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. 2012;8(12):e1003142.
doi: 10.1371/journal.pgen.1003142. Epub 2012 Dec 20.

Dynamic and differential regulation of stem cell factor FoxD3 in the neural crest is Encrypted in the genome

Marcos S Simões-Costa  1 , Sonja J McKeown, Joanne Tan-Cabugao, Tatjana Sauka-Spengler, Marianne E Bronner
Affiliations

Dynamic and differential regulation of stem cell factor FoxD3 in the neural crest is Encrypted in the genome

Marcos S Simões-Costa et al. PLoS Genet. 2012.

Abstract

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Endogenous FoxD3 in the neural crest is reflected by activity of two enhancers, NC1 and NC2.
(A) Expression of FoxD3 in premigratory neural crest cells at HH8−. (B) At HH8+, FoxD3 expression extends to the midbrain and hindbrain neural folds. (C) At HH9, FoxD3 is expressed by premigratory and migrating neural crest cells, at cranial, vagal and trunk levels with the exception of rhombomere 3 (dotted arrow). (D) FoxD3 transcripts are detected in migrating cranial crest cells at stage HH10. (E–F) Double fluorescent in situ hybridization for Sox10 (red) and FoxD3 (green) reveals differences in the expression domains of these neural crest specifiers at stages HH9 (E) and HH10 (F). Expression of Sox10 begins only as cells leave the neural tube at all axial levels. (G–H) Expression of eGFP driven by enhancer NC1 at stages HH8+ and HH10. Bar indicates approximate level of transverse section shown in L. (I) In situ hybridization of FoxD3 at stage HH12. (J) Expression of eGFP driven by enhancer NC1. (K) eGFP driven by enhancer NC2. (L–N) Transverse sections through embryos shown in H, J, K. Arrows indicate migratory neural crest expressing eGFP. HNK-1-positive cells shown are in blue in M and N. (O) Genomic region of FoxD3 in chick showing regions tested for enhancer activity between the flanking genes Atg4c and Alg6 (blue boxes). Boxes indicate regions that were tested for enhancer activity: black boxes indicate no detectable activity in the neural crest; green boxes indicate enhancers active in the neural crest. Coding regions are indicated by blue boxes. anf: anterior neural fold, not: notochord, nf: neural fold, nc: neural crest, ncc: neural crest cells, nt: neural tube, ot: otic placode, R: rhombomere.
Figure 2
Figure 2. Chick NC1 and NC2 enhancers drive expression that overlaps with endogenous FoxD3 expression, function in reverse orientation, and are conserved with mouse NC1 and NC2.
(A–C) Immunostaining with anti-FoxD3 antibody (red) of embryos electroporated with NC1:eGFP (green) shows overlap of enhancer activity and endogenous expression of FoxD3 in early cranial neural crest cells. (D–F) Migrating cranial neural crest cells express FoxD3 and eGFP driven by the NC2 enhancer in stage HH11 embryos. (G–I) Vagal and trunk neural crest cells that are positive for FoxD3 also express eGFP driven by the FoxD3 enhancer. (J–K) Reversing the orientation of NC1 and NC2 does not alter their ability to drive eGFP expression. (L–M) Genomic regions homologous to the NC1 and NC2 enhancers cloned from mouse (mNC1 and mNC2) drive expression of eGFP in a manner identical to chick NC1 and NC2. cnc: cranial neural crest, vnc: vagal neural crest, tnc: trunk neural crest.
Figure 3
Figure 3. Dynamic regulation of FoxD3 and Sox10 enhancers in the cranial neural crest.
(A–D) Selected images from a time lapse sequence showing activity of the NC1 (green) and NC2 (blue) FoxD3 enhancers and the Sox10E2 (red) enhancer in a chick cranial slice preparation (see Video S1). (E–H) Images from a time lapse movie of migrating cranial neural crest cells electroporated with NC1:eGFP and NC2:Cherry (see Video S2).
Figure 4
Figure 4. Dissection of the NC1 and NC2 enhancers.
(A, H) Diagram of deletions and substitutions made to uncover critical enhancer regions. Each numbered bar represents a region of the enhancer that was tested via whole embryo electroporation. The relative level of expression in cranial and vagal/trunk neural crest (NC) is indicated on the right for each region ranging from + indicating weak expression to +++++ indicating strong expression. Gray bars indicate those enhancer regions that drove activity in the neural crest. Red bars indicate a lack of activity. Black fragments of the enhancer indicate substitution with GFP coding sequence. NC1.3 and NC1.4 contain the core region of the enhancer NC1 (dashed box). (B–G, I–L) Whole mount dorsal view of examples of the different constructs and effects of mutations. eGFP expression (green) indicates enhancer activity in electroporated (red) cells. (B,C) NC1.1 directs expression of eGFP in the same pattern as full-length NC1. (D,E) NC1.2 drives weak expression of eGFP in cranial neural crest. (F,G) NC1.1 M7 only drives weak eGFP expression in a small number of cells in no discernable pattern. (I, J) NC2.9 directs expression of eGFP in the same pattern as full-length NC2. (K,L) NC2.9 M11, containing a deletion of the Zic site, fails to drive eGFP in the neural crest. nc: neural crest, R: rhombomere.
Figure 5
Figure 5. Knockdown of several putative inputs affects activity of NC1 and NC2 enhancers.
(A,F) The left side of each embryo was electroporated with control morpholino plus a construct containing either NC1 (A–E) or NC2 (F–J) driving Cherry. The right side of each embryo was electroporated with the same construct plus indicated antisense morpholino. Knockdown of Pax7 (B), Msx1+2 (C), or Ets1 (D) results in dramatic loss of NC1.1 activity, whereas Zic1 morpholino (E) has no effect. In contrast, Pax7 (G), Msx1/2 (H) and Zic1 (J) have a strong effect on activity of the NC2 enhancer in the trunk, while Ets1 has no effect (I). (K, L) Percentage of embryos that showed either mild or strong reduction of Cherry expression on the side electroporated with antisense morpholino.
Figure 6
Figure 6. Effect of morpholino-mediated knockdown and chromatin immunoprecipitation.
(A–I) Effect of morpholino-mediated knockdown of Pax7, Msx1/2, Ets1 and Zic1 on the endogenous expression of FoxD3. Knockdown of Pax7 (A), Msx1/2 (B) and Ets1 (C) results in reduction of endogenous expression of FoxD3 in the cranial neural crest. In trunk neural crest cells, electroporation of morpholinos to Pax7 (D), Msx1/2 (E) and Zic1 (F) results in reduction of FoxD3 expression. (G–J) Chromatin immunoprecipitation shows direct binding of Pax7, Ets1, Msx1 to the NC1 enhancer and Zic1 to NC2 enhancer. Immunoprecipitation of chromatin isolated from the midbrain dorsal neural tubes of chicken embryos using Pax7 (G), Msx1 (H) or Ets1 (I) antibodies was used in site-specific QPCR, with primers designed to amplify fragments within the NC1 region. The results reveal significant enrichment of the NC1 region amplicon, expressed as a percent of the total input chromatin, compared to negative control regions. (J) Immunoprecipitation of chromatin isolated from the trunk dorsal neural tubes of chicken embryos using Zic1 antibody reveals direct binding of this transcription factor to the NC2 enhancer compared to negative control regions.
Figure 7
Figure 7. Model for differential regulation of FoxD3 in cranial and trunk neural crest cell populations.
FoxD3 expression is controlled by distinct inputs and enhancers at different axial levels. Ets1 is critical for activating NC1 at cranial levels, while the neural plate border specifier, Zic1, is required for NC2 activity in the trunk. Both Zic1 and Ets1 transcription factors appear to act in concert with Pax7 and Msx1/2 that are expressed along the entire neural axis.
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References

    1. Sauka-Spengler T, Barembaum M (2008) Gain- and loss-of-function approaches in the chick embryo. Methods Cell Biol 87: 237–256. - PubMed
    1. Betancur P, Bronner-Fraser M, Sauka-Spengler T (2010) Assembling neural crest regulatory circuits into a gene regulatory network. Annu Rev Cell Dev Biol 26: 581–603. - PMC - PubMed
    1. Meulemans D, Bronner-Fraser M (2004) Gene-regulatory interactions in neural crest evolution and development. Dev Cell 7: 291–299. - PubMed
    1. Basch ML, Bronner-Fraser M, Garcia-Castro MI (2006) Specification of the neural crest occurs during gastrulation and requires Pax7. Nature 441: 218–222. - PubMed
    1. Betancur P, Bronner-Fraser M, Sauka-Spengler T (2010) Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest. Proc Natl Acad Sci U S A 107: 3570–3575. - PMC - PubMed

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