Gbx2 Directly Restricts Otx2 Expression to Forebrain and Midbrain, Competing with Class III POU Factors

Abstract

bOtx2 plays essential roles in rostral brain development, and its counteraction with Gbx2 has been suggested to determine the midbrain-hindbrain boundary (MHB) in vertebrates. We previously identified the FM enhancer that is conserved among vertebrates and drives Otx2 transcription in forebrain/midbrain from the early somite stage. In this study, we found that the POU homeodomain of class III POU factors (Brn1, Brn2, Brn4, and Oct6) associates with noncanonical target sequence TAATTA in the FM enhancer. MicroRNA-mediated knockdown of Brn2 and Oct6 diminished the FM enhancer activity in anterior neural progenitor cells (NPCs) differentiated from P19 cells. The class III POU factors associate with the FM enhancer in forebrain and midbrain but not in hindbrain. We also demonstrated that the Gbx2 homeodomain recognizes the same target TAATTA in the FM enhancer, and Gbx2 associates with the FM enhancer in hindbrain. Gbx2 misexpression in the anterior NPCs repressed the FM enhancer activity and inhibited Brn2 association with the enhancer, whereas Gbx2 knockdown caused ectopic Brn2 association in the posterior NPCs. These results suggest that class III POU factors and Gbx2 share the same target site, TAATTA, in the FM enhancer and that their region-specific binding restricts Otx2 expression at the MHB. One of the most important events in the initial brain regionalization is the formation of the midbrain-hindbrain boundary (MHB), where isthmus, a local organizer for midbrain and hindbrain development, is formed. A homeobox transcription factor gene, Otx2, plays essential roles in the formation and patterning of the rostral brain, while a homeobox gene, Gbx2, does so in caudal brain development (2,12,56,65). Their expression initially overlaps, but segregates around the 6-somite stage; the mutual suppression between them has been suggested to establish the MHB at embryonic day 8.5 (E8.5) and to maintain the isthmic structure at later stages (23, 53); however, the details of the molecular mechanisms of the suppressive interaction between the Otx2 and Gbx2 genes remain to be determined.We have made efforts to elucidate the molecular mechanisms of the transcriptional regulation of the Otx2 gene and identified a series of cis-regulatory elements (26,(28)(29)(30)(31). Among these, the Otx2 expression in the anterior neural plate at the presomite stage is regulated by the AN enhancer, which is located 90 kb upstream (31, 60); its activity covers the entire anterior neural plate, the caudal limits being obscured and overlapping with the anterior part of the Gbx2 expression. The AN enhancer loses its activity at an early somite stage, and the subsequent Otx2 expression in rostral brain is regulated by the FM and FM2 enhancers, which are located 75 kb upstream and 115 kb downstream, respectively (28). The FM and FM2 enhancers lack activities in the most rostral part that correspond to future telencephalon and hypothalamus, and the caudal limit of their activities coincides with the MHB. Genetic analysis by enhancer mutants sugge...

Reconstructed cell fate–regulatory programs in stem cells reveal hierarchies and key factors of neurogenesis

“…The negatively regulated coexpression path 10 associated with the self-renewal and pluripotency factors NANOG, POU5F1, ZFP42, SOX2, or SALL4 or with GBX2 and OTX2, TFs expressed very early during neuroectoderm development (Millet et al 1999). Note that RA induction of GBX2 negatively regulates the expression of OTX2 in the anterior brain (Li and Joyner 2001;Inoue et al 2012), corroborating their inverse expression patterns (Fig. 4D).…”

“…3E,F; Voronova et al 2011;Huang et al 2012Huang et al , 2015; Gata3 [Martinez-Monedero et al 2008]). Interestingly, however, the expression of some P19-specific TGs was already affected during the first hours of RA-treatment, among them, the TFs Gbx2 (Bouillet et al 1995;Inoue et al 2012;Nakayama et al 2013), and Tal2, which is essential for midbrain neurogenesis (Achim et al 2013) and contains an intronic RA response element (Kobayashi et al 2014(Kobayashi et al , 2015. We identified two additional RXRA binding sites proximal to Tal2-a constitutive RXRA binding site ∼3 kb downstream from the coding region and a second site upstream of the transcription start site (TSS) (∼5 kb), which is similarly occupied in the absence of ligand but persists only until 6 h after initiating RA treatment (Fig.…”

Oct transcription factors in development and stem cells: insights and mechanisms

“…In neural progenitor cells of the forebrain and midbrain the members of this classOct6, Brn2, Brn1 and Brn4 -all associate with an Otx2 upstream enhancer. By contrast, an antagonistic transcription factor, Gbx2, associates and represses this locus in hindbrain (Inoue et al, 2012). These results reveal a complex interplay between multiple Oct proteins and other factors at a single enhancer binding site.…”

Genomic Control Processes in Adult Body Part Formation

“…4.7(C4), i.e., the transcriptional antagonism between otx2 and gbx2 expression. An enhancer of otx2 which displays extremely conserved sequence features has recently been found which explains how this antagonism is programmed in the genomic cis-regulatory sequence (Inoue et al, 2012). This enhancer recreates otx2 spatial expression in the anterior neuroepithelium of transgenic mice (Fig.…”