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Proneural basic helix-loop-helix (bHLH) transcription factors are critical positive regulators of neuronal differentiation in a variety of species and are required for proper differentiation of various subtypes of neurons. Although bHLH factors demonstrate some unique functions during neural development, they share the ability to regulate neuronal differentiation, potentially by targeting overlapping sets of genes. To assess this, we performed a screen in ectodermanimal captissue to identify direct transcriptional targets shared by two Xenopus ato-related bHLH factors, Xath5 and XNeuroD. Candidate target genes identified in this screen include several transcriptional regulators (Xebf2, Xebf3, XETOR and NKL), an RNA binding protein (elrC), a cell cycle component (Xgadd45gamma) and several novel genes. Overexpression of either Xath5 or XNeuroD induced ectopic in vivo expression of these candidate target genes. Conversely, blocking ato-related bHLH activity prevented endogenous nervous system expression of these genes. Therefore, we have identified a set of genes that can be regulated by multiple ato-related bHLH factors and may function as critical effectors of proneural bHLH-mediated differentiation.
Fig. 1. Identification of shared Xath5-hGR and XNeuroD-hGR gene targets by Fluorescent Differential Display. (A) RT-PCR for the indicated genes was first performed to ensure transcriptional activation of the hGR fusion proteins with DEX treatment and to monitor quality of the isolated animal caps. Animal caps were isolated from stage 8 embryos and cultured for 3 h in the presence (+) or absence (−) of hormone DEX and in the presence (+) of the protein synthesis inhibitor CHX. EF1α was used for normalization. Water was used in place of cDNA in no-template control (NTC) samples, and cDNA from stage 18 to 25 whole embryos was used for positive controls (pos). (A) XBrn3d was upregulated in Xath5-hGR-injected animal caps treated with DEX even in the presence of CHX, but not in uninduced caps or in caps injected with hGR domain. (B) Similarly, DEX treatment of XNeuroD-hGR-injected animal caps resulted in Xebf3 upregulation even with CHX co-treatment, but Xebf3 was not detected in control samples. (C) XNF-M was not detectable in CHX-treated caps, confirming inhibition of protein synthesis, and the mesoderm-specific gene Xbra was also undetectable. (D) Animal caps were injected and treated with DEX and CHX as indicated in the table, and transcript profiles were compared by FDD. Example image depicts a portion of a larger FDD gel showing the PCR products generated from duplicate reactions of a single primer set. Arrow indicates a band present in DEX-treated animal caps corresponding to a candidate common target of Xath5-hGR and XNeuroD-hGR.
Fig. 2. Xath5 or XNeuroD overexpression induces ectopic expression of candidate target genes identified by FDD. Xath5 or XNeuroD mRNA was injected into 1 cell of 2-cell stage embryos with GFP mRNA as an injection marker. Whole mount in situ hybridization was performed to visualize expression of candidate target genes. Xath5 overexpression induces significant ectopic broad expression of elrC (A) and XETOR (B) on the injected side of the embryo. The novel gene SBT1 is expressed in the developing nervous system (arrows in panels C and J) and displays a scattered pattern of ectopic expression on the Xath5-injected side. Xath5 overexpression results in upregulation of Xgadd45γ (D), Xebf2 (E) and Xebf3 (F) within the developing nervous system, with weaker ectopic expression also induced in lateral ectodermal regions. At early tailbud stages, Xath5 induces ectopic expression of NKL (G) and SVOP (H) on the injected side of the embryo. NeuroD overexpression also induces broad robust ectopic elrC expression (I) and scattered ectopic expression of SBT1 (J). At early tailbud stages, NeuroD injection promotes scattered ectopic expression of NKL (K) and SVOP (L) on the injected side. Ectopic gene expression is indicated with brackets. Embryos in panels (A) and (I) are presented as dorsal views with anterior to the top and injected side on the left. Tailbud stage embryos in panels (H) and (L) are shown as lateral views with anterior to the right.
Fig. 3. Dominant-negative Xath5 inhibits proneural function of ato-related bHLH factors. mRNA for indicated transcription factor(s) was co-injected into 1 cell of 2-cell stage embryos with mRNA encoding β-galactosidase (β-gal) or GFP. Arrowheads indicate endogenous expression of indicated genes visualized by whole mount in situ hybridization. Xath5 (A), XNeuroD (C), Xath3 (E) and X-Ngnr-1 (G) induce ectopic neurogenesis, indicated by visualization of ectopic N-tubulin (A, C, E) or XNeuroD (G) (ectopic expression indicated by brackets). The proneural activity of these bHLH factors is blocked by co-injection of Xath5EnR (B, D, F, H). However, proneural activity of Xash3, indicated by increased X-Delta-1 expression on the injected side (I), is not inhibited by Xath5EnR co-injection (J), while the ability of XNeuroD to induce expression of X-Delta-1 (K) is efficiently suppressed by coexpression of Xath5EnR (L). Injection of Xath5EnR alone suppresses primary neurogenesis on the injected side (bracket in M) but does not disrupt expression of the neural plate marker Sox2 (N). Panels (A) and (K) are dorsal views with anterior to the top. Panels (I) and (J) are anterior views with dorsal to the top. Injected side is on the right in all images, and pink staining (B, F, N) and light blue staining (D) denote expression of β-gal injection tracer.
Fig. 4. Dominant-negative Xath5 inhibits expression of Xath5/XNeuroD shared target genes. Injection of Xath5EnR inhibits endogenous expression of elrC (A), SBT1 (B), Xebf2 (C), XETOR (D), Xebf3 (E), SVOP (F) and Xgadd45γ (G) in mid-neurula and tailbud stage embryos. Xath5EnR injection also blocks endogenous expression of NKL (H) at tailbud stages. Endogenous expression of target genes is indicated by arrowheads, and loss of or reduced expression of genes on Xath5EnR injected side is indicated by brackets. All embryos are shown as dorsal views with anterior to the top and Xath5EnR-injected side on the left, except for the NKL probed embryo (H), which is shown as a lateral view with anterior to the right. Pink staining (A) and light blue staining (B) correspond to β-galactosidase injection tracer. Embryos in remaining panels (C) were presorted for GFP to identify injected side. Abbreviations: sc, spinal cord; e, eye.
Fig. 5. Proneural bHLH target genes are expressed in late progenitors or post-mitotic neurons of the X. laevis retina. (A) Schematic of stage 41/42 X. laevis retinal cross-section. The central retina contains post-mitotic cells stratified into 3 major layers: retinal ganglion cell (RGC) layer, inner nuclear layer (INL) and outer nuclear layer (ONL). The ciliary marginal zone (CMZ) contains retinal stem cells within the most peripheral region (per) and early differentiating progenitors within the central region (cntl). The entire neural retina is surrounded by retinal pigment epithelium (RPE). (B) In situ hybridizations were performed on paraffin sections of stage 41/42 X. laevis embryos. (B) Xath5 expression is restricted to the central CMZ (bracket) and excluded from the most peripheral CMZ. (C) SBT1, Xgadd45γ and XETOR, respectively, are also restricted to progenitors and early differentiating neurons within the central CMZ (bracket). (F) elrC is expressed in differentiating neurons in the most central CMZ region (arrow in panel F) and persists in post-mitotic cells of the RGC layer and a subset of cells within the INL. (G) Xebf3 expression is also present in differentiating neurons of the central CMZ (arrow in panel G) and maintained in mature RGCs.
