Buitrago-Delgado E et al. (2018), A transition from SoxB1 to SoxE transcription f...

XB-ART-55216

Dev Biol 2018 Dec 15;4442:50-61. doi: 10.1016/j.ydbio.2018.08.008.

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A transition from SoxB1 to SoxE transcription factors is essential for progression from pluripotent blastula cells to neural crest cells.

Buitrago-Delgado E , Schock EN , Nordin K , LaBonne C .

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The neural crest is a stem cell population unique to vertebrate embryos that gives rise to derivatives from multiple embryonic germ layers. The molecular underpinnings of potency that govern neural crest potential are highly conserved with that of pluripotent blastula stem cells, suggesting that neural crest cells may have evolved through retention of aspects of the pluripotency gene regulatory network (GRN). A striking difference in the regulatory factors utilized in pluripotent blastula cells and neural crest cells is the deployment of different sub-families of Sox transcription factors; SoxB1 factors play central roles in the pluripotency of naïve blastula and ES cells, whereas neural crest cells require SoxE function. Here we explore the shared and distinct activities of these factors to shed light on the role that this molecular hand-off of Sox factor activity plays in the genesis of neural crest and the lineages derived from it. Our findings provide evidence that SoxB1 and SoxE factors have both overlapping and distinct activities in regulating pluripotency and lineage restriction in the embryo. We hypothesize that SoxE factors may transiently replace SoxB1 factors to control pluripotency in neural crest cells, and then poise these cells to contribute to glial, chondrogenic and melanocyte lineages at stages when SoxB1 factors promote neuronal progenitor formation.

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Species referenced: Xenopus laevis
Genes referenced: a2m chrd foxd3 id3 krt12.4 pou5f3.2 snai2 sox10 sox11 sox17a sox2 sox3 sox8 sox9 tbxt tfap2a ventx2.2 wnt8a
GO keywords: neural crest cell development [+]
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	Fig. 1. Expression ofSoxB1 andSoxEfactors inXenopusembryos.(A)In situhybridization examiningSox2andSox3expression in wildtypeXenopusembryos collectedbetween blastula and late neurula stages. (B)In situ hybridization examiningSox8, Sox9,and Sox10 expression in wildtype Xenopus embryos collected between blastula and late neurula stages
	Fig. 2. SoxE factors interference with pluripotency gene expression depends upon DNA-binding capabilities.(A)In situhybridization examiningVent2,Oct25,Id3, andTF-AP2 in blastula stage (stage 9) embryos injected unilaterally with Sox2, Sox3, Sox9 or Sox10. (B) Quantification of phenotypes associated with SoxB1 and SoxE factor overexpression(C) Schematic of Sox9 and Sox10 protein domains. (D)In situ hybridization examining Vent2 and Id3 expression in blastula stage (stage 9) embryos injected unilaterally with wildtype or mutant forms of Sox9. (D)In situ hybridization examining Vent2 and Id3 expression in blastula stage (stage 9) embryos injected unilaterally with wildtype or mutant forms of Sox10. (E)Quantification of phenotypes associated with overexpression of SoxE factors and SoxE functional variants. Asterisk (*) denotes injected side of the embryo as marked by staining of the lineage tracerβ-galactosidase (red) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 3. Inducing SoxB1 and SoxE protein activities at gastrulation leads to differential effects on neural crest formation, but similar effects on the epidermis.(A)Schematic of experimental overview. (B)In situ hybridization assaying expression of neural crest factors Snai2, FoxD3, and Sox10 in neurula (stage 15) embryos injected with inducible Sox2, Sox3, Sox9, or Sox10 constructs. (C)In situ hybridization assaying the expression of epidermal factor EPKin neurula (stage 15) embryos injected with inducible Sox2, Sox3, Sox9, or Sox10 constructs. (D) Quantification of phenotypes associated with overexpression of inducible SoxB1 and SoxE factors. Asterisk (*) denotes injected side of the embryo as marked by staining of the lineage tracer β-galactosidase (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 4. Overexpression of SoxB1 or SoxE factors impairs the formation of mesoderm and endoderm in animal pole explant assays.(A) Schematic of experimental overview. (B)In situ hybridization examining Brachyury expression in animal pole explants injected with Sox2, Sox3, Sox9 or Sox10 mRNA and cultured with or without a low dose of activin. (C)In situhybridization examiningSox17expression in animal pole explants injected with Sox2, Sox3, Sox9 or Sox10 mRNA and cultured with or without a high dose of activin.(D) Quantification of induction potential following SoxB1 or SoxE factor overexpression in morphant animal caps.
	Fig. 5. SoxB1 and SoxE factors can both restore potential to animal pole explants following morpholino-mediated depletion of Sox2 and Sox3.(A) Schematic of experimental overview. (B) In situ hybridization examining Brachyury expression in animal pole explants injected with Sox2 and Sox3 morpholino and either Sox2, Sox3, Sox9 or Sox10mRNA that were cultured with or without a low dose of activin. (C)In situ hybridization examining Endoderm in expression in animal pole explants injected with Sox2 and Sox3 morpholino and either Sox2, Sox3, Sox9 or Sox10 that were cultured with or without a high dose of activin. (D) Quantification of induction potential following SoxB1 and SoxE factoroverexpression in morphant animal caps.
	Fig. 6. SoxE factors cannot fully replace the function of SoxB1 factors in animal pole explants induced to a neuronal lineage.(A) Schematic of experimental overviewfor knockdown experiments. (B)In situ hybridization examining Sox2 expression in animal pole explants injected with Sox2 and Sox3 morpholino and chordin. (C)In situhybridizationexamining Sox3 expression in animal pole explants injected with Sox2 and Sox3 morpholino and chordin. (D) Schematic of experimental overview for rescue experiments. (E)In situ hybridization examining Sox11 expression in animal pole explants injected with Sox2 and Sox3 morpholino, chordin, and either Sox2 or Sox9 mRNA. (F)In situhybridization examining Sox11 expression in animal pole explants co-injected with Sox2 and Sox3 morpholino, chordin, and either Sox3 or Sox10 mRNA. (G) Quantification of induction potential following SoxB1 and SoxE factor overexpression in morphant animal caps
	Fig. 7. SoxB1 factors cannot replace the function of SoxE factors in animal pole explants induced to a neural crest state.(A) Schematic of experimental overview for knockdown experiments. (B)In situ hybridization examining Sox9, Sox10,or FoxD3 expression in animal pole explants injected with Sox10 morpholino, Wnt8, and chordin. (C)Schematic of experimental overview for rescue experiments. (D)In situ hybridization examining FoxD3 expression in animal pole explants injected with Sox10 morpholino, Wnt8,chordin, and either Sox2 or Sox9 mRNA. (E)In situ hybridization examining FoxD3 expression in animal pole explants injected with Sox10 morpholino, Wnt8, chordin, and either Sox3or Sox10 mRNA. (F) Quantification of induction potential following SoxB1 and SoxE factor overexpression in morphant animal caps.
	Fig. S1. Western blots to validate protein levels. (A) Western blot associated with Fig. 2A,D,E. (B) Western blot associated with Fig. 3. (C) Western blot associated with Fig. 4B,C. (D) Western blot associated with Fig. 5B,C. (E) Western blot associated with Fig. 6E,F. (F) Western blot associated with Fig. 7D,E.

References [+] :

Abdelalim, The SOX transcription factors as key players in pluripotent stem cells. 2014, Pubmed