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

Abstract

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 Determines the Early Patterning of the Diencephalon by Regulating Shh

“…Recently published studies have identified a Shh ZLI enhancer of evolutionary origin and have demonstrated that BARHL2 directly binds to this enhancer in E10.5 mouse embryo brains [30]. Moreover, studies in Xenopus have shown that Barhl2 restricts the dorsal expansion of the ZLI by affecting the competence of neuroepithelial cells to respond to the secreted form of SHH from the alar plate [31]. Previous studies have also shown that Gbx2 expression is regulated by Shh and that blocking Shh signaling in the dorsal portion of ZLI attenuates Gbx2 expression [6].…”

Expression of Barhl2 and its relationship with Pax6 expression in the forebrain of the mouse embryo

“…Morpholino knockdown of Barhl2 expression in Xenopus results in a failure of the ZLI to develop [20], while the loss of functional Pax6 in mice causes the ZLI to undergo an expansion along the rostrocaudal axis of the diencephalon [22–24]. Together these findings suggest that Barhl2 is required for ZLI initiation, while Pax6 may play a later role in the shaping of the ZLI.…”

“…Studies in Xenopus have shown that the Xenopus BarH2 homologue, Xbarhl2 [5], promotes the formation of the zona limitans intrathalamica (ZLI) [20], a forebrain organizer region that patterns the diencephalon via the secretion of morphogens including Sonic hedgehog (Shh) [21, 22]. Another transcription factor, paired-box 6 (Pax6), has an opposite effect on the ZLI, limiting its size [22–24].…”

Early development of the neural plate: New roles for apoptosis and for one of its main effectors caspase‐3

“…The ANR expresses Fgf8 and specifies the forebrain (Houart et al, 1998). The zona limitans intrathalamica (ZLI) is defined by the alar plate expression of Shh, a secreted morphogen that mediates regionalization of the prethalamus anteriorly and the thalamus posteriorly (Juraver-Geslin et al, 2014;Scholpp et al, 2006;Vieira et al, 2005). The midbrainhindbrain boundary (or isthmic organizer) separates the anterior and posterior neural tube and secretes Wnt1 and Fgf8 (Crossley and Martin, 1995).…”

“…In both Xenopus and mice, barhl2 is expressed in the developing neuroepithelium from early gastrulation onward, at a time period overlapping with the initial steps of neural plate patterning (Fig. A) (Juraver‐Geslin et al ., ; Offner et al ., ; Patterson et al ., ). The investigation of BarH proteins activities in nematode, Xenopus , and mice revealed the roles for these transcription factors in the developmental control of cell death and as cell type‐specific regulators of caspase‐3 nonapoptotic functions (Juraver‐Geslin et al ., ; Li et al ., ; Nehme et al ., ; Offner et al ., ; Peden et al ., ; Schwartz and Horvitz, ).…”