Sox21 regulates the progression of neuronal differentiation in a dose-dependent manner

Abstract

Members of the SoxB transcription factor family play critical roles in the regulation of neurogenesis. The SoxB1 proteins are required for the induction and maintenance of a proliferating neural progenitor population in numerous vertebrates, however the role of the SoxB2 protein, Sox21, is less clear due to conflicting results. To clarify the role of Sox21 in neurogenesis, we examined its function in the Xenopus neural plate. Here we report that misexpression of Sox21 expands the neural progenitor domain, and represses neuron formation by binding to Neurogenin (Ngn2) and blocking its function. Conversely, we found that Sox21 is also required for neuron formation, as cells lacking Sox21 undergo cell death and thus are unable to differentiate. Together our data indicate that Sox21 plays more than one role in neurogenesis, where a threshold level is required for cell viability and normal differentiation of neurons, but a higher concentration of Sox21 inhibits neuron formation and instead promotes progenitor maintenance.

Keywords: Differentiation; Neural progenitor; Neurogenesis; Sox; Xenopus.

Fig. 1. Sox21 functions as a repressor to expand neural progenitors and inhibit neuronal differentiation

(A) Diagram of Sox21 proteins used in overexpression studies. (B) In situ hybridization (ISH) of neurula stage embryos injected with sox21 mRNA on right side and analyzed for sox3, ngn2, neuroD or n-tubulin expression. (C) ISH of neurula stage embryos injected with sox21-EnR or sox21-VP16 mRNA on right side and analyzed for sox3, ngn2 or n-tubulin expression. (D) ISH of embryos injected with hormone-inducible GR-sox21 mRNA and analyzed for sox3 or n-tubulin expression. Right: Transverse section of GR-sox21 injected tailbud embryo following ISH for sox3. (E) ISH of embryos injected with chick sox21 mRNA and analyzed for sox3 or n-tubulin expression.

Fig. 2. Sox21 facilitates neural progenitor induction by Noggin

(A) Quantitative RT-PCR analysis of sox2 and sox3 expression in neurula stage ectodermal explants isolated from embryos injected with low (+) or high (++) levels of noggin mRNA with or without sox21 mRNA at the 1-cell stage. * indicates significance by Student’s t-test (p<0.05) compared to both sox21 and low noggin alone. (B) Graph of secondary axis phenotype for embryos injected with low (+) or high (++) nogginA3 mRNA with or without sox21 into one ventral blastomere, and analyzed by ISH for sox3. (C) ISH of a single neurula stage embryo injected with sox21 mRNA and analyzed for epi-keratin expression. (D) ISH of embryos injected into one ventral blastomere with hormone-inducible GR-sox21 mRNA, and analyzed for sox3 or epi-keratin expression. * indicates reduced expression.

Fig. 3. Sox21 is required for the induction and maintenance of sox2 expression

(A) RT-PCR analysis of sox21 expression in neurula stage ectodermal explants isolated from untreated embryos and those injected with noggin at the 1-cell stage. Reference gene ef1a was used as a loading control. (B) Quantitative RT-PCR analysis of sox2, sox3, and epi-keratin expression in mid-neurula stage ectodermal explants isolated from embryos injected with noggin, Sox21MO2, mismatch MO (mmMO) or in combination (as indicated) at the 1-cell stage. * indicates significance by Student’s t-test (p<0.05) compared to noggin alone. (C) Quantitative RT-PCR analysis of sox2 expression in early gastrula (stage 11) ectodermal explants isolated from embryos injected as in B.

Fig. 4. Sox21 inhibits the expression of neuronal differentiation genes and binds to Ngn2 to counteract its function

(A) Quantitative RT-PCR analysis of neuroD, myT1, n-tubulin and sox2 expression in neurula stage ectodermal explants isolated from embryos injected with ngn2, sox21, or ngn2+sox21. * indicates significance by Student’s t-test (p<0.05) compared to ngn2 alone. (B) Co-Immunoprecipitation (co-IP) of in vitro translated (IVT) Sox21-FLAG protein with Ngn2-myc or mCherry-myc proteins (top), or Sox2-HA (bottom) protein.

Fig. 5. The loss of Sox21 reduces neuron formation and increases apoptosis in embryos

(A) ISH of neurula stage embryos injected on right side with Sox21 morpholino MO1 (top) or fluorescent morpholino MO2 (bottom) and analzed for expression of neurogenesis genes. Asterisks indicate a loss of expression. (B) Quantitative RT-PCR analysis of sox2, ngn2 and epi-keratin expression in gastrula stage embryos injected with Sox21MO2. * indicates significance by Student’s t-test (p<0.05) compared to uninjected embryos. (C) Quantitative RT-PCR analysis of neuroD, myT1, and n-tubulin expression in neurula stage ectodermal explants isolated from embryos injected with ngn2 mRNA, Sox21MO1, or the combination as indicated. * indicates significance by Student’s t-test (p<0.05) compared to ngn2 alone. (D) ISH of neurula stage embryos injected on right side with ngn2 mRNA and MO1 (as indicated) and analyzed for n-tubulin expression. (E) TUNEL staining of embryos injected with MO1 or MO2.

Fig. 6. Sox21 affects neural gene expression in a dose-dependent manner

Embryos were injected with the indicated amounts of GR-sox21 mRNA and analyzed by ISH for changes in sox3 and n-tubulin expression. Graphs are data from a single representative experiment, which was performed three times and displayed the same trend. n = total number of injected embryos analyzed in one experiment. * Indicates significance by Chi-square test (p<0.05) compared to uninjected (0 pg) alone. * Indicates significance by Chi-square test (p<0.05) compared to other doses (25–200 pg).

Fig. 7. Model for function of Sox21 during neurogenesis

Sox21 is induced by Noggin and maintains cells in a progenitor state by binding to Ngn2 to inhibit neuronal differentiation and expand the expression of the soxB1 genes when expressed at a high level.