Wang JC and Harris WA (2005), The role of combinational coding by homeodomain...

XB-ART-1599

Dev Biol 2005 Sep 01;2851:101-15. doi: 10.1016/j.ydbio.2005.05.041.

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The role of combinational coding by homeodomain and bHLH transcription factors in retinal cell fate specification.

Wang JC , Harris WA .

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Two major families of transcription factors (TFs), basic helix-loop-helix (bHLH) and homeodomain (HD), are known to be involved in cell fate identity. Some recent findings suggest that these TFs are used combinatorially to code for cellular determination in the retina. However, neither the extent nor the efficiency of such a combinatorial coding mechanism has been tested. To look systematically for interactions between these two TF types that would address these questions, we used a matrix analysis. We co-expressed each of six retinally expressed bHLH TFs (XNeuroD; XNgnr-1; Xath3; Xath5; Xash1; Xash3) with each of eight retinally expressed HD TFs (XRx1; XOptx2; XSix3; XPax6; XOtx2; XOtx5b; XBH; XChx10) in retinal progenitors of Xenopus laevis using targeted lipofection. The effects of each of these combinations were assayed on the six major cell types in the retina: Retinal ganglion cells (GCs), Amacrines (ACs), Bipolars (BCs), Horizontals (HCs), Photoreceptors (PRs), and Muller cells (MCs), creating 288 result categories. Multiple-way ANOVA indicated that in 14 categories, there were interactions between the two TFs that produced significantly more or less of a particular cell type than either of the components alone. However, even the most effective combinations were incapable of generating more than 65% of any particular cell type. We therefore used the same techniques to misexpress selected combinations of three TFs in retinal progenitors, but found no further enhancements of particular cell fates, indicating that other factors are probably involved in cell type specification. To test whether particular combinations were essential for horizontal fates, we made VP16 and EnR fusion constructs of some of the factors to provide dominant negative transcriptional activities. Our results confirmed that normal activities of certain combinations were sufficient, and that individually these activities were important for this fate.

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Species referenced: Xenopus laevis
Genes referenced: ascl1 ascl3 atoh7 barhl2 crx neurod1 neurod4 neurog2 otx2 pax6 rax six3 six6 vsx2

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	Fig. 1. In situ hybridizations for HD and bHLH TFs at stage 32 (upper panel) and stage 41 (lower panel). (A) For homeodomain transcription factors, all are expressed in neural retina at stage 32 except XChx10. XSix3, XOptx2, and XPax6 are also expressed in the lens. By stage 41, all HD TFs are expressed in the CMZ but only XRx1 and XSix3 extend to the most peripheral region. Distinctive expression patterns are also observed in the retinal layers: ONL and outer INL for XRx1 and XOtx5b; outer INL for XOtx2 and XChx10; GCL and inner INL for XOptx2, XPax6, and XBH; GCL and INL for XSix3. (B) For bHLH transcription factors, all are expressed in the neural retina by stage 32 and localized in the CMZ at stage 41. XNeuroD is the only bHLH gene whose expression is maintained at stage 41. bHLH = basic helix–loop–helix; HD = homeodomain; ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer; TF = transcription factor.
	ascl1 (achaete-scute complex homolog 1) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization. Shown are histological sections of the eye.
	ascl3 (achaete-scute complex homolog 3) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization. Shown are histological sections of the eye.