Cytoplasmic protein methylation is essential for neural crest migration

As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration.     

Bmp and Fgf signaling are essential for liver specification in zebrafish

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.     

Msx1 and Msx2 have shared essential functions in neural crest but may be dispensable in epidermis and axis formation in Xenopus

The homeodomain factors Msx1 and Msx2 are expressed in essentially identical patterns in the epidermis and neural crest of Xenopus embryos during neurula stages. Disruption of Msx1 and Msx2 RNA splicing with antisense morpholino oligonucleotides shows that both factors are also required for expression of the neural crest gene Slug. Loss of Msx1 can be compensated by overexpression of Msx2 and vice versa. Loss of Msx factors also leads to alterations in the expression boundaries for neural and epidermal genes, but does not prevent or reduce expression of epidermal keratin in ventrolateral ectoderm, nor is there a detectable effect on dorsal mesodermal marker gene expression. These results indicate that Msx1 and Msx2 are both essential for neural crest development, but that the two genes have the same function in this tissue. If Msx genes have important functions in epidermis or axial mesoderm induction, these functions must be shared with other regulatory proteins.     

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1^−/− embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect.     

BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.     

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.     

The Wnt-activated Xiro1 gene encodes a repressor that is essential for neural development and downregulates Bmp4

In the early Xenopus embryo, the Xiro homeodomain proteins of the Iroquois (Iro) family control the expression of proneural genes and the size of the neural plate. We report that Xiro1 functions as a repressor that is strictly required for neural differentiation, even when the BMP4 pathway is impaired. We also show that Xiro1 and Bmp4 repress each other. Consistently, Xiro1 and Bmp4 have complementary patterns of expression during gastrulation. The expression of Xiro1 requires Wnt signaling. Thus, Xiro1 is probably a mediator of the known downregulation of Bmp4 by Wnt signaling.     

The actin depolymerizing factor n-cofilin is essential for neural tube morphogenesis and neural crest cell migration

Cofilin/ADF proteins are a ubiquitously expressed family of F-actin depolymerizing factors found in eukaryotic cells including plants. In vitro, cofilin/ADF activity has been shown to be essential for actin driven motility, by accelerating actin filament turnover. Three actin depolymerizing factors (n-cofilin, m-cofilin, ADF) can be found in mouse and human. Here we show that in mouse the non-muscle-specific gene-n-cofilin-is essential for migration of neural crest cells as well as other cell types in the paraxial mesoderm. The main defects observed in n-cofilin mutant embryos are an impaired delamination and migration of neural crest cells, affecting the development of neural crest derived tissues. Neural crest cells lacking n-cofilin do not polarize, and F-actin bundles or fibers are not detectable. In addition, n-cofilin is required for neuronal precursor cell proliferation and scattering. These defects result in a complete lack of neural tube closure in n-cofilin mutant embryos. Although ADF is overexpressed in mutant embryos, this cannot compensate the lack of n-cofilin, suggesting that they might have a different function in embryonic development. Our data suggest that in mammalian development, regulation of the actin cytoskeleton by the F-actin depolymerizing factor n-cofilin is critical for epithelial-mesenchymal type of cell shape changes as well as cell proliferation.     

Extracellular cleavage of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest cell migration

Cell adhesion molecules such as cadherins alternate their expression throughout cranial neural crest (CNC) development, yet our understanding of the role of these molecules during CNC migration remains incomplete. The “mesenchymal” cadherin-11 is expressed in the CNC during migration yet prevents migration when overexpressed in the embryo, suggesting that a defined level of cadherin-11–mediated cell adhesion is required for migration. Here we show that members of the meltrin subfamily of ADAM metalloproteases cleave the extracellular domain of cadherin-11 during CNC migration. We show that a fragment corresponding to the putative shed form of cadherin-11 retains biological activity by promoting CNC migration in vivo, in a non-cell–autonomous manner. Additionally, cleavage of cadherin-11 does not affect binding to β-catenin and downstream signaling events. We propose that ADAM cleavage of cadherin-11 promotes migration by modifying its ability to support cell–cell adhesion while maintaining the membrane-bound pool of β-catenin associated with the cadherin-11 cytoplasmic domain.     

The protooncogene c-myc is an essential regulator of neural crest formation in xenopus

The neural crest, a population of multipotent progenitor cells, is a defining feature of vertebrate embryos. Neural crest precursor cells arise at the neural plate border in response to inductive signals, but much remains to be learned about the molecular mechanisms underlying their induction. Here we show that the protooncogene c-Myc is an essential early regulator of neural crest cell formation in Xenopus. c-myc is localized at the neural plate border prior to the expression of early neural crest markers, such as slug. A morpholino-mediated "knockdown" of c-Myc protein results in the absence of neural crest precursor cells and a resultant loss of neural crest derivatives. These effects are not dependent upon changes in cell proliferation or cell death. Instead, our findings reveal an important and unexpected role for c-Myc in the specification of cell fates in the early ectoderm.     

Regulation of trunk myogenesis by the neural crest: a new facet of neural crest-somite interactions

It is well established that the somitic mesoderm regulates early stages of neural crest development and further segmentation of crest-derived peripheral ganglia. The possibility that neural crest progenitors feed back on the somites was, however, not explored. Two recent studies provide evidence that the neural crest regulates somite-derived myogenesis by distinct mechanisms.