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Development
2010 Aug 01;13716:2623-31. doi: 10.1242/dev.047159.
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Conserved and novel roles for the Gsh2 transcription factor in primary neurogenesis.
Winterbottom EF
,
Illes JC
,
Faas L
,
Isaacs HV
.
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The Gsx genes encode members of the ParaHox family of homeodomain transcription factors, which are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. Here, we show that manipulation of Gsx function disrupts the differentiation of primary interneurons. We demonstrate that, despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, we report the identification of Dbx1 as a direct target of Gsh2-mediated transcriptional repression, and show that a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus.
Fig. 1. Effect of Gsx on primary interneuron development. (A-F) Whole-mount in situ hybridisation to show expression of (A-C) Lbx1 or (D-F) N-tubulin in stage 14 X. tropicalis embryos unilaterally injected with (A,D) 5 pg Gsh2; (B,E) 15 pg Gsh2-VP16-GR with addition of dexamethasone at stage 11, or (C,F) 20 ng Gsh2-AMO (T2). Dorsal views, anterior towards the left. Asterisks indicate injected side. Arrowheads indicate interneuron column on injected side. i, intermediate column (primary interneurons); l, lateral column (primary sensory neurons); m, medial column (primary motoneurons). (G,H) Expression of Lbx1 in stage 25 X. tropicalis embryos unilaterally injected with (G) 5 pg Gsh2 or (H) 10 ng Gsh2-AMO (T2). Main pictures are dorsal views with anterior towards the left; insets show cross-sections at the hindbrain level. Asterisks indicate injected side. (I) Western blot to show ability of Gsh2-AMO (T2) to inhibit translation of a myc-tagged Gsh2 mRNA in stage 10.5 X. tropicalis embryos. Embryos were injected with Gsh2 mRNA and Gsh2-AMO (T2) or a control AMO as indicated, and blotted with antibodies against Myc or GAPDH as a loading control. cMO, control morpholino.
Fig. 2. Interactions between Gsx, Msx and Nkx factors. (A) Schematic cross-section diagrams to show expression of homeobox genes Msx1, Gsh2 and Nkx6.1 in Xenopus open neural plate, and msh, ind and vnd in the Drosophila neuroectoderm. Uni-directional repressive interactions in Drosophila and putative equivalent interactions in Xenopus are indicated by solid and broken lines, respectively. (B-D,F,G) Whole-mount in situ hybridisation of stage 14 and (E) stage 13 X. tropicalis embryos to show expression of (B) Msx1 in 5 pg Gsh2-injected embryos; (C) Msx1 in 15 pg Gsh2-VP16-GR-injected embryos, plus dexamethasone at stage 11; (D) Gsh2 in 10 pg Msx1-injected embryos; (E) Gsh2 in 20 pg Nkx6.1-injected embryos; (F) Nkx6.1 in 5 pg Gsh2-injected embryos; and (G) Nkx6.1 in 15 pg Gsh2-VP16-GR-injected embryos, plus dexamethasone at stage 11. All embryos are unilaterally injected; asterisks indicate injected side. Dorsal views, anterior towards the left.
Fig. 3. Gsh2 and Dbx1 in the open neural plate. (A-C) Whole-mount in situ hybridisation to show expression of Dbx1 in stage 14 X. tropicalis embryos unilaterally injected with (A) 5 pg Gsh2; (B) 15 pg Gsh2-VP16-GR, plus dexamethasone at stage 11; or (C) 10 ng Gsh2-AMO (T2). Dorsal views, anterior towards the left, asterisks indicate injected side. (D) Dbx1 expression in a cross-section at the hindbrain level of an embryo unilaterally injected with 10 ng Gsh2-AMO (T2). (E) RT-PCR analysis of Dbx1 expression in animal cap explants from X. laevis control embryos or embryos injected with 20 pg Gsh2-VP16-GR, and treated with cycloheximide (CHX) and/or dexamethasone (DEX) as indicated. L8 is a loading control. (F) Double whole-mount in situ hybridisation to show adjacent expression of Gsh2 and Dbx1 in the open neural plate. Inset shows a half cross-section at hindbrain level. (G) Schematic cross-section diagram to show relative positions of homeobox gene expression domains in the Xenopus open neural plate.
Fig. 4. Interactions between Gsx, Nkx and Dbx factors. (A-C) Whole-mount in situ hybridisation of stage 14 X. tropicalis embryos to show expression of (A) Nkx6.1 and (B) Gsh2 in 5 pg Dbx1-injected embryos, or (C) Dbx1 in 20 pg Nkx6.1-injected embryos. Asterisks indicate injected side. (D) Proposed model of bidirectional repressive interactions between Gsh2, Dbx1 and Nkx6.1 transcription factors in the Xenopus open neural plate.
