Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Zic2 and Zic3 synergistically control neurulation and segmentation of paraxial mesoderm in mouse embryo

Zic family zinc-finger proteins play various roles in animal development. In mice, five Zic genes (Zic1-5) have been reported. Despite the partly overlapping expression profiles of these genes, mouse mutants for each Zic show distinct phenotypes. To uncover possible redundant roles, we characterized Zic2/Zic3 compound mutant mice. Zic2 and Zic3 are both expressed in presomitic mesoderm, forming and newly generated somites with differential spatiotemporal accentuation. Mice heterozygous for the hypomorphic Zic2 allele together with null Zic3 allele generally showed severe malformations of the axial skeleton, including asymmetric or rostro-caudally bridged vertebrae, and reduction of the number of caudal vertebral bones, that are not obvious in single mutants. These defects were preceded by perturbed somitic marker expression, and reduced paraxial mesoderm progenitors in the primitive streak. These results suggest that Zic2 and Zic3 cooperatively control the segmentation of paraxial mesoderm at multiple stages. In addition to the segmentation abnormality, the compound mutant also showed neural tube defects that ran the entire rostro-caudal extent (craniorachischisis), suggesting that neurulation is another developmental process where Zic2 and Zic3 have redundant functions.     

Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.     

Xenopus Meis3 protein lies at a nexus downstream to Zic1 and Pax3 proteins, regulating multiple cell-fates during early nervous system development

In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.     

Loss of Gbx2 results in neural crest cell patterning and pharyngeal arch artery defects in the mouse embryo

Several syndromes characterized by defects in cardiovascular and craniofacial development are associated with a hemizygous deletion of chromosome 22q11 in humans and involve defects in pharyngeal arch and neural crest cell development. Recent efforts have focused on identifying 22q11 deletion syndrome modifying loci. In this study, we show that mouse embryos deficient for Gbx2 display aberrant neural crest cell patterning and defects in pharyngeal arch-derived structures. Gbx2(-/-) embryos exhibit cardiovascular defects associated with aberrant development of the fourth pharyngeal arch arteries including interrupted aortic arch type B, right aortic arch, and retroesophageal right subclavian artery. Other developmental abnormalities include overriding aorta, ventricular septal defects, cranial nerve, and craniofacial skeletal patterning defects. Recently, Fgf8 has been proposed as a candidate modifier for 22q11 deletion syndromes. Here, we demonstrate that Fgf8 and Gbx2 expression overlaps in regions of the developing pharyngeal arches and that they interact genetically during pharyngeal arch and cardiovascular development.     

A novel member of the Xenopus Zic family, Zic5, mediates neural crest development

We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.     

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.     

Multiple developmental programs are altered by loss of Zic1 and Zic4 to cause Dandy-Walker malformation cerebellar pathogenesis

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. Here, we show that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, we show that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1(+/-);Zic4(+/-) background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1(-/-);Zic4(-/-) mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlated with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype was Shh independent. In Zic1(+/-);Zic4(+/-);Shh(+/-), we observed normal cerebellar anlage patterning and foliation. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, our data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM.     

An essential and highly conserved role for Zic3 in left-right patterning, gastrulation and convergent extension morphogenesis

Mutations in ZIC3 result in X-linked heterotaxy in humans, a syndrome consisting of left-right (L-R) patterning defects, midline abnormalities, and cardiac malformations. Similarly, loss of function of Zic3 in mouse results in abnormal L-R patterning and cardiac development. However, Zic3 null mice also exhibit defects in gastrulation, neural tube closure, and axial patterning, suggesting the hypothesis that Zic3 is necessary for proper convergent extension (C-E) morphogenesis. To further investigate the role of Zic3 in early embryonic development, we utilized two model systems, Xenopus laevis and zebrafish, and performed loss of function analysis using antisense morpholino-mediated gene knockdown. Both Xenopus and zebrafish demonstrated significant impairment of C-E in Zic3 morphants. L-R patterning was also disrupted, indicating that the role of Zic3 in L-R axis development is conserved across species. Correlation of L-R patterning and C-E defects in Xenopus suggests that early C-E defects may underlie L-R patterning defects at later stages, since Zic3 morphants with moderate to severe C-E defects exhibited an increase in laterality defects. Taken together, these results demonstrate a functional conservation of Zic3 in L-R patterning and uncover a previously unrecognized role for Zic3 in C-E morphogenesis during early vertebrate development.