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. 2017 Feb 1;422(1):47-57.
doi: 10.1016/j.ydbio.2016.12.004. Epub 2016 Dec 22.

Tissue Specific Regulation of the Chick Sox10E1 Enhancer by Different Sox Family Members

Free PMC article

Tissue Specific Regulation of the Chick Sox10E1 Enhancer by Different Sox Family Members

Christina Murko et al. Dev Biol. .
Free PMC article


The transcription factor Sox10 is a key regulator of vertebrate neural crest development and serves crucial functions in the differentiation of multiple neural crest lineages. In the chick neural crest, two cis-regulatory elements have been identified that mediate Sox10 expression: Sox10E2, which initiates expression in cranial neural crest; Sox10E1 driving expression in vagal and trunk neural crest. Both also mediate Sox10 expression in the otic placode. Here, we have dissected and analyzed the Sox10E1 enhancer element to identify upstream regulatory inputs. Via mutational analysis, we found two critical Sox sites with differential impact on trunk versus otic Sox10E1 mediated reporter expression. Mutation of a combined SoxD/E motif was sufficient to completely abolish neural crest but not ear enhancer activity. However, mutation of both the SoxD/E and another SoxE site eliminated otic Sox10E1 expression. Loss-of-function experiments reveal Sox5 and Sox8 as critical inputs for trunk neural crest enhancer activity, but only Sox8 for its activity in the ear. Finally, we show by ChIP and co-immunoprecipitation that Sox5 directly binds to the SoxD/E site, and that it can interact with Sox8, further supporting their combinatorial role in activation of Sox10E1 in the trunk neural crest. The results reveal important tissue-specific inputs into Sox10 expression in the developing embryo.

Keywords: Chick: Sox10; Neural crest; Otic.


Figure 1
Figure 1. Activity of the Sox10 E1 enhancer
EGFP expression driven under control of the Sox10E1 enhancer fragment in stage HH18 chick embryos (A, C). Endogenous Sox10 expression pattern at the corresponding developmental stage: In situ hybridization to detect Sox10mRNA levels (B) and antibody staining (D) on electroporated embryos (merge in E). Note that while endogenous Sox10 protein is also expressed in the delaminating NC cells (arrowhead in D), the GFP signal is only visible in the migratory cells that have detached from the neural tube. Comparison of Sox10E-GFP (including Sox10E2, F) with Sox10E1-mCherry activity (G) and endogenous Sox10 expression levels (H, in situ) at stage HH12. Sox10E1 is restricted to the otic placode at that stage and not active in the cranial neural crest (arrowheads in F).
Figure 2
Figure 2. Sox10E1 sequence conservation and analysis
Output of the ECR genome browser showing the sequence conservation of chicken Sox10E1 throughout different species (A). The element is found on chromosome 1, position 53010774-53011395. Summary of the deletion constructs generated in this study is shown in (B). Corresponding positions of the mutations are indicated. Conservation across species was analyzed using the UCSC genome browser (C).Mutated sites are indicated.
Figure 3
Figure 3. Defining essential regions mediating trunk expression
Serial deletions of the Sox10E1 fragment were tested for their ability to drive GFP in ovo. While So10E1Δ1 still drives robust GFP expression in the trunk neural crest (A) and in the otic vesicle (B), Sox10E1Δ8 has lost trunk neural crest activity (C) while still being active in the otic vesicle (D). In all cases, the deleted version was coinjected with the full length Sox10E1mCherry. Merged images are shown.
Figure 4
Figure 4. Two SOX sites have differential regulatory function
Mutation of a potential SoxD/E binding site (M4) omits expression in the trunk (A), but is still active in the otic vesicle (B–C). Mutating a putative SoxE binding site (M6) has no effect on trunk expression (D) but reduces expression in the otic vesicle (E–F). Mutating both sites completely abolishes expression in both tissues (G–I). In all cases, mutant constructs are marked by GFP and co electroporated with the wild type version of the Sox10E1 enhancer tagged with mCherry. C, F and I show representative sections through the otic vesicle. Merged images are shown. Quantified otic fluorescence for M4 and M6 are shown in (J). P= 0.06.
Figure 5
Figure 5. Expression of Sox5 and Sox8 during stages of Sox10E1 enhancer activity
In situ hybridization showing expression of Sox5 during vagal and trunk neural crest migration (AB). Sox5 is strongly expressed in the migratory crest cells streams surrounding the otic vesicle but not in the otic vesicle itself (A). Later on, it is present in the trunk neural crest, somites and surrounding mesoderm (B). In situ hybridization for Sox8 shows expression in the otic vesicle and in the R6 stream at HH13 (C). During trunk neural crest migration, Sox8 is expressed at lower levels in the migratory crest (D).
Figure 6
Figure 6. Sox5 and Sox8 regulate trunk Sox10E1 enhancer activity
Knockdown efficiency of the Sox5 morpholino was confirmed by Western blot (A). Samples were taken from stage HH4 electroporated embryos. Tubulin was used as loading control. Co electroporation of Sox10E1 (in red, Cherry) with a control morpholino (in green, FITC) in the trunk exhibits strong mCherry signal in the migratory crest cells (B). In contrast, Sox10E1 activity (as measured by mCherry signal intensity) is significantly weaker compared to the morpholino signal in the presence of a Sox5 morpholino (C). A similar reduction in Sox10E1 expression is observed upon knockdown of Sox8 (D). Sox10 morphant embryos partially exhibit a reduction in Sox10E1 expression (E). Merged images are shown. Summary and quantification of fluorescence intensity is shown in (F). * indicate P< 0.05. Western blots from pull downs of FLAG Sox8 electroporated embryos (G). The immunoprecipitation reaction was carried out with either FLAG (blot 1) or Sox5 (blot 2) antibody, and the respectively other antibody was used for the immunoblot detection. ChIP assay was performed on trunk regions of WT chick embryos (H). Sox5 is specifically bound to the M4 binding site.
Figure 7
Figure 7. Sox8 is involved in regulating otic Sox10E1 activity
Co-electroporation of Sox10E1 (red, Cherry) with a control morpholino (green, FITC) leads to robust reporter expression in the otic vesicle (A–A′). Knockdown of Sox8 exhibits stage specific effects on reporter expression (B, C). While a knockdown at stage HH8 or earlier reduces reporter expression (B–B′), a knockdown of HH9 has only mild effects on Sox10E1 activity (C–C′). Knockdown of Sox9 in the otic vesicle (D–D′). Some embryos exhibit a reduction at early stages. Sox5 knockdowns do not effect Sox10E1 expression (E–E′). Merged images and individual channels of Sox10E1 reporter are shown.

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