limb bud of Bhlha9-knockout mice; TP63 (Trp63) was regulated by Bhlha9 on the basis of in vitro analysis. These observations suggest that Bhlha9 regulates AER formation during limb/finger development by regulating the expression of some AER-formation-related genes and abnormal expression of Bhlha9 leads to SHFM and SHFLD via dysregulation of AER formation and associated gene expression.
... FGF8 is expressed throughout the AER, indicating its important role in limb development. [120][121][122] Mice with deleted Fgf8 show early embryonic lethality before limb development. 123-124 Lewandoski et al. generated mice with targeted deletion of Fgf8 124 (via Msx2-cre) in limb bud. ...
Article
Full-text available
Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling plays essential roles in bone development and diseases. Missense mutations in FGFs and FGFRs in humans can cause various congenital bone diseases, including chondrodysplasia syndromes, craniosynostosis syndromes and syndromes with dysregulated phosphate metabolism. FGF/FGFR signaling is also an important pathway involved in the maintenance of adult bone homeostasis. Multiple kinds of mouse models, mimicking human skeleton diseases caused by missense mutations in FGFs and FGFRs, have been established by knock-in/out and transgenic technologies. These genetically modified mice provide good models for studying the role of FGF/FGFR signaling in skeleton development and homeostasis. In this review, we summarize the mouse models of FGF signaling-related skeleton diseases and recent progresses regarding the molecular mechanisms, underlying the role of FGFs/FGFRs in the regulation of bone development and homeostasis. This review also provides a perspective view on future works to explore the roles of FGF signaling in skeletal development and homeostasis.
... FGF8 is one of several FGF isoforms expressed in the AER responsible for maintaining the outgrowth and differentiation of the mesenchymal cells making up the limb bud (Mariani et al., 2008). FGF8 and Shh cross-talk to regulate limb bud development (Mahmood et al., 1995;Probst et al., 2011). Therefore, we examined FGF8 expression in the fore-and hind-limbs by in situ hybridization after the embryos were treated with various high dose of glucose. ...
... -------------------------------------11 Fibroblast growth factors (FGFs) play an important role in the development of hormonal cancers (Dorkin et al. 1999. In healthy adults, low expression of FGF8 is found mainly in reproductive organs , Valve et al. 1997) but during embryogenesis FGF8 has an important role in gastrulation, development of the nervous system, the heart, skeleton and limbs (Crossley, Martin 1995, Mahmood et al. 1995, Sun et al. 1999). FGF8b has found to be the most malignant of the four isoforms found in humans (MacArthur et al. 1995b). ...
... mRNA in situ hybridization was performed as previously described (Sive et al., 2000; Wilkinson et al., 1989). The following mRNA probes were used: mouse Fgf8 (Mahmood et al., 1995), mouse erm (Hippenmeyer et al., 2002), mouse Pea3 (Livet et al., 2002), X. laevis Fgf8 (Monsoro-Burq et al., 2003), and X. laevis twist (Hopwood et al., 1989). Primary antibodies used for immunohistochemistry: anti-rabbit phospho-histone H3 (PH3) (BDH, 1:200) or anti-rabbit b-catenin (Sigma, 1:200). ...
... Fgf8 is an important factor for limb development that is expressed in the dorsoventral boundary of the limb ectoderm (Heikinheimo et al., 1994;Ohuchi et al., 1994;Mahmood et al., 1995). It is known that ectodermal Fgf8 expression and secreted FGF8 protein are essential for Shh expression (Crossley et al., 1996;Vogel et al., 1996). ...
Article
Background: The organizing center, which serves as a morphogen source, has crucial functions in morphogenesis in animal development. The center is necessarily located in a certain restricted area in the morphogenetic field, and there are several ways in which an organizing center can be restricted. The organizing center for limb morphogenesis, the ZPA (zone of polarizing activity), specifically expresses the Shh gene and is restricted to the posterior region of the developing limb bud. Results: The pre-pattern along the limb anteroposterior axis, provided by anterior Gli3 expression and posterior Hand2 expression, seems insufficient for the initiation of Shh expression restricted to a narrow, small spot in the posterior limb field. Comparison of the spatiotemporal patterns of gene expression between Shh and some candidate genes (Fgf8, Hoxd10, Hoxd11, Tbx2, and Alx4) upstream of Shh expression suggested that a combination of these genes' expression provides the restricted initiation of Shh expression. Conclusions: Taken together with results of functional assays, we propose a model in which positive and negative transcriptional regulatory networks accumulate their functions in the intersection area of their expression regions to provide a restricted spot for the ZPA, the source of morphogen, Shh. Developmental Dynamics 246:417-430, 2017. © 2017 Wiley Periodicals, Inc.
... A deficiency of Fgf8 and 4 (from the AER) (Boulet et al., 2004) or Fgf10 (from limb mesoderm) (Min et al., 1998) in mice results in the complete absence of fore-and hindlimbs. Interrupting AER-related FGF function either by AER removal in chick wings or conditional Fgf receptor knockouts at various developmental stages, truncates limbs along the proximal-distal axis corresponding to its progressive stage of distalization (Saunders, 1948;Mahmood et al., 1995;Lu et al., 2008;Yu and Ornitz, 2008). In addition, maintenance of AER integrity and FGF secretion is important for proper digit formation. ...
Chapter
Exfoliation syndrome (XFS) is a genetically determined systemic condition characterized by the production and accumulation of an abnormal protein, the exfoliative material. Though XFS is a systemic elastosis, the most significant clinical consequences occur in the eye (nuclear cataract formation, Zinn’s zonule fragility, increased risk for cataract surgery complications and exfoliative glaucoma XFG). XFG is the most common type of secondary open-angle glaucoma, and is characterized by elevated intraocular pressure with high fluctuation, and rapid progression. XFG is typically a disease of the elderly. In many cases XFG starts as a clinically unilateral disease in eyes with XFS. This chapter summarizes the current knowledge on genetics, environmental factors, clinical characteristics, medical, laser and surgical treatment options. It also addresses the significance of systemic vascular diseases associated with XFS and XFG, and the need for consultation between the ophthalmologist and cardiologist or general practitioner during long-term management of the disease.
... Similarly to Fgf8 deletion in mice, Fgf10-knockout mice show a complete lack of forelimb and hindlimb development; limb bud initiation still occurs in these mice but limb bud outgrowth is impaired [147][148][149] . FGF8 is also expressed in osteoblasts, specifically in the cortical bone of the calvarium 150,151 . Addition of FGF8 to bone cell culture medium results in increased differentiation of the cells into osteoblasts and increased in vitro bone formation 152 . ...
Article
Bone development occurs through a series of synchronous events that result in the formation of the body scaffold. The repair potential of bone and its surrounding microenvironment - including inflammatory, endothelial and Schwann cells - persists throughout adulthood, enabling restoration of tissue to its homeostatic functional state. The isolation of a single skeletal stem cell population through cell surface markers and the development of single-cell technologies are enabling precise elucidation of cellular activity and fate during bone repair by providing key insights into the mechanisms that maintain and regenerate bone during homeostasis and repair. Increased understanding of bone development, as well as normal and aberrant bone repair, has important therapeutic implications for the treatment of bone disease and ageing-related degeneration.
... In amniote embryos, induction of early limb buds is, at least in part, dependent on signaling between the somites and the lateral plate mesoderm (LPM; Duboc & Logan, 2011;Duester, 2008;Zeller et al., 2009;Zhao et al., 2009), while the maintenance of limb development depends on the activity of the AER (Mahmood et al., 1995). ...
Article
Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb‐reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake‐like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B. lukbani in early stages of development, on the other hand, show a truncated but well‐developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole‐mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B. lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well‐developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well‐developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well‐developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that “limbless” lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B. lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade‐specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.
... The apical ectodermal ridge is a thickening of the distal epithelium that maintains limb outgrowth (17)(18)(19)(20) and is marked by the expression of Fgf8. However, the proliferative growth of the chick wing is an autonomous property of the mesoderm and is associated with the precise regulation of cell cycle parameters (G1-S-phase entry), in both the distal tip and in the polarising region (3,21) -a region of posterior-distal mesoderm that produces Sonic hedgehog . ...
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How development is timed between differently sized species is a fundamental question in biology. To address this problem, we compared wing development in the quail and the larger chick. We reveal that developmental timing is faster in the quail than in the chick, and is associated with pattern specification, proliferation, organiser duration, differentiation and apoptosis. However, developmental timing is independent of the growth rate, which is equivalent between both species, and therefore scales pattern to the size of the wing. We reveal that developmental timing can be either maintained or reset in interspecies tissue grafts, and we implicate retinoic acid as the resetting signal. Accordingly, retinoic acid can switch species developmental timing and rescale pattern, both between the quail and chick, and the chick and the larger turkey. We suggest that the scaling of pattern to wing bud size is achieved by the modulation of developmental timing against a comparable rate of growth. Summary - We show that developmental timing scales wing patterning
... Based on the morphological similarity between apical epidermal ridge (AER) in developing limb bud and AEC in regenerating limb, the two structures are thought to share common roles in growth and patterning in limb formation in the respective situation, limb development or regeneration. FGF molecules, especially FGF8, are assumed to have a pivotal role in both processes, as the instructive role of fgf8 in limb development is evident in several vertebrate species (Crossley, Minowada, MacArthur, & Martin, 1996;Mahmood et al., 1995) and fgf8 is expressed in AEC during limb regeneration in amphibians (Christen & Slack, 1997;Han et al., 2001;Yokoyama et al., 2007). On the other hand, it was reported that MARCKS-like protein (MLP) was expressed in WE and induced cell proliferation during axolotl limb and tail regeneration, but its counterparts in other species, including Xenopus laevis, are not likely to be secreted (Sugiura, Wang, Barsacchi, Simon, & Tanaka, 2016). ...
Article
Wound epidermis (WE) and the apical epithelial cap (AEC) are believed to trigger regeneration of amputated appendages such as limb and tail in amphibians by producing certain secreted signaling molecules. To date, however, only limited information about the molecular signatures of these epidermal structures is available. Here we used a transgenic Xenopus laevis line harboring the enhanced green fluorescent protein (egfp) gene under control of an es1 gene regulatory sequence to isolate WE/AEC cells by performing fluorescence-activated cell sorting during the time course of tail regeneration (day 1, day 2, day 3 and day 4 after amputation). Time-course transcriptome analysis of these isolated WE/AEC cells revealed that more than 8,000 genes, including genes involved in signaling pathways such as those of reactive oxygen species, fibroblast growth factor (FGF), canonical and non-canonical Wnt, transforming growth factor β (TGF β) and Notch, displayed dynamic changes of their expression during tail regeneration. Notably, this approach enabled us to newly identify seven secreted signaling molecule genes (mdk, fstl, slit1, tgfβ1, bmp7.1, angptl2 and egfl6) that are highly expressed in tail AEC cells. Among these genes, five (mdk, fstl, slit1, tgfβ1 and bmp7.1) were also highly expressed in limb AEC cells but the other two (angptl2 and egfl6) are specifically expressed in tail AEC cells. Interestingly, there was no expression of fgf8 in tail WE/AEC cells, whose expression and pivotal role in limb AEC cells have been reported previously. Thus, we identified common and different properties between tail and limb AEC cells.
... The mesenchyme-to-ectoderm signal is in fact strong enough to initiate an ectopic AER in lateral ectoderm that would not normally produce a limb, and thereby cause an ectopic limb (Kieny, 1968). There are a number of putative intercellular signalling molecules expressed in the AER which may be responsible for this signal to the PZ: bone morphogenetic proteins (which are members of the TGF-b superfamily) BMP-2 (Lyons et ai, 1990) and BMP-4 (Jones et a l, 1991) and fibroblast growth factors: FGF-2 , FGF-4 (Niswander and Martin, 1992) and FGF-8 (Crossley and Martin, 1995;Mahmood et al., 1995;Crossley et a l, 1996). So far, the strongest evidence supporting any of these candidates is for FGF-4: In short-term organ cultures it can maintain distal outgrowth of mouse limb buds that have been stripped of their AER (Niswander etal., 1993), and outgrowth of chick wing buds whose AERs have been removed can be rescued by application of beads soaked in FGF-4 protein. ...
Thesis
The vertebrate Hox genes are essential for embryo development, and are thought to specify positional information along the anteroposterior (A-P) axis. In this study, murine transgenic technology was used to further our understanding of how Hox genes are regulated, and to test a hypothesis which might explain their clustered organisation. Two regions of the Hoxb complex were studied: the Hoxb-9 region, and the intergenic stretch betfween Hoxb-5 and Hoxb-4. In the first study, constructs were created which contained regulatory elements from near the Hoxb-9 gene coupled to the reporter gene LacZ. These allowed identification of two regulatory regions which appear to be important for normal expression of the Hoxb-9 gene. Previous work on the regulation of the Hoxb-5 gene had suggested that two regulatory elements which are probably important for Hoxb-5 may also interact with the Hoxb-4 promoter. In the second study, a double-reporter system was developed, with which it was possible to monitor the expression from two different promoters in the same construct. Constructs were created spanning the Hoxb-5-Hoxb-4 region, in which the LacZ gene was inserted into the Hoxb-5 coding region, and the human placental alkaline phosphatase (PLAP) gene was inserted into the Hoxb-4 coding region. Using this construct, and two versions in which the regulatory regions had been deleted, it was possible to show that certain elements in this stretch of DNA may be able to activate both promoters. During the course of these transgenic experiments, an insertional mutant was created which displayed preaxial polydactyly. In the last part of this study, the mutant strain was characterised with regard to its developmental and skeletal phenotype, the chromosomal localisation of the transgene, expression of the gene Sonic hedgehog in the developing limb bud, and expression of the transgenic PLAP reporter construct. Using these data the strain was compared to previously known polydactylous mutants from the hemimelia-luxate group.
... 58 FGF10 induces WNT, BMP, and FGF8 signals in the ectoderm, establishing the overlying Apical Ectodermal Ridge (AER). 59,60 The AER forms as a distal thickening of epithelium and is an essential signaling center necessary for limb bud outgrowth and patterning. 4,11,61 FGF8 is secreted into the underlying mesenchyme to further activate FGF10 and establish a positive feedback loop which maintains the limb bud mesenchyme in a proliferative state ( Figure 1E). ...
Article
The vertebrate limb is a dynamic structure that has evolved into many diverse forms to facilitate complex behavioural adaptations. The principle molecular and cellular processes that underlie development of the vertebrate limb are well characterised. However, how these processes are altered to drive differential limb development between vertebrates is less well understood. Several vertebrate models are being utilized to determine the developmental basis of differential limb morphogenesis, though these typically focus on later patterning of the established limb bud and may not represent the complete developmental trajectory. Particularly, heterochronic limb development can occur prior to limb outgrowth and patterning but receives little attention. This review summarises the developmental regulation of vertebrate forelimb diversity, with particular focus on wing reduction in the flightless emu as a model for studying limb heterochrony. These studies highlight that wing reduction is complex, with heterochronic cellular and genetic events influencing the major stages of limb development. Together, these studies provide a broader picture of how different limb morphologies may be established during development. This article is protected by copyright. All rights reserved.
... However, mice hom ozygous for a null m utation in Fgf2 show no overt defects in kidney m orphogenesis (Dono et al., 1998;Ortega et al, 1998;Zhou et al, 1998), dem onstrating that simple failure of FGF2 signalling is not an adequate explanation for the Hs2st m utant phenotype. Since other members of the FGF family (including FGF7 (Finch et al., 1995) and FGF8 (Mahmood et al., 1995; data not shown)) are expressed in the developing kidney, the phenotype observed in H s2 st"/embryos m ay be due to perturbation of m ore than one FGF signalling pathw ay. ...
Thesis
The acquisition of pattern and form by metazoan organisms relies on highly orchestrated cell-cell communications. In an attempt to learn more about the molecules that are required for these processes, a gene trap screen has been employed that is designed to identify, mutate and report on the embryonic expression pattern of genes expressed in mouse embryonic stem cells. This thesis deals with the initial characterisation of gene trap integrations from this screen and the detailed analysis of one of these insertions, termed ST125. The gene disrupted in this cell line encodes the previously unidentified mouse heparan sulphate 2-sulphotransferase (HS2ST). This enzyme catalyses a specific modification of the glycosaminoglycan chains of heparan sulphate proteoglycans (HSPGs), a class of molecule that has recently been implicated in the reception of a number of secreted signalling molecules important for development. Transgenic mice have been generated from embryonic stem cells harbouring this insertion. lacZ reporter gene activity in heterozygous embryos has demonstrated that the gene is expressed differentially during embryogenesis, presumably directing dynamic changes in heparan sulphate structure. Moreover, mice homozygous for the Hs2st gene trap allele die perinatally and exhibit bilateral renal agenesis and defects of the eye, skeleton and female genital system. Analysis of metanephric kidney development in Hs2st mutants reveals that the gene is not required for two early events; ureteric bud outgrowth from the Wolffian duct and initial induction of the metanephric mesenchyme. However, it is required subsequently for condensation of the mesenchyme around the ureteric bud and initiation of branching morphogenesis. Finally, the results of in vitro manipulations of Hs2st mutant kidneys are consistent with a crucial role for HSPGs in intercellular signalling during metanephric development. These data provide the first genetic evidence that specific glycosaminoglycan structures are required for highly selective morphogenetic events, presumably by facilitating the action of specific signalling molecules.
... Fgf-8 localisation has perhaps the best correlation with the morphological ridge (Crossley and Martin, 1995;Mahmood et al., 1995), 2ls fgf-4 is expressed in a more restricted domain of the posterior ridge (Niswander and Martin, 1992). ...
Thesis
I have investigated the phenomenon of neural crest regneration in the chick embryonic hindbrain using cellular and molecular markers as a model for the study of dorso-ventral patterning. By comparing and contrasting the expression of a number of genes - Pax-3, Pax-6, slug, dorsalin-1, Bmp-4, Bmp-7 and examination of the neural structures of the hindbrain in normal and ablated hindbrain, the critical events of repair and re-patterning were determined. The predominant finding was that the dorso-ventral pattern of the hindbrain was reproduced despite the removal of the dorsal third of the neural tissue. In agreement with other studies this indicates that dorsal and ventral signals coordinate to produce the spectrum of cell types in this axis. Additionally I found that slug and Pax-3 marked apparently independent lineages. Despite the normal re-patterning of the hindbrain after ablation this does not appear to have occurred by a recapitulation of normal development. That is, the sequence of gene expression (slug and Pax-3) resembles the trunk rather than the head, with slug expression being dependent upon closure of the ablated neural tube. This up-regulation of slug in cells of the dorsal midline correlated with the down-regulation of Pax-3 in the same population of dorsal midline cells, indicating that the exclusivity of these two genes may be obligate. To investigate this observation evidence of slug expression in the chick limb and other regions of the embryo was utilised. In the course of a range of experiments (in collaboration with K. Kostakopoulou) I found that slug expression was up-regulated by fgf-4 protein and down-regulated by ridge removal or retinoic acid addition in the wing bud. Thus, slug expression correlated positively with growth and the undifferentiated state and negatively with the onset of differentiation (condensation of progress zone cells). To conclude, slug and Pax-3 are expressed in non-overlapping, but neighbouring domains in the neural tube and represent lineage markers with very different characteristics; Pax-3 marks predominantly myogenic and neurogenic lineages and slug pre-skeletogenic.
Article
How can a group of undifferentiated cells develop into complex structures and highly organized and differentiated tissues? What is the nature of the signals that dictate these events? Which developmental processes act so that different species can develop similar structures adapted to the environment where they live? A model that partly answers these questions is the embryonic limb. Limb bud studies have suggested interesting paradigms that have changed the perception of development as an isolated and not very dynamic process. The present work reviews the studies that have led to understand the induction of the limb bud and the establishment of the organizing centers that control limb morphogenesis, as well as the skeletal pattern that includes cartilage differentiation and cell death.
Article
The analysis of vertebrate limb bud development provides insight of general relevance into the signaling networks that underlie the controlled proliferative expansion of large populations of mesenchymal progenitors, cell fate determination and initiation of differentiation. In particular, extensive genetic analysis of mouse and experimental manipulation of chicken limb bud development has revealed the self-regulatory feedback signaling systems that interlink the main morphoregulatory signaling pathways including BMPs and their antagonists. It this review, we showcase the key role of BMPs and their antagonists during limb bud development. This review provides an understanding of the key morphoregulatory interactions that underlie the highly dynamic changes in BMP activity and signal transduction as limb bud development progresses from initiation and setting-up the signaling centres to determination and formation of the chondrogenic primordia for the limb skeletal elements.
Thesis
Embryonic wound repair is both rapid and perfect. In this thesis, I describe my studies investigating the molecular mechanisms underlying this process. I report my experiments searching for genes which are upregulated at the wound site in a mouse embryo model, and explore the hypothesis that the cellular and molecular mechanisms that underlie tissue repair are the same ones that drive natural morphogenetic tissue movements. My studies have focussed on two animal models: the E11.5 mouse embryo and the Drosophila embryo. In the mouse I have used two approaches to identify "wound-induced" genes (WIGs) upregulated in response to wounding: 1. An informed guesswork approach, looking for expression of genes which seem likely candidates for upregulation at the wound site; and 2. A subtractive hybridisation approach, to identify unknown WIGs. My predictive approach reveals a dramatic upregulation of the zinc-finger transcription factors krox-20 and krox-24, within minutes of wounding, following an identical timecourse of expression as c-fos, the first embryonic WIG ever identified. My subtractive search reveals a number of exciting WIGs, including a protective antioxidant. Non-selenium glutathione peroxidase, which has previously been identified in a cell-based search for wound genes. Alongside these mouse studies, I have characterised two wound healing models in the fruitfly, Drosophila melanogaster: one in late stage embryos and the other in wing imaginal discs. I have shown that the tissue movements of morphogenesis, particularly dorsal closure, and wound repair are remarkably similar, both in their gross appearance and the cytoskeletal machinery they employ. I have demonstrated that Drosophila embryos and imaginal discs use exactly the same cytoskeletal machinery to close a wound as vertebrate embryos, assembling a contractile actin cable within minutes of wounding, suggesting that mechanisms of embryonic wound repair are remarkably well conserved. I have also shown that it is possible to investigate gene expression at the wound site in imaginal discs, and serendipitously struck upon another wound healing model in the larval epidermis, where the tissue polarity gene Dfz2, a homologue of which was identified in my mouse wound subtractive screen, is shown to be upregulated following wounding. The characterisation of the Drosophila wound models described in this thesis will allow further genetic dissection of the molecular events governing wound repair, in a genetically tractable model. These future studies will allow us to determine whether the cellular and molecular tools facilitating tissue repair are indeed the same ones that drive natural morphogenetic tissue movements in the embryo.
Article
Gastrulation is the process in which the three germ layers are formed that contribute to the formation of all major tissues in the developing embryo. We here review mouse genetic models in which defective gastrulation leads to mesoderm insufficiencies in the embryo. Depending on severity of the abnormalities, the outcomes range from incompatible with embryonic survival to structural birth defects, such as heart defects, spina bifida, or caudal dysgenesis. The combined evidence from the mutant models supports the notion that these congenital anomalies can originate from perturbations of mesoderm specification, epithelial–mesenchymal transition, and mesodermal cell migration. Knowledge about the molecular pathways involved may help to improve strategies for the prevention of major structural birth defects. Birth Defects Research (Part A), 2014. © 2014 Wiley Periodicals, Inc.
Article
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Fibroblast growth factors (FGF) and their receptors serve many functions in both the developing and adult organism. Humans contain 18 FGF ligands and four FGF receptors (FGFR). FGF ligands are polypeptide growth factors that regulate several developmental processes including cellular proliferation, differentiation, and migration, morphogenesis, and patterning. FGF-FGFR signaling is also critical to the developing axial and craniofacial skeleton. In particular, the signaling cascade has been implicated in intramembranous ossification of cranial bones as well as cranial suture homeostasis. In the adult, FGFs and FGFRs are crucial for tissue repair. FGF signaling generally follows one of three transduction pathways: RAS/MAP kinase, PI3/AKT, or PLCγ. Each pathway likely regulates specific cellular behaviors. Inappropriate expression of FGF and improper activation of FGFRs are associated with various pathologic conditions, unregulated cell growth, and tumorigenesis. Additionally, aberrant signaling has been implicated in many skeletal abnormalities including achondroplasia and craniosynostosis. The biology and mechanisms of the FGF family have been the subject of significant research over the past 30 years. Recently, work has focused on the therapeutic targeting and potential of FGF ligands and their associated receptors. The majority of FGF-related therapy is aimed at age-related disorders. Increased understanding of FGF signaling and biology may reveal additional therapeutic roles, both in utero and postnatally. This review discusses the role of FGF signaling in general physiologic and pathologic embryogenesis and further explores it within the context of skeletal development.
Thesis
Bone morphogenetic protein-4 (Bmp-4) is a member of the Transforming Growth Factor-β (TGF-β) superfamily of secreted signalling molecules. In this work, I have investigated its spatiotemporal distribution and possible function in the developing chick eye. The chick homologue of Bmp-4 is expressed throughout the developing chicken optic vesicle between Hamburger and Hamilton stages 13 and 17. It then shows a distinctive restriction to the dorsal quadrant of the eye until stage 30, when expression begins to fade, becoming undetectable by stage 35. Transcripts of the homeobox-containing gene GH6 show a similar dorsal restriction to Bmp-4, but between stages 20-35. Overexpression of Bmp-4 in vivo results in no detectable change in eye morphology, but enlarges the expression domain of GH6, indicating that the two genes may form part of a gene cascade involved in patterning the eye. In vitro, BMP-4 appears to stimulate retinal cell proliferation. It has no effect on retinal cell differentiation in vitro. In the chick, the patterning activities of sonic hedgehog (shh) are known to involve activation of Bmp expression in at least two instances; the development of the limb and midgut. I report here that shh is not expressed in the chick eye at all during development, but is found in the ventral portion of the diencephalon (forebrain). The available data suggests a number of roles for BMP-4 in chick eye development. BMP-4 may specify dorso-ventral retinal polarity required for proper retinal axon pathfinding, or establish a dorso-ventral gradient of cell proliferation in the developing optic cup. Alternatively, it may act as a hedgehog family member antagonist in establishing the retinal identity of the most dorsal portion of the eyecup.
Thesis
This thesis investigates the cellular and molecular basis of patterning of the antero-posterior axis of the chick limb by Sonic Hedgehog (Shh). Shh is expressed in the polarizing region, which is localised at the posterior margin of the limb bud. Shh expression is stable when the polarizing region is transplanted to the anterior margin of another chick limb bud and additional digits are specified, but Shh is not induced in host tissue. Application of Shh at the anterior of the chick limb bud also gives rise to digit duplications. High Shh concentrations result in shoulder girdle defects mediated by activation of Bmps. Shh acts in a dose dependent manner specifying digits in an anterior to posterior sequence, with anterior digits being specified first. DiI and DiA labelling shows that the fate of cells up to 300?m away from the Shh source can be influenced and cells that would not normally contribute to digit formation, can now give rise to an extra digit 2 or 3 according to the length of time that they have been exposed to Shh. Molecules such as Bmp-2, Fgf-4, Hoxd-13 and Hoxa-13 involved in antero-posterior patterning are induced by Shh at 16 hours after application. The tumor suppressor gene patched (ptc), the putative receptor for Shh is induced as early as 2-4 hours indicating a rather fast response. However, it is not until 24 hours after Shh application that irreversible digit pattern duplications are produced and this correlates with irreversible ectopic Bmp-2 expression. Application of Bmp-2, at 16 hours can not replace the effects of Shh, but can enhance significantly the polarizing activity, especially if combined with Fgf-4. In talpid3 chick mutant, ptc is not expressed at high levels even after exogenous application of Shh, suggesting that induction of Bmp-2 and Hoxd-13 is independent of ptc. A COS cell line expressing a membrane tethered Shh construct was prepared but these cells did not produce pattern changes when grafted to chick wing buds.
Article
The species-specific morphology of digits in the tetrapod limb, including the length and number of metacarpal, metatarsal, and phalangeal bones, suggests that a common developmental mechanism for digit formation is modified in a species-specific manner. Here, we examined the function of the AP-2β transcription factor in regulating digit length in the chicken autopod. Mutations in the gene encoding AP-2β are associated with Char syndrome, a human autosomal dominant disorder. Char syndrome patients exhibit autopod skeletal defects, including loss of phalanges and shortened fingers, suggestive of a function for AP-2β in normal digit development. The ectopic expression of two different dominant-negative forms of chick AP-2β, equivalent to mutant forms associated with human Char syndrome, in the developing chick hindlimb bud resulted in defective digit formation, including reductions in the number and length of phalanges and metatarsals. A detailed analysis of the AP-2β expression pattern in the limb bud indicated a correlation between the pattern/duration of AP-2β expression in the limb mesenchyme and digit length in three amniote species, the chicken, mouse and gecko. In addition, we found that AP-2β expression was downstream of Fgf signals from the apical ectodermal ridge, which is crucial in digit morphogenesis, and that excessive AP-2β function resulted in dysregulated digit length. Taken together, these results suggest that AP-2β functions as a novel transcriptional regulator for digit morphogenesis. Copyright © 2015. Published by Elsevier Inc.
Article
The vertebrate olfactory epithelium (OE) is a system in which behavior of neuronal progenitor cells can be observed and manipulated easily. It is morphologically and functionally similar to embryonic germinal neuroepithelia, but is simpler in that it produces large numbers of a single type of neuron, the olfactory receptor neuron (ORN). The OE is amenable to tissue culture, gene transfer, and in vivo surgical approaches, and these have been exploited in experiments aimed at understanding the characteristics of OE neuronal progenitor cells. This has led to the realization that the ORN lineage contains at least three distinct stages of proliferating neuronal progenitor cells (including a stem cell), each of which represents a point at which growth control can be exerted. Neurogenesis proceeds continually in the OE, and studies in vivo have shown that this is a regulated process that serves to maintain the number of ORNs at a particular level. These studies suggest that OE neuronal progenitors—which are in close physical proximity to ORNs—can “read” the number of differentiated neurons in their environment and regulate production of new neurons accordingly. Putative neuronal stem cells of the OE have been identified in vitro, and studies of these cells indicate that ORNs produce a signal that feeds back to inhibit neurogenesis. This inhibitory signal may be exerted at the level of the stem cell itself. Recent studies to identify this signal, as well as endogenous stimulatory signals that may be important in regulating OE neurogenesis, are also discussed. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 190–205, 1998
Article
In this article, an adjunct to a platform presentation at the Winternational 2000 Symposium, we summarize the recent findings of this group concerning the regulation and functions of FGF8 expressed at the isthmus of the developing brain. We show that several different FGF8 isoforms, ectopically expressed in midbrain or posterior forebrain, are able to mimic the proliferative and patterning functions previously attributed to the isthmus in tissue grafting studies. Moreover, we also show that FGF8 protein is sufficient to induce an ectopic isthmic organiser (Fgf-8+, Gbx2+) in anterior midbrain. We also provide evidence that isthmic FGF8 patterns anterior hindbrain, repressing Hox-a2 expression and setting aside a territory of the brain that includes the cerebellar anlage. We show that these effects of FGF8 are likely to be mediated via FGFR1 and be modulated by the putative FGF antagonist, Sprouty2, identified using a differential display screen. Finally, we provide evidence that the onset of Fgf8 expression is regulated by En1 and that its expression at the isthmus is subsequently maintained by a specific and direct interaction between rhombomere 1 and midbrain.
Chapter
Of vertebrate organ systems, the developing limb has been especially well characterized. Embryological studies combined with molecular manipulations have yielded a wealth of information about the control of pattern formation during limb outgrowth. A number of key signaling pathways have been implicated in the control of numerous aspects of limb development, including the establishment of the early limb field, determination of limb identity, elongation of the limb bud, specification of digit pattern, and sculpting of the digits. Although there is clear evidence that specific signaling pathways that operate in the limb field and early limb bud control the specification of pattern, little is known about how these signals interface with the cell biology of limb development (1). One instance where some progress has been made concerns the role of FGF4 signaling by the apical ectodermal ridge (AER) in the limb bud.
Chapter
The developing vertebrate limb is one of the most carefully studied model systems for examining how complex patterns of differentiated tissues form in secondary embryonic fields. The mature vertebrate limb contains a large number of distinct cell types that develop side by side in a highly coordinated and orchestrated manner, and it is the molecular underpinnings of this patterning mechanism that lie at the heart of our understanding of how genetic alterations and toxicological insults result in developmentally defective organs. The limb is an excellent experimental model system because it is a nonessential organ that can be easily manipulated. Because limb-specific defects do not generally result in embryolethality, a large number of mutations in man, mouse, and chicken that alter limb morphogenesis have been characterized, and in some cases studied in some detail. For similar reasons, the limb is also a highly visible target for toxicology studies. In addition, the developing limb bud has been used extensively as a source of cells to study issues concerning cellular differentiation, the control of cell proliferation, and the production of morphogenetic signals; thus the behavior of limb bud cells in culture is reasonably well characterized.
Thesis
Drosophila snail, a gene encoding a zinc-finger protein, is essential for the correct specification of ventral cell types, the future mesodermal cells, of the Drosophila embryo. This gene is also implicated in the maintenance of wing development later in Drosophila embryogenesis. This work characterises the expression pattern and attempts to elucidate the functions of a chick gene related to Drosophila snail (cSnR). The gene is first expressed briefly throughout the presumptive neurectoderm just before it forms the neural plate at the full length streak stage. At head fold stages this ectodermal expression is replaced by expression in the heart forming mesoderm. On the formation of two or three somites, cSnR expression becomes markedly asymmetrical in the lateral plate mesoderm; on the right hand side of the embryo only, it is expressed in regions containing material that will migrate into the heart. This asymmetry continues until approximately twelve somites have formed. Expression is also found in the lateral edges of the somites on their formation, followed shortly by expression throughout the whole ventral region of the somite. After regionalisation of the somite, transcripts are found in the myotome and sclerotome, but are absent from the dermatome. Expression of cSnR is also found in the gut endoderm, a subset of neural crest derivatives, and the limb mesenchyme from the time of limb bud initiation. I have used phosphorothioated antisense oligodeoxynucleotide treatment to disrupt cSnR function. Consistent with the asymmetrical nature of the expression pattern of this gene, treatment during the hours immediately preceding heart formation leads to randomisation of heart handedness and the associated embryo torsion. Laterally implanted ectopic sources of Hedgehog and activin proteins, known to cause heart situs randomisation, also result in altered expression of cSnR in the lateral plate mesoderm. The results provide evidence that cSnR also has a role in left-right asymmetry, reading and acting on left-right information rather than initiating the information itself. The antisense oligonucleotide interference also leads to abnormal segmentation. Somites are another region of cSnR expression, suggesting that cSnR also plays an important role in segmentation. Implanting ectopic sources of bFGF into the flank regions of chick embryos in vivo, a procedure known to lead to ectopic limb formation, leads to very early induction of cSnR, implicating cSnR at an early point in limb specification.
Article
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Although decades of studies have produced a generalized model for tetrapod limb development, urodeles deviate from anurans and amniotes in at least two key respects: their limbs exhibit preaxial skeletal differentiation and do not develop an apical ectodermal ridge (AER). Here, we investigated how Sonic hedgehog (Shh) and Fibroblast growth factor (Fgf) signaling regulate limb development in the axolotl. We found that Shh-expressing cells contributed to the most posterior digit, and that inhibiting Shh-signaling inhibited Fgf8 expression, anteroposterior patterning, and distal cell proliferation. In addition to lack of a morphological AER, we found that salamander limbs also lack a molecular AER. We found that amniote and anuran AER-specific Fgfs and their cognate receptors were expressed entirely in the mesenchyme. Broad inhibition of Fgf-signaling demonstrated that this pathway regulates cell proliferation across all three limb axes, in contrast to anurans and amniotes where Fgf-signaling regulates cell survival and proximodistal patterning.
Article
Fibroblast growth factor (FGF) family members are important regulators of cell growth, proliferation, differentiation, and regeneration. The abnormal expression of certain FGF family members can cause skeletal diseases, including achondroplasia, craniosynostosis syndrome, osteoarthritis and Kashin‐Beck disease. Accumulating evidence shows that FGFs play a crucial role in the growth and proliferation of bone and in the pathogenesis of certain bone‐related diseases. Here, we review the involvement of FGFs in bone‐related processes and diseases; FGF1 in the differentiation of human bone marrow mesenchymal stem cells and fracture repair; FGF2, FGF9 and FGF18 in osteoarthritis; FGF6 in bone and muscle injury; FGF8 in osteoarthritis and Kashin‐Beck disease; and FGF21 and FGF23 on bone regulation. These findings indicate that FGFs are targets for novel therapeutic interventions for bone‐related diseases. This article is protected by copyright. All rights reserved. Here, we review the biological relevance of FGF1, FGF2, FGF9,FGF18,FGF6,FGF8,FGF21,FGF23 in bone‐related diseases.These findings potentiate the targeting of FGFs family members for novel therapeutic interventions in bone‐releated diseases and may also hold valuable clinical poterntial to improve the care of patients with bone‐related diseases.
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The elongating mouse anteroposterior axis is supplied by progenitors with distinct tissue fates. It is not known whether these progenitors confer anteroposterior pattern to the embryo. We have analysed the progenitor population transcriptomes in the mouse primitive streak and tail bud throughout axial elongation. Transcriptomic signatures distinguish three known progenitor types (neuromesodermal, lateral/paraxial mesoderm and notochord progenitors; NMPs, LPMPs and NotoPs). Both NMP and LPMP transcriptomes change extensively over time. In particular, NMPs upregulate Wnt, Fgf and Notch signalling components, and many Hox genes as progenitors transit from production of the trunk to the tail and expand in number. In contrast, the transcriptome of NotoPs is stable throughout axial elongation and they are required for normal axis elongation. These results suggest that NotoPs act as a progenitor niche whereas anteroposterior patterning originates within NMPs and LPMPs.
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In many respects, reptile hatchlings are fully functional, albeit miniature, adults. This means that the adult morphology must emerge during embryonic development. This insight emphasizes the connection between the mechanisms that generate phenotypic variation during embryonic development and the action of selection on post-hatching individuals. To determine when species-specific differences in limb and tail lengths emerge during embryonic development, we compared allometric patterns of early limb growth of four distantly related species of lizards. The major questions addressed were whether early embryonic limb and tail growth is characterized by the gradual (continuous allometry) or by the abrupt emergence (transpositional allometry) of size differences among species. Our observations supported transpositional allometry of both limbs and tails. Species-specific differences in limb and tail length were exhibited when limb and tail buds first protruded from the body wall. Genes known to be associated with early limb development of tetrapods are obvious targets for studies on the genetic mechanisms that determine interspecific differences in relative limb length. Broadly comparative studies of gene regulation would facilitate understanding of the mechanisms underlying adaptive variation in limb size, including limb reduction and loss, of squamate reptiles.
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Ribosomal RNA (rRNA) transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies; however, the cellular and molecular reasons for these defects are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. ncl mutant (ncl−/−) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observed that ncl−/− mutants exhibited decreased rRNA synthesis and p53-dependent apoptosis, consistent with a role in ribosome biogenesis. However, we found that Nucleolin also performs functions not associated with ribosome biogenesis. We discovered that the half-life of fgf8a mRNA was reduced in ncl−/− mutants, which perturbed Fgf signaling, resulting in misregulated Sox9a-mediated chondrogenesis and Runx2-mediated osteogenesis. Consistent with this model, exogenous FGF8 treatment significantly rescued the cranioskeletal phenotype in ncl−/− zebrafish, suggesting that Nucleolin regulates osteochondroprogenitor differentiation. Our work has therefore uncovered tissue-specific functions for Nucleolin in rRNA transcription and post-transcriptional regulation of growth factor signaling during embryonic craniofacial development.
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A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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An androgen-dependent mouse mammary carcinoma cell line (SC-3) requires androgen for growth stimulation. We have shown previously that androgen acts on SC-3 cells to induce secretion of a fibroblast growth factor (FGF)-like growth factor, which in turn stimulates growth of the cells in an autocrine manner. In this study, the androgen-induced growth factor (AIGF) was purified from a conditioned medium of SC-3 cells stimulated with testosterone. cDNA cloning of AIGF by use of its partial amino acid sequence data revealed that AIGF is a distinctive FGF-like growth factor. An AIGF cDNA (pSC17) encodes a 215-amino acid protein with a putative signal peptide, which shares 30-40% homology with known members of the FGF family. The AIGF mRNA was markedly induced by 10 nM testosterone in Northern blot analysis. Expression of AIGF cDNA in mammalian cells clearly showed remarkable stimulatory effects of AIGF on growth of SC-3 cells in the absence of androgen. Thus, it is clear that the androgen-induced growth of SC-3 cells is mediated in an autocrine manner by AIGF, which is secreted by the tumor cells themselves in response to hormonal stimuli.
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Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.
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The developing mouse fore- and hindlimbs begin as bumps on the flank of the embryo and grow out to form miniature models of the adult limb during a five day period from E9.5 to E14.5. In this paper I show a series of embryos taken at half-day intervals during limb development and outline the timetable of patterning for each of the component tissues of a limb: epidermis, connective tissues, muscle, nerves and blood vessels. Scanning electron micrographs, supplemented by histological sections, are presented to define a set of standard stages for the description of mouse limb development. I discuss my observations of the mouse limb in the light of current theories of vertebrate limb development, which are based on classic manipulation experiments in the chick as well as more recent molecular data from the mouse system. The limb skeletal pattern in a mouse is laid down in a proximodistal direction, as it is in a bird: the E11.5 forelimb reveals the first signs of a humerus and by E14.5 even the most distal phalanges of the hand are formed. At this late stage ossification sleeves are seen around the proximal limb elements as the cartilage template begins to be converted to a bony skeleton. Myogenic cells stainable with the MF20 antibody against early muscle myosin heavy chain are first seen in the mouse forelimb at E11.5, which is also when the first nerve fascicles begin to enter the limb. From E11.5 to E14.5 both muscle and nerve patterns mature to give distinct muscles at all proximodistal levels of the limb, each muscle with its own nerve branch, and a cutaneous nerve plexus that extends to the fingertips. The developing skin of the mouse limb matures from a bi-layered epidermis overlying an avascular, but otherwise nondescript, prospective dermal layer of mesenchyme at E9.5, to a 4- or 5-layered epidermis with early hair placodes and the first signs of a distinct dermal layer at E14.5. Notable differences between mouse and chick limb development lie in the relatively late formation of the apical ectodermal ridge in the mouse and its unexpectedly close relations with blood vessels, in the absence of anterior and posterior necrotic zones and, possibly, in a late migration of myogenic cells into the mouse limb bud.
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During gastrulation in the mouse, the pluripotent embryonic ectoderm cells form the three primary germ layers, ectoderm, mesoderm and endoderm. Little is known about the mechanisms responsible for these processes, but evidence from previous studies in amphibians, as well as expression studies in mammals, suggest that signalling molecules of the Fibroblast Growth Factor (FGF) family may play a role in gastrulation. To determine whether this might be the case for FGF-5 in the mouse embryo, we carried out RNA in situ hybridization studies to determine when and where in the early postimplantation embryo the Fgf-5 gene is expressed. We chose to study this particular member of the FGF gene family because we had previously observed that its pattern of expression in cultures of teratocarcinoma cell aggregates is consistent with the proposal that Fgf-5 plays a role in gastrulation in vivo. The results reported here show that Fgf-5 expression increases dramatically in the pluripotent embryonic ectoderm just prior to gastrulation, is restricted to the cells forming the three primary germ layers during gastrulation, and is not detectable in any cells in the embryo once formation of the primary germ layers is virtually complete. Based on this provocative expression pattern and in light of what is known about the functions in vitro of other members of the FGF family, we hypothesize that in the mouse embryo Fgf-5 functions in an autocrine manner to stimulate the mobility of the cells that contribute to the embryonic germ layers or to render them competent to respond to other inductive or positional signals.
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Fibroblast growth factors (FGFs) are structurally related mitogens that can regulate the differentiation of a wide variety of cells. As a step towards elucidating the developmental roles played by one of these factors, we have used in situ hybridization methods to examine expression of the murine F gf-5 gene during embryogenesis. F gf-5 RNA was detected at seven distinct sites in the developing mouse embryo: (1) postimplantation epiblast (embryonic day 5 1/4-7 1/2), (2) lateral splanchnic mesoderm (E9 1/2-10 1/2), (3) lateral somatic mesoderm (E10 1/2-12 1/2), (4) myotomes (E10 1/2-12 1/2), (5) mastication muscle (E11 1/2-14 1/2), (6) limb mesenchyme (E12 1/2-14 1/2), and (7) acoustic ganglion (E12 1/2-14 1/2). At several of these sites, expression is spatially restricted within the tissues. We offer several hypotheses regarding the roles of FGF-5 in murine development.
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A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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FGF-3 has been implicated in the development of the hindbrain and otocyst in vertebrate embryos. Since the chicken embryo offers a favourable system in which to study the development of these structures, we have isolated and characterised cDNAs for chicken Fgf-3 and determined its pattern of expression in chick embryos from stage 3 (primitive streak) to stage 25 (early organogenesis). Within the developing cranial neural tube, Fgf-3 exhibits dynamic spatial and temporal expression. During extension of the head process, RNA is detected in the midline of the developing neural plate. In neurulating embryos, transcripts are observed initially in rhombomeres 4 and 5 of the hindbrain and later, in rhombomere 6. During hindbrain development, expression is lost from these rhombomeres, but becomes restricted to rhombomere boundaries, providing an intracellular marker which distinguishes a population of cells within boundary regions. Fgf-3 expression is elevated in ventral and medial boundary regions and is greatly reduced in dorsal parts. Studies of regenerating rhombomere boundaries show that Fgf-3 expression is induced in reforming boundaries when even-numbered rhombomere tissue is grafted next to odd, but not when like is juxtaposed to like. Fgf-3 disappears from boundary regions just prior to the loss of the morphological boundaries suggesting a boundary-associated function. Other sites of expression have also been identified. At early stages of development Fgf-3 is expressed in the epiblast and mesendoderm of the primitive streak, in mesoderm lateral to the streak and in Hensen's node. In older embryos transcripts are detected in the endoderm of the pharyngeal pouches, the ectoderm of the second and third pharyngeal arches and the stomodeum. Expression was also detected in the segmental plate and in the posterior half of the three most-recently generated somites.
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We demonstrate that purified fibroblast growth factor (FGF) 3 from Xenopus laevis (XFGF3) activates the mitogen-activated protein kinase pathway and induces DNA synthesis in quiescent cells. To characterize the high affinity cell surface receptors that mediate these responses, the ligand binding domains of different FGF receptors (FGFR) were expressed on COS-1 cells, and their affinity for XFGF3 was determined. Unlabeled XFGF3 efficiently competed with I-FGF1 for binding to the IIIb and IIIc isoforms of FGFR2, giving 50% displacement (ID) at 0.3-0.8 nM. Higher XFGF3 concentrations were needed to displace I-FGF1 from FGFR3 and FGFR1 (ID 4 and 21 nM, respectively), indicating that XFGF3 has a lower affinity for these receptors. No association of XFGF3 with FGFR4 was found using this assay. FGFR2 isoforms isolated from both mouse and Xenopus showed similar high affinity binding of XFGF3 as determined by direct binding assays (K values in the range of 0.2-0.6 nM). These results indicate that the binding specificity of XFGF3 is different from that of other FGFs, and identifies FGFR2 as its high affinity receptor.
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We show by immunohistology that distinct expression patterns of the four muscle regulatory factor (MRF) proteins identify subdomains of mouse somites. Myf-5 and MyoD are, at specific stages, each expressed in both myotome and dermatome cells. Myf-5 expression is initially restricted to dorsal cells in all somites, as is MyoD expression in neck somites. In trunk somites, however, MyoD is initially expressed in ventral cells. Myogenin and MRF4 are restricted to myotome cells, though the MRF4-expressing cells are initially less widely distributed than the myogenin-expressing cells, which are at all stages found throughout the myotome. All somitic myocytes express one or more MRFs. The transiently distinct expression patterns of the four MRF proteins identify dorsal and ventral subdomains of somites, and suggest that skeletal muscle cells in somites originate at multiple sites and via multiple molecular pathways.
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The zone of polarizing activity (ZPA) is a region at the posterior margin of the limb bud that induces mirror-image duplications when grafted to the anterior of a second limb. We have isolated a vertebrate gene, Sonic hedgehog, related to the Drosophila segment polarity gene hedgehog, which is expressed specifically in the ZPA and in other regions of the embryo, that is capable of polarizing limbs in grafting experiments. Retinoic acid, which can convert anterior limb bud tissue into tissue with polarizing activity, concomitantly induces Sonic hedgehog expression in the anterior limb bud. Implanting cells that express Sonic hedgehog into anterior limb buds is sufficient to cause ZPA-like limb duplications. Like the ZPA, Sonic hedgehog expression leads to the activation of Hox genes. Sonic hedgehog thus appears to function as the signal for anteroposterior patterning in the limb.
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Recent results make it possible to postulate credible candidates for each of the known inducing signals that act to determine cell fate during Xenopus early development. Experiments on biological activity, expression patterns and inhibition of function suggest that Vg-1 and Wnt-11 may act as the primary mesoderm-inducing signals,FGF and activin may serve to relay their effects, and noggin may be a major component of the dorsalizing and neural-inducing signals from the organizer.
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Reprinted from THE JOURNAL OF EXPERIMENTAL ZOOLOGY, Vol. 108, No. 3, August, 1948.
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Our culture system appears to represent an in vitro analogue of early chick limb morphogenesis. Organized mesodermal cell accumulations resembling limb buds were derived from a monolayer of limb mesoderm cells when covered by limb ectoderm which included the apical ectodermal ridge (AER). The ridge retained its normal configuration when grown over a limb mesoderm monolayer and the mesoderm cells accumulated under the ridge to form a multilayered structure (10-25 cells in thickness) with the characteristic shape of a limb bud. Ectoderm which did not include the ridge failed to promote the formation of limb-like mesodermal accumulations thus the action of the ridge appears to be specific. The AER-elicited expression of mesodermal cell behaviour leading to early limb outgrowth is discussed in terms of possible morphogenetic mechanisms involved i.e. differential mitosis, cell migration, changes in cell shape and especially the adhesive properties of the cells.
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FORTY-FIVE FIGURES The preparation of a series of normal stages of the chick embryo does not need justification at a time when chick ernbryos are not only widely used in descriptive and experimental embryology but are proving to be increasingly valuable in medical research, as in work on viruses and cancer. The present series was planned in connection with the preparation of a new edition of Lillie’s DeueZopmerzt of the Chick by the junior author. It is being published separately to make it accessible immediately to a large group of workers. Ever since Aristotle “discovered” the chick embryo as the ideal, object for embryological studies, the embryos have been described in terms of the length of time of incubation, and this arbitrary method is still in general use, except for the first three days of incubation during which more detailed characteristics such as the numbers of somites are applied. The shortcomings of a classification based on chronological age are obvious to every worker in this field, for enormous variations may occur in embryos even though all eggs in a setting are plmaced in the incubator at the same time. Many factors are responsible for the lack of correlation between chronological and structural age. Among these are : genetic differences in the rate of development of different breccls (eg., the embryo of the White Leghorn breed develops more 49
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Fibroblast growth factors (FGFs) can influence the growth and differentiation of cultured cells derived from neuroectoderm, ectoderm or mesenchyme. The FGFs interact with a family of at least four closely related receptor tyrosine kinases that are products of individual genes. To investigate the role of FGFs in the growth and differentiation of embryonic tissues and to determine whether the individual FGF receptor genes might have specific functions, we compared the localization of mRNA for two FGF receptor genes, FGFR1 (the flg gene product) and FGFR2 (the bek gene product), during limb formation and organogenesis in mouse embryos (E9.5-E16.5). Although the two genes were coexpressed in some tissues, the differential expression of FGFR1 and FGFR2 in most embryonic tissues was striking. FGFR1 was expressed diffusely in mesenchyme of limb buds, somites and organ rudiments. In contrast, FGFR2 was expressed predominantly in the epithelial cells of embryonic skin and of developing organs. The differential expression of FGFR1 and FGFR2 in mesenchyme and epithelium respectively, suggests the receptor genes are independently regulated and that they mediate different functions of FGFs during development.
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The mouse Fgf2 and Fgf5 genes, two members of the FGF family whose human homologs are on the long arm of chromosome (chr) 4, were localized by using in situ chromosomal hybridization, to mouse Chrs 3 and 5, respectively.
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Fgf-4, initially isolated as a transforming gene from human tumors, is a member of the Fibroblast Growth Factor (FGF) family. It has previously been shown by northern blot hybridization analysis to be expressed in teratocarcinoma and embryonic stem cells, suggesting that it plays a role in embryonic development. We have carried out an RNA in situ hybridization analysis of Fgf-4 expression in the developing mouse embryo, from fertilization through the 14th day of gestation (E14.5). Our results show that Fgf-4 RNA is first detected at the late blastocyst stage in cells that give rise to all of the embryonic lineages (inner cell mass cells). During the early stages of gastrulation, expression becomes restricted to the primitive streak where mesoderm and definitive endoderm are formed. Expression continues in the distal (rostral) two-thirds of the streak through approx. E10, and then is detected in the tail bud, which replaces the streak as the primary source of mesoderm. Additional sites of expression are found after the three primary germ layers are established and organogenesis begins. Fgf-4 RNA is detected transiently in the branchial arch units, the somitic myotome, the apical ectodermal ridge of the developing limb bud and the tooth bud, suggesting that the gene has multiple roles during embryogenesis. These results are compared with the expression patterns of other FGF genes. Taken together, the data suggest that individual members of the gene family are expressed sequentially in developmental pathways such as mesoderm formation and myogenesis, and play a role in specific epithelial-mesenchymal interactions.
Article
Developmental expression of two closely related fibroblast growth factor receptors, bek and flg, is described from early postimplantation until advanced organogenesis. Transcripts of bek and flg were first seen in the primitive ectoderm of egg-cylinder-stage embryos. Later, starting with somitogenesis, and then throughout embryogenesis, they were actively transcribed both in the mesoderm and neuroectoderm. Bek was expressed also in the surface ectoderm and in various epithelia, whereas flg expression was restricted mainly to the mesenchyme. In the limb bud bek transcripts displayed a gradient-like distribution and appeared earlier than flg. The two receptors, in contrast to their almost identical ligand binding specificity, displayed distinct spatial specificities throughout development, suggesting that developmental localization may contribute to functional specificity. The role of bek and flg in gastrulation and in epithelial-mesenchymal interactions of organogenesis will be discussed.
Article
We have used the polymerase chain reaction to clone from fetal cerebellar RNA a novel member of the fibroblast growth factor receptor family, FGFR-4. cDNAs encoding a full-length receptor were isolated and RNA expression examined in adult and fetal tissues by RNA blot analysis. Transcripts were detected in adult lung, liver and kidney and in fetal RNAs from 11.5 to 16.5 days post coitum (p.c.). In situ hybridization was performed to examine developmental expression. FGFR-4 RNA was expressed in definitive endoderm of the developing gut and extraembryonic endoderm of the yolk-sac from 8.5 to 14.5 days p.c. At early somite stages, FGFR-4 was also expressed in the myotomal component of the somite, and by 14.5 days p.c. in the myotomally derived skeletal muscle. No expression was seen at any stage in cardiac muscle. Several endodermal derivatives, the liver, lung and pancreas, expressed FGFR-4 at 14.5 days p.c. In addition, FGFR-4 RNA was detected in the adrenal cortex, collecting tubules of the kidney and condensing cartilage at this time. These results suggest that FGFR-4 is likely to have diverse roles in development, which may include regulation of definitive endoderm and skeletal muscle lineages.
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Mouse embryonal carcinoma cell lines that differ in their patterns of expression of the potential oncogene int-2 have been exploited in a structural analysis of the multiple RNA transcripts characteristic of this gene. Ribonuclease protection experiments indicate that four major classes of int-2 RNA initiate at heterogeneous cap sites within two distinct promoter regions, P1 and P2, spanning approximately 50 and 150 bp respectively. The more downstream promoter P2 is located in a region of the DNA that constitutes an intron in RNA transcripts that initiate at the upstream promoter P1. Otherwise, all four RNA structures share the same splice donor and acceptor sites that define the boundaries of the second and third exons. Further complexity arises through usage of two distinct polyadenylation signals, both variants of the normal consensus, that are separated by 1100 bp. Despite these structural variations, the results suggest that all four major classes of RNA encode the same protein product which shows significant homology to the family of heparin-binding proteins typified by basic fibroblast growth factor (FGF).
Article
Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.
Article
The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.
Article
Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.
Article
The effect of removal of the apical ectodermal ridge from the early chick limb bud is reexamined using a new quantitative method of analysis of results. The concept of the proximodistal sequence of laying down of parts is confirmed and evidence is presented that this proceeds as a continuous process, there being a gradual change in the level specified from one cell to another at a more distal level. The results are then interpreted in terms of the 'progress zone' model to show that they are both consisted with the model and that they provide an assay for one of its parameters, the rate of change of positional value with time at the tip.
Article
Pattern formation by the cells of a growing organism depends on coordination of changes in space and time. Evidence is presented here for the control of morphogenesis by positional information specified by an autonomous timing mechanism that operates in a ``progress zone'' at the tip of the limb bud.
Article
While the apical ectodermal ridge (AER) is well known for its required role in the development of distal parts of the limb and for its ability to stimulate limb duplications, the mechanism of its action is unknown. In this study we use a culture system previously developed by M. Globus and S. Vethamany-Globus (1976, Differentiation6, 91–96) in which an AER grafted onto a high-density cell culture of limb mesenchyme stimulates the formation of an outgrowth. Time-lapse movies taken during the outgrowth period demonstrated no cellular activities other than cell division. Both the mitotic index and labeling index in the mesenchyme were significantly elevated under the AER as compared to that without AER, indicating that the AER provides a growth-promoting stimulus which increases the proportion of dividing cells. On the other hand, nonridge ectoderm had no detectable effect on the mitotic index. Treatment of cultures with cytosine arabinoside both inhibited DNA synthesis and prevented AER-induced outgrowth. These results demonstrate a mitogenic capacity of AER tissue and suggest that mesenchymal outgrowth requires this activity. The mitogenic property of the AER is considered in relation to limb outgrowth in situ.
Article
Fibroblast growth factors are believed to play many distinct roles in vertebrate development, owing to their ability to stimulate cell growth, prevent cell death, determine cell fate, and inhibit terminal differentiation in a variety of in vitro culture systems. We have used in situ hybridization to localize fibroblast growth factor-4 (FGF-4, also termed HST and K-FGF) gene expression in 7.5 to 16.5 day gestation mouse embryos. Seven discrete sites of gene expression were detected: (1) primitive streak (E7.5-8.5); (2) paraxial presomitic mesoderm in the trunk (E7.5-11.5); (3) primitive neuroectoderm (E8.0-8.5); (4) pharyngeal pouch endoderm (E8.5-9.5); (5) branchial arch ectoderm (E8.5-9.5); (6) limb apical ectoderm (E10.5-12.5), and (7) skeletal myoblast groups (E9.5-13.5). FGF-4 gene expression is spatially restricted within many of these sites. The profile of FGF-4 gene expression among skeletal muscle groups is overlapping, but distinct, from that of FGF-5, thereby revealing myoblast heterogeneity at the molecular level and suggesting distinct roles for multiple FGFs in muscle development.
Article
One of the earliest indications of regional patterning in the CNS is the spatially restricted expression of regulatory genes within the neuroepithelium. Many of these genes encode transcription factors and, although little is known of their downstream targets, it seems likely that they control the identity of cells in different regions of the CNS. This review discusses how the expression of these patterning genes might influence the location at which the first axon pathways in the CNS are pioneered. Evidence is described that suggests that the boundary regions between adjacent domains of regulatory gene expression influence where the first axons will extend.
Article
use of the FGff probe pKGF-SX, and 49 of these (26.7%) were located on Chr 2. The distribution of the grains on this chromosome was not random: 75.5% mapped in the F-G region of Chr 2, allowing us to map the Fgf7 gene to the 2F-2G region of the murine genome. With the Fgf8 probe, there were 172 silver grains associated with chromosomes, and 40 of these (23.2%) were located on Chr. 19. The distribution was not random'. 36 out 40 grains (90%) mapped to the C3-D region of Chr 19 with a maximum in the D band. This result allowed us to map the Fgf8 gene to the chromosomal region 19C3-19D. Kelley and associates (1992) have reported that FGF7 is located on human Chr 15. In addition, related copies of part of the FGF7 gene were found dispersed to multiple chromosomes in human and great apes, but not in mice (Kelley et ai. 1992). Our results are in accordance with these findings. First, mouse Chr 2 presents a conserved region of synteny with region q13-q22 of human Chr 15. Our result, therefore, suggests that human FGF7 maps to 15q13-22. Second, no other significant clusters of silver grains were found in the other chromosomal regions of the mouse genome. Third, hybridization of our murine Fgf7 probe to human metaphase chromosomes showed signals on Chr 15 (not shown). The localization of human FGF8 has not yet been reported. On the basis of the existence of conserved regions of synteny between mouse Chr 19 and human chromosomes (Copeland et ai. 1993), it could be predicted that FGF8 maps to the long man of human Chr 10.
Article
Evidence is accumulating that members of the FGF gene family provide signals that act locally to regulate growth and patterning in vertebrate embryos. In this report, we provide a detailed analysis of the mouse Fgf8 gene. We have mapped the Fgf8 locus to the distal region of mouse chromosome 19, and sequenced the 5' coding region of the gene. Our data identify a new coding exon, and locate multiple splice donor and splice acceptor sites that can be used to produce at least seven transcripts encoding a family of secreted FGF8 proteins with different N termini. From these results, it appears that Fgf8 is structurally the most complex member of the FGF family described to date. In the embryo, many of the regions in which Fgf8 RNA is localized are known to direct outgrowth and patterning, including the apical ectodermal ridge of the limb bud, the primitive streak and tail bud, the surface ectoderm overlying the facial primorida and the midbrain-hindbrain junction, suggesting that FGF8 may be a component of the regulatory signals that emanate from these regions.
Article
Fgf-8 is a member of the fibroblast growth factor (FGF) family that was initially identified as an androgen-inducible growth factor in a mammary carcinoma cell line. Alternative splicing of the primary Fgf-8 transcript results in three messenger RNAs which code for secreted FGF-8 protein isoforms that differ only in their mature amino termini. Fgf-8 RNA is present from day 10 through 12 of murine gestation when analyzed by northern blot analysis, suggesting that Fgf-8 normally functions during post-gastrulation development. To characterize the temporal, spatial and isoform-specific aspects of Fgf-8 expression during mouse development, we performed in situ hybridization and ribonuclease protection assays between the days 8 and 16 of gestation. Fgf-8 expression is first detected at day 9 of gestation in the surface ectoderm of the first branchial arches, the frontonasal process, the forebrain and the midbrain-hindbrain junction. At days 10-12 of gestation, Fgf-8 expression is detected in the surface ectoderm of the forelimb and hindlimb buds, in the nasal pits and nasopharynx, in the infundibulum and in the telencephalon, diencephalon and metencephalon. Fgf-8 expression continues in the developing hindlimbs through day 13 of gestation but is undetectable thereafter. Ribonuclease protection assays reveal that RNAs coding for all three FGF-8 isoforms are present at days 10-12 of gestation. These results reveal a unique temporal and spatial pattern of Fgf-8 expression in the developing mouse and suggest a role for this FGF in multiple regions of ectodermal differentiation in the post-gastrulation mouse embryo.
Article
In the vertebrate embryo, only somatopleural cells in the limb-forming region are released from the mesodermal layer and undergo outgrowth from the embryonic body to form the limb bud. Molecular signals which regulate limb bud induction are unknown to date. In the present study we examined the ability of fibroblast growth factor (FGF) to induce limb bud formation in chicken embryos. A replication-defective retrovirus encoding FGF type 4 with a reporter, bacterial beta-galactosidase, was microinjected into lateral plate mesoderm inside and outside limb-forming regions. Effects of the ectopic and precocious expression of FGF were assessed at various stages after infection. Here we report that somatic mesodermal cells in both flanks and limb-forming regions can respond to FGF and induce limb bud-like outgrowth. The supernumerary limb bud induced within a limb-forming region differentiated into extralimb structures. These results strongly suggest potential roles of FGF signaling for induction of limb bud formation.
Article
Fibroblast growth factors (FGFs) act as signals in the developing limb and can maintain proliferation of limb bud mesenchyme cells. Remarkably, beads soaked in FGF-1, FGF-2, or FGF-4 and placed in the presumptive flank of chick embryos induce formation of ectopic limb buds, which can develop into complete limbs. The entire flank can produce additional limbs, but generally wings are formed anteriorly and legs posteriorly. FGF application activates Sonic hedgehog in cells with polarizing potential to make a discrete polarizing region. Hoxd-13 is also expressed in the ectopic bud, and an apical ectodermal ridge forms. A limb bud is thus established that can generate the appropriate signals to develop into a complete limb. The additional limbs have reversed polarity. This can be explained by the distribution of cells in the flank with potential polarizing activity. The results suggest that local production of an FGF may initiate limb development.
Article
The apical ectodermal ridge permits growth and elongation of amniote limb buds; removal causes rapid changes in mesodermal gene expression, patterned cell death, and truncation of the limb. Ectopic fibroblast growth factor (FGF)-2 supplied to the chick apical bud mesoderm after ridge removal will sustain normal gene expression and cell viability, and allow relatively normal limb development. A bioassay for FGFs demonstrated that FGF-2 was the only detectable FGF in chick limb bud extracts. By distribution and bioactivity, FGF-2 is the prime candidate for the chick limb bud apical ridge growth signal.
Article
We have identified three members of a mouse gene family related to the Drosophila segment polarity gene, hedgehog (hh). Like hh, they encode putative secreted proteins and are thus implicated in cell-cell interactions. One of these, Sonic hh (Shh), is expressed in the notochord, the floor plate, and the zone of polarizing activity, signaling centers that are thought to mediate central nervous system (CNS) and limb polarity. Ectopic expression of Shh in the mouse CNS leads to the activation of floor plate-expressed genes. These results suggest that Shh may play a role in the normal inductive interactions that pattern the ventral CNS.
Article
Fibroblast growth factor 5 (FGF5) is a secreted signaling protein. Mice homozygous for a predicted null allele of the Fgf5 gene, fgf5neo, produced by gene targeting in embryonic stem cells, have abnormally long hair. This phenotype appears identical to that of mice homozygous for the spontaneous mutation angora (go). The fgf5neo and go mutations fail to complement one another, and exon 1 of Fgf5 is deleted in DNA from go homozygotes, demonstrating that go is a mutant allele of Fgf5. Expression of Fgf5 is detected in hair follicles from wild-type mice and is localized to the outer root sheath during the anagen VI phase of the hair growth cycle. These findings provide evidence that FGF5 functions as an inhibitor of hair elongation, thus identifying a molecule whose normal function is apparently to regulate one step in the progression of the follicle through the hair growth cycle.
Article
We show by immunohistology that distinct expression patterns of the four muscle regulatory factor (MRF) proteins identify subdomains of mouse somites. Myf-5 and MyoD are, at specific stages, each expressed in both myotome and dermatome cells. Myf-5 expression is initially restricted to dorsal cells in all somites, as is MyoD expression in neck somites. In trunk somites, however, MyoD is initially expressed in ventral cells. Myogenin and MRF4 are restricted to myotome cells, though the MRF4-expressing cells are initially less widely distributed than the myogenin-expressing cells, which are at all stages found throughout the myotome. All somitic myocytes express one or more MRFs. The transiently distinct expression patterns of the four MRF proteins identify dorsal and ventral subdomains of somites, and suggest that skeletal muscle cells in somites originate at multiple sites and via multiple molecular pathways.
Article
Limb development depends on signals from the apical ectodermal ridge and underlying mesenchyme. Fibroblast growth factor (FGF) can replace the ridge and, because Fgf4 RNA is localized to the mouse posterior ridge, we proposed that FGF4 is the endogenous ridge signal. Ridge signals control limb outgrowth and maintain the zone of polarizing activity (ZPA) at the limb posterior margin, which is important in limb pattering: a ZPA graft to limb anterior mesenchyme causes cell respecification and mirror-image duplications. Sonic hedgehog (SHH) has polarizing activity, and Shh RNA co-localizes with ZPA activity, suggesting SHH is the endogenous polarizing signal. We have investigated the molecular regulation of Fgf4 and Shh expression. We report here that Fgf4 expression in the ridge can be regulated by Shh-expressing cells. Moreover, Shh expression in mesenchyme can be activated by FGF4 in combination with retinoic acid