Schock EN et al. (2024), SoxB1 transcription factors are essential for i...

XB-ART-60760

Development 2024 Jul 15;15114:. doi: 10.1242/dev.202693.

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SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

Schock EN , York JR , Li AP , Tu AY , LaBonne C .

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SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine if SoxB1 transcription factors have a regulatory function in NPB formation. Herein, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show their combined activity is needed for normal NPB gene expression. Together, these data identify a novel role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

???displayArticle.pubmedLink??? 38940470
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Species referenced: Xenopus laevis
Genes referenced: dlx5 dlx6 foxd3 foxi2 gapdh klf17 lin28a msx1 myc npb odc1 olig2 pax3 pax7 pou5f3 pou5f3.2 pou5f3.3 prdm1 psmd6 six1 snai2 sox10 sox2 sox3 tfap2a ubc ventx2.2 zic1
GO keywords: Wnt signaling pathway [+]
???displayArticle.antibodies??? Acta1 Ab6 FLAG Ab3 Myc Ab1
???displayArticle.morpholinos??? pou5f3.1 MO5 pou5f3.1 MO6 pou5f3.2 MO8 pou5f3.2 MO9 sox2 MO6 sox3 MO4

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	Fig. 1. Sox3 protein co-localization with pax3 transcripts during gastrulation and co-localization decreases with the onset of neurulation. (A) Whole embryos immunostained for Sox3 (magenta) and probed with HCR oligos for pax3 (green) across multiple developmental stages (blastula through late neurula). DAPI is shown in gray. Diagrams show domains of co-localization (yellow) and at each time point. Sox3 protein. (C) localization is displayed in magenta and pax3 transcripts in green in diagrams. (B) Neural plate border/neural crest-induced explants immunostained for Sox3 (magenta) and probed with HCR oligos for intronic pax3 (green), marking nascent transcripts, across multiple developmental stages (blastula through late neurula). DAPI is shown in gray. Arrows denote cells where nascent pax3 transcripts co-localize with nuclear Sox3 protein. (C) Quantification of co-localization, as shown by violin plots, between Sox3 protein and nascent pax3 transcripts over developmental time. Outlier data points are displayed as hollow circles. Means are displayed as black horizonal bars. (D) Western blot for endogenous Sox3 protein (green) and actin (red) in neural plate border-induced explants, epidermal explants, and neural-induced explants at stages 9, 11, 13, and 15. Bar graph shows the quantification of western signal where the ratio of Sox3 to actin is normalized to stage 9. Error bars show standard deviation. ns=not significant; (***) p<0.001; dorsal (d); anterior (a); posterior (p); blastopore (bp); neural plate border (NPB); epidermis (Epi); neural (Neu)
	Fig. 2. SoxB1 factors are necessary for neural plate border formation. (A) In situ hybridization for pax3, zic1, msx1, and tfap2a in stage 12.5-13 embryos unilaterally injected with sox2 and sox3 morpholino (denotes injected side). Beta-galactosidase (red) was used as a lineage tracer. (B) In situ hybridization for pax3 and msx1 in stage 12.5-13 embryos unilaterally injected with sox2 and sox3 morpholinos or rescued embryos which were co-injected with sox3 mRNA (denotes injected side). Fluorescein dextran was used as a lineage tracer and embryos were presorted for left/right side targeting. (C) In situ hybridization for pax3 and msx1 on wildtype and soxb1 morphant neural plate border-induced explants (stage 12.5). (D) qPCR examining gene expression fold changes in neural plate border-induced explants (stage 12.5) comparing wildtype neural plate border-induced cells (blue) to soxb1 morphant neural plate borderinduced cells (magenta). Error bars show standard deviation. () p<0.05; () p< 0.01; (**) p<0.001; morpholino (MO); neural plate border (NPB)
	Fig. 3. SoxB1 expression blocks the transition from neural plate border to neural crest gene expression. (A-C,E,G) In situ hybridization in embryos unilaterally expressing sox2 or sox3 mRNA (* denotes injected side). Beta-galactosidase (red) was used as a lineage tracer. (A) pax3 and zic1 in early neurula embryos; (B) pax3 and zic1 in late neurula embryos, arrowhead denotes expanded domain of expression; (C) foxd3 and snai2 in late neurula embryos. (D) Hybridization chain reaction for foxd3 (magenta) and pax3 (yellow) in embryos unilaterally expressing sox3 mRNA (* denotes injected side). Myc antibody staining (green) was used as a lineage tracer. Arrowhead denotes region with loss of foxd3 expression and expanded pax3 expression. (E) pax3 and snai2 in stage 17 and stage 19 embryos unilaterally expressing sox3 mRNA. Arrowhead denotes expanded domain of expression. (F) Hybridization chain reaction for sox10 (magenta) and pax3 (green) in embryos unilaterally expressing sox3 mRNA (* denotes injected side). Myc antibody staining (gray) was used as a lineage tracer. (G) In situ hybridization for pax3 and tfap2a in stage 21 in embryos unilaterally expressing sox3 mRNA. (H) Time course western blot with quantification for myc-tagged Sox3 protein levels in the embryo where the ratio of myc to actin is normalized to stage 15. () p<0.05; (**) p<0.001; lineage tracer (lt)
	Fig. 4. ChIP-seq in early neural plate border cells reveals Sox3 enrichment at multiple neural plate border genes. (A) Schematic of experimental design for Sox3 ChIP-seq experiments in neural plate border-induced explants. (B) Genome browser view of Sox3 peaks at loci for neural plate border genes (pax3, zic1, msx1, dlx6, prdm1, and klf17). (C) Representative k-means clusters for transcriptome data for blastula stem cells (stage 9), neural plate border-induced explants (stage 13), and neural crestinduced explants (stage 17) (Adapted from (York et al., 2023)). (D) Percentage of Sox3 peaks (5300 peaks total) associated with k-means cluster genes and accompanying forest plot for odds ratio. (E) Percentage of genes in k-means clusters with associated Sox3 ChIP peaks and accompanying forest plot for odds ratio. Blastula cluster (red); neural plate border (NPB) cluster (blue); neural crest (NC) cluster (yellow); Cluster 10 (C10; gray); n-terminal myc-tag (nMT); Control (Ctrl)
	Fig. 5. Sox3 is enriched upstream of neural plate border and other ectodermal genes in blastula stem cells. (A) Genome browser view of Sox3 ChIP peaks at loci for pluripotency genes (pou5f3.2, ventx2.2, and lin28a). (B) Genome browser view of Sox3 ChIP peaks at loci for neural plate border genes (pax3, zic1, msx1, dlx6, prdm1, and klf17) in blastula stem cells (red) and neural plate border cells (blue). (C) Diagram of Sox3 peaks (red bars) near the transcription start site (TSS) of neural (olig2, otx2), epidermal (krt12.4, trim29), neural crest (NC; foxd3, snai2), and placodal genes (six1, eya1) in blastula stem cells.
	Fig. 5. (D) Graphical summary of genome occupancy in blastula stem cells and neural plate border cells with colored boxes (red/blue) indicating Sox3 enrichment. (E) Venn diagram showing overlap (maroon) between blastula stem cell transcriptome (gray) and Sox3 peaks in blastula stem cells (red). (F) GO term analysis for annotated genes associated with Sox3 peaks that are not expressed in blastula stem cells. (G) Volcano plot for differentially expressed genes between wildtype blastula stem cells and soxb1 morphant blastula stem cells. Genes downregulated in soxb1 morphants are shown in blue and include several neural plate border genes. Upregulated genes are shown in green. (H) Percent of differentially expressed genes with an associated blastula stage Sox3 peak. Neural plate border (NPB); neural crest (NC)
	Fig. 6. Motif analysis identifies POU factors as potential SoxB1 transcriptional partner involved in neural plate border formation. (A) Venn diagram showing overlap (purple) between Sox3 blastula stage peaks (red) and Sox3 neural plate border cell peaks (blue). (B) Dot plot showing motif enrichment for shared category (purple) of Sox3 peaks. Motifs for POU transcription factors are highly enriched. (C) Localization of Sox2, Oct4, and Oct4::Sox2 motifs upstream of neural plate border gene transcription start sites. (D) TPM plots for pou5f3 factor expression from blastula stem cells (stage 9), neural plate border-induced explants (stage 11.5, stage 13) and neural crest-induced explants (stage 17). (E) Co-immunoprecipitation of Sox3 with Pou5f3.1, Pou5f3.2, Pou5f3.3 factors. (F) Wildtype embryo (stage 12) immunostained for Sox3 (yellow) and probed with HCR oligos for pou5f3.1 (magenta) and pax3 (cyan). (G) Wildtype embryo (stage 11) immunostained for Sox3 (yellow) and probed with HCR oligos for pou5f3.2 (magenta) and pax3 (cyan). n-terminal myc tag (nMT); n-terminal flag tag (nFT); immunoblot (IB); immunoprecipitated (IP); zinc finger (ZF)
	Fig. 7. Combined activity of Pou5f3 and SoxB1 transcription factors is required for neural plate border formation. (A) In situ hybridization for pax3, zic1, and msx1 at early neurula embryos injected unilaterally (injected side marked with *) with Sox3 morpholino, Pou5f3.1+Pou5f3.2 morpholinos, or Sox3+Pou5f3.1+ Pou5f3.2 morpholinos. Fluorescein dextran was used as a lineage tracer and embryos were presorted for left/right side targeting. (B) In situ hybridization for pax3 and zic1 on wildtype and triplemorphant neural plate border-induced explants (stage 13). (C) Proposed model for SoxB1-mediated regulation of the neural plate border and neural crest. SoxB1 factors (teal) bind near ectodermal genes in blastula stem cells and promote or poise gene expression. SoxB1 factors, in partnership with Pou5f transcription factors (navy) maintain neural plate border gene (blue) expression throughout gastrulation. Downregulation of SoxB1 in neural plate border cells is required for neural crest (yellow) gene expression. Morpholino (MO); neural plate border (NPB); neural crest (NC)
	Fig. S1. Sox2 and Sox3 have overlapping expression domains. (A) Wildtype stage 13 embryo immunostained for Sox2 (green) and Sox3 (magenta). DAPI is shown in gray. (B) Average TPMs for sox2 (blue) and sox3 (red) in blastula stem cells (St. 9) and neural plate border/neural crest-induced explants (St. 11.5, St. 13, St. 17). (C) Average TPMs for sox2 (blue) and sox3 (red) from dissected neural plate border/neural crest cells (St. 12.5, St.14, St.17). Error bars are standard deviation. Neural plate border (NPB); neural crest (NC)
	Fig. S2. Temporal relationship between Sox3 and neural plate border gene expression. (A) Experimental set up for neural plate border/neural crest induction of blastula stem cell explants using small molecules. (B) Average TPMs for pax3 in epidermal vs neural crest-induced explants (St. 9, 11.5, St. 13, St. 17). Error bars are standard deviation. (C) Nascent pax3 expression (green) and Sox3 protein (red) in epidermal (control) explants from early gastrulation through mid-neurulation. DAPI is shown in blue. (D) Quantification of percent pax3+ cells in epidermal explants. (E) Wildtype stage 15 embryo immunostained for Sox3 (magenta) and probed for snai2 (green) using HCR. DAPI is shown in gray. Neural plate border (NPB); neural crest (NC)
	Fig. S3. Sox2 and Sox3 morpholino validation. (A) Schematic showing morpholino sequences and target regions at sox2 and sox3 alloalleles. (B) Western blot validation of Sox2 and Sox3 morpholinos. C-terminal myc tag (cMT); glucocorticoid receptor (GR); morpholino (MO)
	Fig. S4. soxb1 morphant scoring. (A) Stacked bar graphs with the percent of embryos with changes in gene expression (loss, expansion, no change) for sox2 and sox3 double morphants and rescued morphants (B) Stacked bar graphs with the percent of neural plate border-induced explants expressing pax3 or msx1, indicating induction to a neural plate border state. Wildtype (WT); morpholino (MO)
	Fig. S5. In situ hybridization and scoring for Sox2 and Sox3 expressing embryos. (A) Stacked bar graphs with the percent of embryos with changes in gene expression (loss, expansion, no change) in sox2 or sox3 expressing embryos. (B) In situ hybridization in stage 16 embryos unilaterally expressing sox2 or sox3 mRNA (* denotes injected side) probing for placodal markers (six1 and foxi1c), neural markers (sox11 and nrp1), and epidermal markers (epk and trim29) with associated scoring. Beta-galactosidase (red) was used as a lineage tracer. Stacked bar graphs with the percent of embryos with changes in gene expression (loss, expansion, no change) in sox2 or sox3 expressing embryos.
	Fig. S5. (C) Stacked bar graphs with the percent of sox3 expressing embryos with changes in gene expression (loss, expansion, recovery, no change) at stage 19 and 21.
	Fig. S6. Neural plate border induction assessment for ChIP-seq experiments. (A) Western blot for Sox3 (green) and actin (red) in wildtype and sox3 injected neural plate border-induced explants (St. 11.5). Arrowhead denotes endogenous protein and * deonotes myc-tagged sox3. (B) In situ hybridization for pax3 in neural plate borderinduced explants with and without myc-tagged sox3.
	Fig. S7. Fisher’s exact tests on differentially expressed genes in relation to Sox3 binding. Volcano plot showing differentially expressed genes between stage 13 epidermal and neural plate border-induced explants. Genes differentially expressed in epidermal cells are shown in maroon and in blue for neural plate border cells. Forrest plot displaying the log(odds ratio) for each group of differentially expressed genes (epidermal vs neural plate border) in relation to Sox3 binding in neural plate borderinduced explants (St. 11.5). Odds ratio (or); neural plate border (NPB), epidermis (Epi)
	Fig. S8. Sox3 was expressed at near endogenous levels for blastula (St. 9) ChIP-seq experiments. Western blot for myc (green) and Sox3 (red) in embryos expressing sox3 mRNA.
	Fig. S9. TPMs for genes in blastula stems cells. Average TPMs for pluripotency genes (pou5f3.2, ventx2.2 and lin28a), neural crest genes (foxd3 and snai2), placodal genes (six1 and eya1), neural genes (olig2, otx2), epidermal genes (krt12.4, trim29), and neural plate border genes (pax3, zic1, msx1, dlx6, prdm1, and klf17) in blastula stem cells (stage 9). Error bars are standard deviation.
	Fig. S10. Motif analysis on shared Sox3 ChIP-seq peaks (St.9 and St. 11.5). (A) HOMER motif consensus sequences and associated p-value. (B) Motif enrichment analysis, focusing on prevalence of SOX and POU motifs, on regions of Sox3 binding in the four k-means clusters.
	Fig. S11. pou5f3.1/2 are expressed in neural plate border cells (A) Average TPMs for pou5f3.1 (blue), pou5f3.2 (orange), and pou5f3.3 (green) from dissected neural plate border/neural crest cells (St. 12.5, St.14, St.17). Error bars are standard deviation. (B) Neural plate border-induced explants (stage 11.5) immunostained for Sox3 (magenta) and probed with HCR oligos for pou5f3.1 or pou5f3.2 (green). DAPI is shown in gray. Neural plate border (NPB); neural crest (NC)
	Fig. S12. sox3 morphants have expanded sox2 and sox3 expression. In situ hybridization for sox2 and sox3 in stage 12.5 sox3 morphant embryos (* denotes injected side). Fluorescein dextran was used as a lineage tracer and embryos were presorted for left/right side targeting. Stacked bar graphs with the percent of embryos with changes in gene expression (loss, expansion, no change) for sox3 morphants. Morpholino (MO)
	Fig. S13. Pou5f3.1/2 + sox3 triple morphant scoring. (A) Stacked bar graphs with the percent of embryos with changes in gene expression (loss, expansion, no change) in sox3 morphants, pou5f3.1+pou5f3.2 double morphants, and sox3+pou5f3.1+pou5f3.2 triple morphants (B) Stacked bar graphs with the percent of neural plate border-induced explants expressing pax3 or zic1, indicating induction to a neural plate border state. Wildtype (WT); morpholino (MO)