Mandibular arch muscle identity is regulated by a conserved molecular process during vertebrate development

Vertebrate head muscles exhibit a highly conserved pattern of innervation and skeletal connectivity and yet it is unclear whether the molecular basis of their development is likewise conserved. Using the highly conserved expression of Engrailed 2 (En2) as a marker of identity in the dorsal mandibular muscles of zebrafish, we have investigated the molecular signals and tissues required for patterning these muscles. We show that muscle En2 expression is not dependent on signals from the adjacent neural tube, pharyngeal endoderm or axial mesoderm and that early identity of head muscles does not require bone morphogenetic pathway, Notch or Hedgehog (Hh) signalling. However, constrictor dorsalis En2 expression is completely lost after a loss of fibroblast growth factor (Fgf) signalling and we show that is true throughout head muscle development. These results suggest that head muscle identity is dependent on Fgf signalling. Data from experiments performed in chick suggest a similar regulation of En2 genes by Fgf signalling revealing a conserved mechanism for specifying head muscle identity. We present evidence that another key gene important in the development of mouse head muscles, Tbx1, is also critical for specification of mandibular arch muscle identity and that this is independent of Fgf signalling. These data imply that dorsal mandibular arch muscle identity in fish, chick and mouse is specified by a highly conserved molecular process despite differing functions of these muscles in different lineages.     

RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.     

A Requirement for FGF Signalling in the Formation of Primitive Streak-Like Intermediates from Primitive Ectoderm in Culture

Background

Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.

Methodology/Principal Findings

Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.

Conclusions/Significance

FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.     

FGF8/17/18 functions together with FGF9/16/20 during formation of the notochord in Ciona embryos

Fibroblast growth factor (FGF) signalling has been implicated in the generation of mesoderm and neural fates in chordate embryos including ascidians and vertebrates. In Ciona, FGF9/16/20 has been implicated in both of these processes. However, in FGF9/16/20 knockdown embryos, notochord fate recovers during later development. It is thus not clear if FGF signalling is an essential requirement for notochord specification in Ciona embryos. We show that FGF-MEK-ERK signals act during two distinct phases to establish notochord fate. During the first phase, FGF signalling is required during an asymmetric cell division to promote notochord at the expense of neural identity. Consistently, ERK1/2 is specifically activated in the notochord precursors following this cell division. Sustained activation of ERK1/2 is then required to maintain notochord fate. We demonstrate that FGF9/16/20 acts solely during the initial induction step and that, subsequently, FGF8/17/18 together with FGF9/16/20 is involved in the following maintenance step. These results together with others' show that the formation of a large part of the mesoderm cell types in ascidian larvae is dependent on signalling events involving FGF ligands.     

Specification and maintenance of the spinal cord stem zone

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes. Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.     

Regulated expression of the retinoblastoma gene product by fibroblast growth factor but not by activin during mesoderm induction in Xenopus

 The retinoblastoma (RB) gene is a tumor suppressor gene that plays an important role in cell cycle arrest and in the terminal differentiation of skeletal myoblasts. Differentiation into muscle occurs in Xenopus embryo explants during mesoderm induction by fibroblast growth factor (FGF) or activin A. We examined expression of the RB gene product (pRB) during mesoderm induction in vivo and in vitro. We show that hypo- and hyper-phosphorylated forms of pRB are present during early development and that expression of both forms increases significantly during the blastula stage, concomitant with mesoderm induction. Further investigation revealed that pRB is enriched in the presumptive mesoderm of the blastula stage embryo. In animal cap explants induced by Xenopus bFGF (XbFGF), pRB expression levels increased approximately tenfold while no increase was observed in explants induced by activin. However, when explants were induced by XbFGF in the presence of sodium orthovanadate, a compound previously shown to synergize with FGF to produce more dorsal ”activin-like” inductions than FGF alone, only a slight increase in pRB expression was observed. Furthermore, upregulation of pRB during mesoderm induction in vitro displayed an inverse correlation with expression of XFKH1, a marker for notochord. These results suggest that pRB may be important for patterning along the dorsoventral axis. Received: 22 February 1996 / Accepted: 20 September 1996     

Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity

Abstract Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.     

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.     

FGF signalling and the mechanism of mesoderm spreading in Drosophila embryos

FGF signalling is needed for the proper establishment of the mesodermal cell layer in Drosophila embryos. The activation of the FGF receptor Heartless triggers the di-phosphorylation of MAPK in the mesoderm, which accumulates in a graded fashion with the highest levels seen at the dorsal edge of the mesoderm. We have examined the specific requirement for FGF signalling in the spreading process. We show that only the initial step of spreading, specifically the establishment of contact between the ectoderm and the mesoderm, depends upon FGF signalling, and that unlike the role of FGF signalling in the differentiation of heart precursors this function cannot be replaced by other receptor tyrosine kinases. The initiation of mesoderm spreading requires the FGF receptor to possess a functional kinase domain, but does not depend upon the activation of MAPK. Thus, the dispersal of the mesoderm at early stages is regulated by pathways downstream of the FGF receptor that are independent of the MAPK cascade. Furthermore, we demonstrate that the activation of MAPK by Heartless needs additional cues from the ectoderm. We propose that FGF signalling is required during the initial stages of mesoderm spreading to promote the efficient interaction of the mesoderm with the ectoderm rather than having a long range chemotactic function, and we discuss this in relation to the cellular mechanism of mesoderm spreading.     

Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that can phosphorylate phosphaditylinositides leading to the cell type-specific regulation of intracellular protein kinases. PI3Ks are involved in a wide variety of cellular events including mitogenic signalling, regulation of growth and survival, vesicular trafficking, and control of the cytoskeleton. Some of these enzymes also act downstream of receptor tyrosine kinases or G-protein-coupled receptors. Using two strategies to inhibit PI3K signalling in embryos, we have analysed the role of PI3Ks during early Xenopus development. We find that a class 1A PI3K catalytic activity is required for the definition of trunk mesoderm during the blastula stages, but is less important for endoderm and prechordal plate mesoderm induction or for organiser formation. It is required in the FGF signalling pathway downstream of Ras and in parallel to the extracellular signal-regulated kinase (ERK) MAP kinases. In addition, our results show that ERKs and PI3Ks can synergise to convert ectoderm into mesoderm. These data provide the first evidence that class 1 PI3Ks are required for a specific set of patterning events in vertebrate embryos. Furthermore, they bring new insight into the FGF signalling cascade in Xenopus.     

Retinoic acid-mediated patterning of the pre-pancreatic endoderm in Xenopus operates via direct and indirect mechanisms

Early patterning of the endoderm as a prerequisite for pancreas specification involves retinoic acid (RA) as a critical signalling molecule in gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce pancreas-specific gene expression in dorsal but not ventral endoderm. The differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm is important for the distinct responsiveness of dorsal versus ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the ventral endoderm of gastrula stage Xenopus embryos. An additional requirement for mesoderm suggests the production of one or more further pancreas inducing signals by this tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.     

FGF signalling controls the timing of Pax6 activation in the neural tube

We have recently demonstrated that Pax6 activation occurs in phase with somitogenesis in the spinal cord. Here we show that the presomitic mesoderm exerts an inhibitory activity on Pax6 expression. This repressive effect is mediated by the FGF signalling pathway. The presomitic mesoderm displays a decreasing caudorostral gradient of FGF8, and grafting FGF8-soaked beads at the level of the neural tube abolishes Pax6 activation. Conversely, when FGF signalling is disrupted, Pax6 is prematurely activated in the neural plate. We propose that the progression of Pax6 activation in the neural tube is controlled by the caudal regression of the anterior limit of FGF activity. Hence, as part of its posteriorising activity, FGF8 downregulation acts as a switch from early (posterior) to a later (anterior) state of neural epithelial development.