Dynamic and Differential Regulation of Stem Cell Factor FoxD3 in the Neural Crest Is Encrypted in the Genome

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.

Establishing neural crest identity: a gene regulatory recipe

“…Neural crest expression of FoxD3 is regulated by the neural plate border specifiers Pax3/7 and Msx1, which directly bind to two regulatory regions that drive differential expression in the cranial and trunk neural crest cells of the chick embryo (Simoes-Costa et al, 2012). Moreover, Pax3/7 and Msx1 are also required for the expression of Ets1 (Barembaum and Bronner, 2013), highlighting their importance during the early steps of neural crest specification.…”

“…In X. laevis, for example, Zic1 together with Pax3/7 (Milet et al, 2013) bind directly to the Snai1 and Snai2 promoters (Plouhinec et al, 2014). They also directly regulate FoxD3 expression in the chick trunk neural crest (Simoes-Costa et al, 2014). Furthermore, experiments in the lamprey suggest that the requirement of Pax3/7, Msx1 and Zic1 in neural crest specification is evolutionarily conserved, as the knockdown of these three neural plate border specifiers results in loss of both FoxD and SoxE1 expression (Sauka-Spengler et al, 2007).…”

“…Such interactions are difficult to parse out given that they take place in a short period of time as the cells quickly transition to the next regulatory state (Nikitina et al, 2008). A few examples of direct regulatory interactions between neural crest specifier genes include the cranial-specific control of FoxD3 by Ets1 (Simoes- Costa et al, 2012). Nevertheless, it is clear that positive regulatory loops result in the consolidation of the neural crest specifier regulatory state.…”

“…However, cis-regulatory analysis reveals important differences between the GRNs of the premigratory and migratory states. For instance, FoxD3 expression in the premigratory cranial neural crest is controlled by an early enhancer, NC1, which initiates FoxD3 expression in the newly closed cranial neural tube but quickly shuts down as cells delaminate (Simoes-Costa et al, 2012). Maintenance of FoxD3 expression in migratory cells is accomplished by the activation of a different enhancer (NC2) (Simoes-Costa et al, 2012) that requires distinct regulatory inputs.…”

Prdm1a directly activates foxd3 and tfap2a during zebrafish neural crest specification

“…Studies in chick and mouse suggest that the role of foxd3 in NCC development is highly conserved (Kos et al, 2001;Teng et al, 2008) and that foxd3 is required for NCCs to maintain their pluripotency (Mundell and Labosky, 2011). Recent work in chick embryos has further identified genomic enhancers that drive Foxd3 expression specifically in the developing neural crest and determined potential transcription factors that bind to and regulate these regions (Simões-Costa et al, 2012); however, the direct regulation of foxd3 in zebrafish NCCs has not previously been studied. Another gene known to be upstream of foxd3 in zebrafish is tfap2a, a well-known neural crest specifier.…”