Chang Soo Hong

Fgf8a induces neural crest indirectly through the activation of Wnt8 in the paraxial mesoderm

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 β-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.

Functional analysis of Sox8 during neural crest development in Xenopus

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.

Identification of Pax3 and Zic1 targets in the developing neural crest

The neural crest (NC) is a multipotent population of migratory cells unique to the vertebrate embryo, contributing to the development of multiple organ systems. Transcription factors pax3 and zic1 are among the earliest genes activated in NC progenitors, and they are both necessary and sufficient to promote NC fate. In order to further characterize the function of these transcription factors during NC development we have used hormone inducible fusion proteins in a Xenopus animal cap assay, and DNA microarray to identify downstream targets of Pax3 and Zic1. Here we present the results of this screen and the initial validation of these targets using quantitative RT-PCR, in situ hybridization and morpholinos-mediated knockdown. Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. We also obtained several factors with no known function in Xenopus NC, which represent novel regulators of NC fate. The comprehensive characterization of Pax3 and Zic1 targets function in the NC gene regulatory network, are essential to understanding the mechanisms regulating the emergence of this important cell population.

Developmental expression and regulation of the chemokine CXCL14 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.

Sf3b4-depleted Xenopus embryos: A model to study the pathogenesis of craniofacial defects in Nager syndrome

Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause of Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4.

Early development of the thymus in Xenopus laevis

Background: Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3, and foxn1, and evaluated the neural crest contribution to thymus development. Results: In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3‐negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T‐cell differentiation in frogs. Conclusions: These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms. Developmental Dynamics, 2012.

Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus

Lower vertebrates develop a unique set of primary sensory neurons located in the dorsal spinal cord. These cells, known as Rohon-Beard (RB) sensory neurons, innervate the skin and mediate the response to touch during larval stages. Here we report the expression and function of the transcription factor Xaml1/Runx1 during RB sensory neurons formation. In Xenopus embryos Runx1 is specifically expressed in RB progenitors at the end of gastrulation. Runx1 expression is positively regulated by Fgf and canonical Wnt signaling and negatively regulated by Notch signaling, the same set of factors that control the development of other neural plate border cell types, i.e. the neural crest and cranial placodes. Embryos lacking Runx1 function fail to differentiate RB sensory neurons and lose the mechanosensory response to touch. At early stages Runx1 knockdown results in a RB progenitor-specific loss of expression of Pak3, a p21-activated kinase that promotes cell cycle withdrawal, and of N-tub, a neuronal-specific tubulin. Interestingly, the pro-neural gene Ngnr1, an upstream regulator of Pak3 and N-tub, is either unaffected or expanded in these embryos, suggesting the existence of two distinct regulatory pathways controlling sensory neuron formation in Xenopus. Consistent with this possibility Ngnr1 is not sufficient to activate Runx1 expression in the ectoderm. We propose that Runx1 function is critically required for the generation of RB sensory neurons, an activity reminiscent of that of Runx1 in the development of the mammalian dorsal root ganglion nociceptive sensory neurons.

Transcription factor AP2 epsilon (Tfap2e) regulates neural crest specification in Xenopus

Transcription factors Pax3 and Zic1 are two important regulators of cell fate decision at the neural plate border, where they act synergistically to promote neural crest (NC) formation. To understand the role of these factors in NC development, we performed a microarray analysis to identify downstream targets of Pax3 and Zic1 in Xenopus embryos. Among the genes identified was a member of transcription factor activator protein 2 (Tfap2) family, Tfap2 epsilon (Tfap2e). Tfap2e is first expressed at early neurula stage in NC progenitors and Rohon–Beard sensory neurons, and persists in a subset of migrating cranial NC cells as they populate the pharyngeal arches. This is in contrast to other species in which Tfap2e is not detected in the early NC lineage. Tfap2e morpholino‐mediated knockdown results in a loss of NC progenitors and an expansion of the neural plate. Tfap2e is also sufficient to activate NC‐specific genes in animal cap explants, and gain‐of‐function experiments in the whole embryo indicate that Tfap2e can promote NC formation. We propose that Tfap2e is a novel player in the gene regulatory network controlling NC specification in Xenopus downstream of Pax3 and Zic1.

Xhe2 is a member of the astacin family of metalloproteases that promotes Xenopus hatching

Transcription factors Pax3 and Zic1 are among the earliest genes activated at the neural plate border. In Xenopus, they are necessary and sufficient to promote the formation of multiple neural plate border cell types, including the neural crest, cranial placodes, and hatching gland. Pax3 is especially critical for the formation of the hatching gland, a group of cells that produce proteolytic enzymes essential to digest the egg vitelline envelope and jelly coat in order to release the tadpole into the environment. In a screen designed to identify downstream targets of Pax3, we isolated a member of the astacin family of metalloproteases, related to Xenopus hatching enzyme (Xhe), that we named Xhe2. Xhe2 is exclusively expressed in hatching gland cells as they first emerge at the lateral edge of the anterior neural plate, and persists in this tissue up to the tadpole stage. Knockdown experiments show that Xhe2 expression depends entirely on Pax3 function. Gain‐of‐function studies demonstrate that Pax3 can induce premature hatching through the upregulation of several proteolytic enzymes including Xhe2. Interestingly, Xhe2 overexpression is sufficient to induce early hatching, indicating that Xhe2 is one of the key components of the degradation mechanism responsible for breaking down the vitelline membrane. genesis 52:946–951, 2014.

Znf703, a novel target of Pax3 and Zic1, regulates hindbrain and neural crest development in Xenopus

The transcription factors Pax3 and Zic1 are critical to specify the neural plate border and to promote neural crest formation. In a microarray screen designed to identify genes regulated by Pax3 and Zic1 in Xenopus we isolated Znf703/Nlz1 a transcriptional repressor member of the NET (NocA/Nlz, Elbow, and TLP‐1) protein family. At early neurula stage znf703 is expressed in the dorsal ectoderm, spanning the neural plate and neural plate border, with an anterior boundary of expression corresponding to rhombomeres 3 and 4 (r3/r4) in the prospective hindbrain. As a bonafide target of Pax3 and Zic1, znf703 is activated by neural plate border inducing signals, and its expression depends on Pax3 and Zic1 function in the embryo. Znf703 morpholino‐mediated knockdown expanded several posterior hindbrain genes, while Znf703 overexpression completely obliterated the expression of these segmental genes, signifying that the transcriptional repressor activity of Znf703 is critical to pattern the hindbrain. Furthermore, snai2 and sox10 expression was severely impaired upon manipulation of Znf703 expression levels in the embryo suggesting that Znf703 participates in neural crest formation downstream of Pax3 and Zic1 in Xenopus.