ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

Abstract

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations.

Fibroblast growth factor 10 gene regulation in the second heart field by Tbx1, Nkx2-5, and Islet1 reveals a genetic switch for down-regulation in the myocardium

“…BAC deletion analysis shows that this regulatory region is necessary, as well as sufficient, for directing expression to these cardiac progenitor cells. This is distinct from a recently described sequence in the first intron of the human FGF10 gene, which was shown to bind Islet1 in extracts of embryonic hearts but did not direct robust cardiac/ SHF expression in vivo (30). The element that we describe is required for expression in the pharyngeal mesoderm of the SHF but not in the mesodermal core of the first two pharyngeal arches, which depends on a −127/−62-kb region of the Fgf10 gene, as well as the −62/+138-kb region.…”

Characterization of a novel Fibroblast growth factor 10 (Fgf10) knock-in mouse line to target mesenchymal progenitors during embryonic development

“…For RNA preparation, whole embryos were used at E11.5 (n = 5) and E13.5 (n = 3) whereas lungs were used at E18.5 (n = 7), P2 (n = 2), P12 (n = 4) and P161 (n = 3). gene [26]. Altogether, these data suggest the presence of key regulatory elements in intron 1 critical for the maintenance of Fgf10 expression over time.…”

Expression of Isl1 during mouse development

“…Isl1 was originally identified as a protein that can bind to an insulin gene enhancer and regulate its expression (Karlsson et al, 1990). Isl1 regulates gene expression by binding of its homeodomain to AT-rich consensus element containing a core TAAT motif (YTAATGR) (Karlsson et al, 1990; Leonard et al, 1992; Wang and Drucker, 1995; Lien et al, 1999; Dodou et al, 2004; Takeuchi et al, 2005; Liu et al, 2011; Golzio et al, 2012). …”