Faculty Bibliography

The transcription factor Sox9 has been implicated in inner ear formation in several species. To investigate the long-term consequences of Sox9 depletion on inner ear development we analyzed the inner ear architecture of Sox9-depleted Xenopus tadpoles generated by injection of increasing amounts of Sox9 morpholino antisense oligonucleotides. We found that Sox9-depletion resulted in major defects in the development of vestibular structures, semicircular canals and utricle, while the ventrally located saccule was less severely affected in these embryos. Consistent with this phenotype, we observed a specific loss of the dorsal expression of Wnt3a expression in the otic vesicle of Sox9 morphants, associated with an increase in cell death and a reduction in cell proliferation in the region of the presumptive otic epithelium. We propose that, in addition to its early role in placode specification, Sox9 is also required for the maintenance of progenitors in the otic epithelium.

The valves and septa of the mature heart are derived from the cardiac cushions, which develop from discrete swellings in two regions of developing heart tube: the atrioventricular (AV) canal and the ventricular outflow tract (OFT). In higher vertebrates, three distinct lineages contribute to the heart valves and septa, the endocardium, the myocardium, and the cardiac neural crest that will populate the cardiac jelly of the OFT. Very little is known about cardiac cushions development in amphibians. Here, we describe the expression of eight genes during key stages of cardiac cushion development in Xenopus. Among these genes, the Wnt antagonist Frzb1 and the transcription factors Xl-Fli, Sox8, Sox9, and Sox10 are differentially expressed in the mesenchyme of the OFT and AV cushions. These genes can be used in combination with lineage-tracing experiments to determine the embryonic origin of the cardiac cushions mesenchyme in Xenopus.

Chemokines are a family of proteins originally identified for their activity promoting the recruitment of leukocytes to inflammatory sites. Recent evidence indicates that chemokines and their receptors may also regulate key developmental processes. In this paper we report the expression and regulation of the chemokine CXCL14 during Xenopus laevis embryogenesis. CXCL14 is first detected in several ectoderm derivatives, the dorsal aspect of the retina, the cement gland and the hatching gland. Later in development, additional domains of expression include the head mesenchyme and the medial ventral aspect of the otic vesicle. CXCL14 expression in the ectoderm is regulated by both Bmp and canonical Wnt signaling. In the hatching gland CXCL14 is co-expressed with the transcription factor Pax3. Using gain of function and knockdown approaches in whole embryos and animal explants we show that Pax3 is both necessary and sufficient for CXCL14 expression in this domain of the ectoderm.

Induction of the otic placode, the rudiment of the inner ear, is believed to depend on signals derived from surrounding tissues, the head mesoderm and the prospective hindbrain. Here we report the first attempt to define the specific contribution of the neuroectoderm to this inductive process in Xenopus. To this end we tested the ability of segments of the neural plate (NP), isolated from different axial levels, to induce the otic marker Pax8 when recombined with blastula stage animal caps. We found that one single domain of the NP, corresponding to the prospective anterior hindbrain, had Pax8-inducing activity in this assay. Surprisingly, more than half of these recombinants formed otic vesicle-like structures. Lineage tracing experiments indicate that these vesicle-like structures are entirely derived from the animal cap and express several pan-otic markers. Pax8 activation in these recombinants requires active Fgf and canonical Wnt signaling, as interference with either pathway blocks Pax8 induction. Furthermore, we demonstrate that Fgf and canonical Wnt signaling cooperate to activate Pax8 expression in isolated animal caps. We propose that in the absence of mesoderm cues the combined activity of hindbrain-derived Wnt and Fgf signals specifies the otic placode in Xenopus, and promotes its morphogenesis into an otocyst.

Two independent signals are necessary for neural crest (NC) induction in Xenopus: a Bmp signal, which must be partially attenuated by Bmp antagonists, and a separate signal mediated by either a canonical Wnt or an Fgf. The mesoderm underlying the NC-forming region has been proposed as a source of this second signal. Wnt8 and Fgf8a are expressed in this tissue around the time of NC induction and are therefore good candidate NC inducers. Loss-of-function studies indicate that both of these ligands are necessary to specify the NC; however, it is unclear whether these signaling molecules are operating in the same or in parallel pathways to generate the NC. Here, we describe experiments addressing this outstanding question. We show that although Wnt8 expression can restore NC progenitors in Fgf8a-deficient embryos, Fgf8a is unable to rescue NC formation in Wnt8-depleted embryos. Moreover, the NC-inducing activity of Fgf8a in neuralized explants is strongly repressed by co-injection of a Wnt8 or a beta-catenin morpholino, suggesting that the activity of these two signaling molecules is linked. Consistent with these observations, Fgf8a is a potent inducer of Wnt8 in both whole embryos and animal explants, and Fgf8a knockdown results in a dramatic loss of Wnt8 expression in the mesoderm. We propose that Fgf8a induces NC indirectly through the activation of Wnt8 in the paraxial mesoderm, which in turn promotes NC formation in the overlying ectoderm primed by Bmp antagonists.

The Dmrt genes encode a large family of transcription factors whose function in sexual development has been well studied in invertebrates and vertebrates. Their expression pattern is not restricted to the developing gonads, indicating that Dmrt genes might regulate other developmental processes. Here we review the expression pattern of several members of this family across species and summarize recent findings on the function of a subset of these genes in non-gonadal tissues.

In Xenopus, the neural plate border gives rise to at least three cell populations: the neural crest, the preplacodal ectoderm, and the hatching gland. To understand the molecular mechanisms that regulate the formation of these lineages, we have analyzed the role of two transcription factors, Pax3 and Zic1, which are among the earliest genes activated in response to neural plate border-inducing signals. At the end of gastrulation, Pax3 and Zic1 are coexpressed in the neural crest forming region. In addition, Pax3 is expressed in progenitors of the hatching gland, and Zic1 is detected in the preplacodal ectoderm. Using gain of function and knockdown approaches in whole embryos and animal explants, we demonstrate that Pax3 and Zic1 are necessary and sufficient to promote hatching gland and preplacodal fates, respectively, whereas their combined activity is essential to specify the neural crest. Moreover, we show that by manipulating the levels of Pax3 and Zic1 it is possible to shift fates among these cells. These findings provide novel information on the mechanisms regulating cell fate decisions at the neural plate border.

Among the families of transcription factors expressed at the neural plate border, Sox proteins have been shown to regulate multiple aspects of neural crest development. Sox8, Sox9 and Sox10, exhibit overlapping expression domains in neural crest progenitors, and studies in mouse suggest that Sox8 functions redundantly with Sox9 and Sox10 during neural crest development. Here, we show that in Xenopus, Sox8 accumulates at the lateral edges of the neural plate at the mid-gastrula stage; in contrast to its mouse and chick orthologs, Sox8 expression precedes that of Sox9 and Sox10 in neural crest progenitors. Later in development, Sox8 expression persists in migrating cranial crest cells as they populate the pharyngeal arches and in trunk neural crest cells, in a pattern that recapitulates both Sox9 and Sox10 expression domains. Although morpholino-mediated knockdown of Sox8 protein did not prevent the formation of neural crest progenitors, the timing of their induction was severely affected. This delay in neural crest specification had dramatic consequences on the development of multiple lineages of the neural crest. We demonstrate that these defects are due to the inability of neural crest cells to migrate into the periphery, rather than to a deficiency in neural crest progenitors specification and survival. These results indicate that the control of Sox8 expression at the neural plate border is a key process in initiating neural crest formation in Xenopus, and highlight species-specific differences in the relative importance of SoxE proteins during neural crest development.

Among the families of transcription factors expressed at the neural plate border in response to neural crest-inducing signals, Sox proteins have emerged as important players in regulating multiple aspects of neural crest development. Here, we summarize the expression of six Sox genes, namely Sox8, Sox9, Sox10, LSox5, Sox4 and Sox11, in neural crest progenitors and their derivatives, and review some aspects of their function pertaining to neural crest development in several species.

The Dmrt genes encode a large family of transcription factors whose function in sexual development has been well studied. However, their expression pattern is not restricted to the gonad, suggesting that Dmrt genes might regulate other developmental processes. Here, we report the expression and functional analysis of one member of this family: Xenopus Dmrt4 (XDmrt4). XDmrt4 is initially expressed in the anterior neural ridge and then becomes progressively restricted to part of the telencephalon and the olfactory placode/epithelium. XDmrt4 is induced at the anterior neural plate by a balance of neural inducers and caudalizing factors. Interference with XDmrt4 function by injection of a morpholino oligonucleotide or an inhibitory mutant resulted in a similar phenotype, the specific disruption of the olfactory placode expression of Xebf2 without affecting the expression of other placodal markers. Xebf2 belongs to a family of helix-loop-helix transcription factors implicated in neuronal differentiation, and later in embryogenesis XDmrt4-deficient embryos show impaired neurogenesis in the olfactory epithelium. Consistent with this finding, XDmrt4 is sufficient to activate neurogenin, Xebf2, and neural cell adhesion molecule expression in animal explants and is required for Noggin-mediated neuralization. Altogether, these results indicate that XDmrt4 is an important regulator of neurogenesis in the olfactory system upstream of neurogenin and Xebf2.