January 1, 2009;
Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives.
-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3
function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3
function in detail in Xenopus early neural development and compared it to that of Sox2
. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3
functions as an activator to induce expression of the early neural genes, sox2
in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2
. As a result, Sox3
increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest
formation to expand the neural plate
. Our studies indicate that Sox3
and 2 have many similar functions in this process including the ability to activate expression of geminin
in naïve ectodermal explants. However, there are some differences; Sox3
activates the expression of sox2
, while Sox2
does not activate expression of sox3
is uniquely expressed throughout the ectoderm
prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3
, we demonstrate that it is required for induction of neural tissue
by BMP inhibition. Together these data indicate that Sox3
has multiple roles in early neural development including as a factor required for nogginmediated neural induction.
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Fig. 1. Sox3 expands the expression of early neural markers and delays the expression of proneural and neuronal markers. (A) WISH of stage 17 neurulae for early neural markers as indicated in lower right corner. (B) WISH of stage 14 neurulae and stage 23 tailbud embryos for the pan-neuronal marker: n-tub and pro-neural marker: neurogenin r-1 ngn. Embryos are injected with sox2 or sox3 (400 or 500 pg) and lacZ (100 pg) mRNA in 1 of 2-cells. Images are a dorsal view with anterior to the bottom and the injected side on the right marked by a black asterisk in the first panel.
Fig. 2. Sox3 activates geminin and sox2 expression in the absence of protein synthesis. (A) WISH of stage 12 embryos injected with sox3, sox3-VP16, or sox3-EnR mRNA (200–800 pg) at the 1-cell stage. Sox3 and Sox3-VP16 expand expression of sox2 and gem (arrows) and Sox3-EnR induces gem in stage 12 embryos. Images are ventral view with the anterior to the top. (B) High levels of Sox2 and Sox3 induce expression of gem in ectodermal explants. RT-PCR analysis of ectodermal explants for sox2, gem and ef1α expression from embryos injected with noggin (25 pg), sox2, sox3 (500 or 800 pg) and sox3-VP16 (200 pg). Sibling embryos were stage 12. (C) Sox3-GR induces expression of sox2 (Dex, n = 22/30; Dex + CHX, n = 12/20) and gem (Dex, n = 31/35; Dex + CHX, n = 46/54). (D) Sox2-GR induces gem in the absence of protein synthesis (Dex, n = 25/43; Dex + CHX, n = 35/47). WISH of embryos injected with either Sox2-GR or Sox3-GR (400 pg) and incubated without or with Dex (1 μM), CHX (10 μM), or Dex + CHX at either stage 9.5 or 11 and collected at stage 12. Hatched rectangle indicates region of flat mount high magnification shown below whole embryo images.
Fig. 4. Sox3, Sox2 and Sox3-VP16 inhibit Xvent2 expression and epidermal formation. (A) WISH for epi-k and Xp63 in embryos injected with 400–800 pg of sox3 or sox2 mRNA with lacZ (100 pg) mRNA into one of 2-cells. Uninjected (UI) side in top row, injected (I) side in bottom with arrow indicating injected area. Anterior is to the left and dorsal to the top. (B) Sox3, sox3-EnR, or sox3-VP16 mRNA (200–400 pg) was injected with lacZ mRNA into one of 2 blastomeres and gastrula or neurula embryos were assayed for bmp4, Xvent2 or epi-k expression by WISH. Bmp4 and Xvent2 (st. 12) embryos are animal pole view with the dorsal side to the top and epi-k embryos (st. 17) are a lateral view with anterior to the left. (C) WISH of stage 12 embryos injected with Sox3-GR into one blastomere of 2-cell embryos and untreated or treated with Dex at stage 9.5. Embryos are an animal pole views with dorsal to the top. (D) Diagram of BMP positive feedback loop, BRE-luciferase construct and relative luciferase activities in bar graph form. One-cell embryos were injected in the animal pole with vector or BRE-luciferase with sox3, sox2, sox11, sox3-EnR (400 pg), or sox3-VP16 mRNA (200 pg) and 50 pg CABR mRNA (top: lanes 3–9, and bottom: 2–5). The graph represents one experiment done in triplicate. Horizontal lines represent standard deviations. *p < 0.05 indicates values that differ significantly from lane 2 and **p < 0.01 indicates values that differ significantly from lane 6, Student’s t-test.
Fig. 6. Sox3 interferes with neural crest formation and migration. (A) Overexpression of sox3-GR induces ectopic pigment (129/181) and expands the cement gland but does not induce ectopic cement gland as indicated by WISH of Xag-1 (n = 49/77) in two right panels. (B) WISH of sox9 (n = 47/68), slug (n = 36/57), and sox10 (n = 46/56) in embryos injected with Sox3-GR and lacZ mRNA. Dorsal view of stages 13–21 embryos with anterior to the top (top row). Asterisk marks injected side. After stage 21, expression is expanded (bottom row) (sox9, n = 33/38, slug, n = 50/52, sox10, n = 50/50), but migration is inhibited (Xtwist, n = 96/130). Uninjected and injected sides of same embryo shown. (C) Development of branchial cartilage is disrupted by Sox3 and Sox3-VP16 as demonstrated by cartilage staining using Alcian blue. Embryos are dorsal view with anterior to the top and cartilage is ventral view with anterior to the top. (D) Two representative embryos demonstrating that Sox3 induces apoptosis in the head region as marked by TUNEL staining (n = 13/22). Left embryo injected in the left side and right embryo injected in the right side to account for potential asymmetric cell death. All embryos injected in 1 of 2 cells with sox3 (400 pg), sox3-VP16 (100 pg) or sox3-GR (400 pg) and lacZ (100–200 pg) or GFP (300 pg) mRNA.