A novel spalt gene expressed in branchial arches affects the ability of cranial neural crest cells to populate sensory ganglia

Cranial neural crest cells differentiate into diverse derivatives including neurons and glia of the cranial ganglia, and cartilage and bone of the facial skeleton. Here, we explore the function of a novel transcription factor of the spalt family that might be involved in early cell-lineage decisions of the avian neural crest. The chicken spalt4 gene (csal4) is expressed in the neural tube, migrating neural crest, branchial arches and, transiently, in the cranial ectoderm. Later, it is expressed in the mesectodermal, but not neuronal or glial, derivatives of midbrain and hindbrain neural crest. After over-expression by electroporation into the cranial neural tube and neural crest, we observed a marked redistribution of electroporated neural crest cells in the vicinity of the trigeminal ganglion. In control-electroporated embryos, numerous, labeled neural crest cells ([similar]80% of the population) entered the ganglion, many of which differentiated into neurons. By contrast, few ([similar]30% of the population) spalt-electroporated neural crest cells entered the trigeminal ganglion. Instead, they localized in the mesenchyme around the ganglionic periphery or continued further ventrally to the branchial arches. Interestingly, little or no expression of differentiation markers for neurons or other cell types was observed in spalt-electroporated neural crest cells

Spalt4 mediates invagination and otic placode gene expression in cranial ectoderm

Vertebrate placodes are regions of thickened head ectoderm that contribute to paired sensory organs and cranial ganglia. We demonstrate that the transcription factor Spalt4 (also known as Sall4) is broadly expressed in chick preplacodal epiblast and later resolves to otic, lens and olfactory placodes. Ectopic expression of Spalt4 by electroporation is sufficient to induce invagination of non-placodal head ectoderm and prevent neurogenic placodes from contributing to cranial ganglia. Conversely, loss of Spalt4 function in the otic placode results in abnormal otic vesicle development. Intriguingly, Spalt4 appears to initiate a placode program appropriate for the axial level but is not involved in later development of specific placode fates. Fgfs can regulate Spalt4, since implantation of Fgf2 beads into the area opaca induces its expression. The results suggest that Spalt4 is involved in early stages of placode development, initiating cranial ectodermal invagination and region-specific gene regulatory networks

Identification and dissection of a key enhancer mediating cranial neural crest specific expression of transcription factor, Ets-1

Neural crest cells form diverse derivatives that vary according to their level of origin along the body axis, with only cranial neural crest cells contributing to facial skeleton. Interestingly, the transcription factor Ets-1 is uniquely expressed in cranial but not trunk neural crest, where it functions as a direct input into neural crest specifier genes, Sox10 and FoxD3. We have isolated and interrogated a cis-regulatory element, conserved between birds and mammals, that drives reporter expression in a manner that recapitulates that of endogenous Ets-1 expression in the neural crest. Within a minimal Ets-1 enhancer region, mutation of putative binding sites for SoxE, homeobox, Ets, TFAP2 or Fox proteins results in loss or reduction of neural crest enhancer activity. Morpholino-mediated loss-of-function experiments show that Sox9, Pax7, Msx1/2, Ets-1, TFAP2A and FoxD3, all are required for enhancer activity. In contrast, mutation of a putative cMyc/E-box sequence augments reporter expression, consistent with this being a repressor binding site. Taken together, these results uncover new inputs into Ets-1, revealing critical links in the cranial neural crest gene regulatory network

Stage-dependent plasticity of the anterior neural folds to form neural crest

The anterior neural fold (ANF) is the only region of the neural tube that does not produce neural crest cells. Instead, ANF cells contribute to the olfactory and lens placodes, as well as to the forebrain and epidermis. Here, we test the ability of the ANF to form neural crest by performing heterotopic transplantation experiments in the chick embryo. We find that, at the neurula stage (HH stage 7), the chick ANF retains the ability to form migrating neural crest cells when transplanted caudally to rostral hindbrain levels. This ability is gradually lost, such that by HH9, this tissue appears to no longer have the potential to form neural crest. In contrast to the ANF, hindbrain dorsal neural folds transplanted rostrally fail to contribute to the olfactory placode but instead continue to generate neural crest cells. The transcription factor GANF is expressed in the ANF and its morpholino-mediated knock-down expands the neural crest domain rostrally and results in the production of migratory cells emerging from the ANF; however, these cells fail to express the HNK1 neural crest marker, suggesting only partial conversion. Our results show that environmental factors can imbue the chick anterior neural folds to assume a neural crest cell fate via a mechanism that partially involves loss of GANF

Pax2 and Pea3 synergize to activate a novel regulatory enhancer for spalt4 in the developing ear

The transcription factor spalt4 is a key early-response gene in otic placode induction. Here, we characterize the cis-regulatory regions of spalt4 responsible for activation of its expression in the developing otic placode and report the isolation of a novel core enhancer. Identification and mutational analysis of putative transcription factor binding sites reveal that Pea3, a downstream effector of FGF signaling, and Pax2 directly activate spalt4 during ear development. Morpholino-mediated knock-down of each factor reduces or eliminates reporter expression. In contrast, combined over-expression of Pea3 and Pax2 drives ectopic reporter expression, suggesting that they function synergistically. These studies expand the gene regulatory network underlying early otic development by identifying direct inputs that mediate spalt4 expression

Lunatic fringe causes expansion and increased neurogenesis of trunk neural tube and neural crest populations

Both neurons and glia of the PNS are derived from the neural crest. In this study, we have examined the potential function of lunatic fringe in neural tube and trunk neural crest development by gain-of-function analysis during early stages of nervous system formation. Normally lunatic fringe is expressed in three broad bands within the neural tube, and is most prominent in the dorsal neural tube containing neural crest precursors. Using retrovirally-mediated gene transfer, we find that excess lunatic fringe in the neural tube increases the numbers of neural crest cells in the migratory stream via an apparent increase in cell proliferation. In addition, lunatic fringe augments the numbers of neurons and upregulates Delta-1 expression. The results indicate that, by modulating Notch/Delta signaling, lunatic fringe not only increases cell division of neural crest precursors, but also increases the numbers of neurons in the trunk neural crest

The Mych Gene Is Required for Neural Crest Survival during Zebrafish Development

Background: Amomg Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role: of zebrafish Myc genes. Principal Findings: We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to sever defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain. Significance: Mych is a novel factor required for neural crest cell survival in zebrafish

Early steps in neural crest specification

The neural crest is a multipotent cell population that arise at the border of the neural plate and non-neural ectoderm. Studies conducted in a number of model organisms including chickens, frogs, zebrafish and mice have been instrumental in elucidating this molecular mechanisms underlying neural crest formation. Signaling molecules of the Wnt, BMP, and FGF families and their downstream effectors have been shown to mediate neural crest induction. Transcription factors including members of the Snail and SoxE gene families as well as FoxD3, c-Myc and others have been implicated in specification of the neural crest. These studies represent an important step in understanding the regulatory interactions involved in generating this complex and interesting cell type

A systems level approach reveals new gene regulatory modules in the developing ear

The inner ear is a complex vertebrate sense organ, yet it arises from a simple epithelium, the otic placode. Specification towards otic fate requires diverse signals and transcriptional inputs that act sequentially and/or in parallel. Using the chick embryo, we uncover novel genes in the gene regulatory network underlying otic commitment and reveal dynamic changes in gene expression. Functional analysis of selected transcription factors reveals the genetic hierarchy underlying the transition from progenitor to committed precursor, integrating known and novel molecular players. Our results not only characterize the otic transcriptome in unprecedented detail, but also identify new gene interactions responsible for inner ear development and for the segregation of the otic lineage from epibranchial progenitors. By recapitulating the embryonic programme, the genes and genetic sub-circuits discovered here might be useful for reprogramming naïve cells towards otic identity to restore hearing loss

Noelin-1 is a secreted glycoprotein involved in generation of the neural crest

The vertebrate neural crest arises at the border of the neural plate during early stages of nervous system development; however, little is known about the molecular mechanisms underlying neural crest formation. Here we identify a secreted protein, Noelin-1, which has the ability to prolong neural crest production. Noelin-1 messenger RNA is expressed in a graded pattern in the closing neural tube. It subsequently becomes restricted to the dorsal neural folds and migrating neural crest. Over expression of Noelin-1 using recombinant retroviruses causes an excess of neural crest emigration and extends the time that the neural tube is competent to generate as well as regenerate neural crest cells. These results support an important role for Noelin-1 in regulating the production of neural crest cells by the neural tube