Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

Pax6 regulates regional development and neuronal migration in the cerebral cortex

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.     

A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.     

Conserved regulation of the Caenorhabditis elegans labial/Hox1 gene ceh-13

Caenorhabditis elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, but unlike in flies not all of them, are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, we present a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes, and find that the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. We have identified two enhancer fragments that are involved in different aspects of the embryonic ceh-13 expression pattern. We show that important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. Our data show that the molecular nature of Hox1 class gene autoregulation has been conserved between worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos.     

Ectopic Pax2 expression in chick ventral optic cup phenocopies loss of Pax2 expression

Pax2 is essential for the development of the urogenital system, neural tube, otic vesicle, optic cup and optic tract [Dressler, G.R., Deutsch, U., et al., 1990. PAX2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109 (4), 787-795; Nornes, H.O., Dressler, G.R., et al., 1990. Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109 (4), 797-809; Eccles, M.R., Wallis, L.J., et al., 1992. Expression of the PAX2 gene in human fetal kidney and Wilms’ tumor. Cell Growth Differ 3 (5), 279-289]. Within the visual system, a loss-of-function leads to lack of choroid fissure closure (known as a coloboma), a loss of optic nerve astrocytes, and anomalous axonal pathfinding at the optic chiasm [Favor, J., Sandulache, R., et al., 1996. The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proc. Natl. Acad. Sci. U. S. A. 93 (24), 13870-13875; Torres, M., Gomez-Pardo, E., et al., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122 (11), 3381-3391]. This study is directed at determining the effects of ectopic Pax2 expression in the chick ventral optic cup past the normal developmental period when Pax2 is found. In ovo electroporation of Pax2 into the chick ventral optic cup results in the formation of colobomas, a condition typically associated with a loss of Pax2 expression. While the overexpression of Pax2 appears to phenocopy a loss of Pax2, the mechanism of the failure of choroid fissure closure is associated with a cell fate switch from ventral retina and retinal pigmented epithelium (RPE) to an astrocyte fate. Further, ectopic expression of Pax2 in RPE appears to have non-cell autonomous effects on adjacent RPE, creating an ectopic neural retina in place of the RPE.