Early development of the neural plate: new roles for apoptosis and for one of its main effectors caspase-3.

Despite its tremendous complexity, the vertebrate nervous system emerges from a homogenous layer of neuroepithelial cells, the neural plate. Its formation relies on the time- and space-controlled progression of developmental programs. Apoptosis is a biological process that removes superfluous and potentially dangerous cells and is implemented through the activation of a molecular pathway conserved during evolution. Apoptosis and an unconventional function of one of its main effectors, caspase-3, contribute to the patterning and growth of the neuroepithelium. Little is known about the intrinsic and extrinsic cues controlling activities of the apoptotic machinery during development. The BarH-like (Barhl) proteins are homeodomain-containing transcription factors. The observations in Caenorhabditis elegans, Xenopus, and mice document that Barhl proteins act in cell survival and as cell type-specific regulators of a caspase-3 function that limits neural progenitor proliferation. In this review, we discuss the roles and regulatory modes of the apoptotic machinery in the development of the neural plate. We focus on the Barhl2, the Sonic Hedgehog, and the Wnt pathways and their activities in neural progenitor survival and proliferation.

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

Barhl2 limits growth of the diencephalic primordium through Caspase3 inhibition of beta-catenin activation.

Little is known about the respective contributions of cell proliferation and cell death to the control of vertebrate forebrain growth. The homeodomain protein barhl2 is expressed in the diencephalons of Xenopus, zebrafish, and mouse embryos, and we previously showed that Barhl2 overexpression in Xenopus neuroepithelial cells induces Caspase3-dependent apoptosis. Here, barhl2 is shown to act as a brake on diencephalic proliferation through an unconventional function of Caspase3. Depletion of Barhl2 or Caspase3 causes an increase in diencephalic cell number, a disruption of the neuroepithelium architecture, and an increase in Wnt activity. Surprisingly, these changes are not caused by decreased apoptosis but instead, are because of an increase in the amount and activation of ß-catenin, which stimulates excessive neuroepithelial cell proliferation and induces defects in ß-catenin intracellular localization and an up-regulation of axin2 and cyclinD1, two downstream targets of ß-catenin/T-cell factor/lymphoïd enhancer factor signaling. Using two different sets of complementation experiments, we showed that, in the developing diencephalon, Caspase3 acts downstream of Barhl2 in limiting neuroepithelial cell proliferation by inhibiting ß-catenin activation. Our data argue that Bar homeodomain proteins share a conserved function as cell type-specific regulators of Caspase3 activities.