Identification and dissection of a key enhancer mediating cranial neural crest specific expression of transcription factor, Ets-1

Abstract

Neural crest cells form diverse derivatives that vary according to their level of origin along the body axis, with only cranial neural crest cells contributing to facial skeleton. Interestingly, the transcription factor Ets-1 is uniquely expressed in cranial but not trunk neural crest, where it functions as a direct input into neural crest specifier genes, Sox10 and FoxD3. We have isolated and interrogated a cis-regulatory element, conserved between birds and mammals, that drives reporter expression in a manner that recapitulates that of endogenous Ets-1 expression in the neural crest. Within a minimal Ets-1 enhancer region, mutation of putative binding sites for SoxE, homeobox, Ets, TFAP2 or Fox proteins results in loss or reduction of neural crest enhancer activity. Morpholino-mediated loss-of-function experiments show that Sox9, Pax7, Msx1/2, Ets-1, TFAP2A and FoxD3, all are required for enhancer activity. In contrast, mutation of a putative cMyc/E-box sequence augments reporter expression, consistent with this being a repressor binding site. Taken together, these results uncover new inputs into Ets-1, revealing critical links in the cranial neural crest gene regulatory network.

Keywords: Enhancer; Ets-1; Neural crest; Pax7; Sox9; TFAP2.

Fig. 1

Ets-1 is expressed in the dorsal neural tube of HH stage 8 embryos (A) and in the migrating crest of HH stage 10 (B) HH stage 12 (C) and HH stage 14 (D) chicken embryos. Arrows point to the Ets-1 expression in the otic pit.

Fig. 2

A. Using the UCSC genome browser, we identified conserved regions near the Ets-1 gene. Conserved regions were cloned into pTK vector and tested by electroporation for neural crest enhancer activity (A). One of these, ECR1 (red arrow) had enhancer activity. ECR1 drives GFP expression in the neural crest of HH stage 9 (B,B′), HH stage 10 (C,C′), HH stage 12 (D,D′) and HH stage 14 (E,E′) embryos. The rectangles in B, C, D, and E correspond to higher magnification images of the midbrain (B′), otic placode (C′), otic pit (D′) and otic vesicle (E′) respectively. Expression of GFP by ECR1 in HH stage 9 embryos (F) recapitulates the Ets-1 expression as seen by in situ hybridization (G). Similarly, expression of GFP by ECR1 in HH stage 11 embryos (H) recapitulates the Ets-1 expression as seen by in situ hybridization (I).

Fig. 3

Conservation of ECR1 using UCSC gene browser (A). The conserved regions are required for ECR1 enhancer activity. The minimal enhancer is depicted as a solid lie at the bottom. There are a number of transcription factor binding consensus sites conserved in the ECR1 region (B) as determined using the JASPAR database. The open rectangles correspond to SoxE consensus sequences whose mutation did alter enhancer activity.

Fig. 4

Putative transcription binding sites were mutated using fusion PCR and cloned into pTK vector. The constructs were co-electroporated with a pTK-ECR1 construct expressing Cherry. Mutations in the SoxE sites (B) reduced enhancer activity versus intact ECR1 driving Cherry expression (A). Mutations in the core Hox2 sequence (D) also reduced enhancer activity compared to control (C ). Mutations in the consensus Ets-1 sequence (F) decrease enhancer activity compared to controls (E). Similarly, the control Cherry construct (G) had higher enhancer activity compared to the TFAP2 consensus site mutation construct (H). Mutations in a Fox consensus sequence reduced neural crest GFP expression but not otic expression (I), compared to control (J). Mutations in a putative cMyc/E-box binding sequence increased enhancer activity in the vagal neural crest only (K) compared to controls (L).

Fig. 5

Morpholino oligos (MO) to transcription factors (right side) and control (left side) were co-electroporated with ECR1-pTK Cherry. MO to Sox9 (A) reduced ECR1 enhancer activity (B). A combination of Msx1 and Msx2 MO (C) reduced enhancer activity in the neural crest (D) compared to the control MO. The Pax7 MO electroplated side (E) had reduced enhancer activity compared to the control MO side (F). FoxD3 MO (G) also reduced the ECR1 enhancer activity compared to the control MO (H). Similarly, the Ets-1 MO (I) reduced enhancer activity versus the control MO (J). The MO to TFAP2A (K) reduced enhancer activity compared to the control MO side (L).

Fig. 6

Schematic of direct interactions in the cranial neural crest on neural crest specifier genes (solid lines) as previously described (Betancur et al., 2010B; Simões-Costa et al., 2012) and the proposed interactions on Ets-1 as determined by morpholino knock-down of putative inputs and effects on Ets-1 enhancer expression (dashed lines).