Abstract
Introduction: The transcription factor Islet-1 (Isl1) is expressed in cardiac mesodermal and neural crest (CNC) lineages during cardiogenesis. A pool of Isl1 + cells persist in the perinatal heart (Isl1 + CPCs), some of which are touted as residual second heart-field (SHF)- derived cardioblasts. However, direct lineage-tracing evidence, supporting a SHF over a CNC origin of Isl1 + CPCs, are lacking. Hypothesis: Isl1 + CPCs are of CNC and not SHF lineage. Methods: The Isl1-nLacZ, Wnt1-Cre;tdTomato and Wnt1::FlpE;RC::Fela mice, and iPSCs derived from Wnt1-Cre;tdTomato mice (iPSC Wnt1 ) were employed to lineage-trace Isl1 + CPCs. Results: Temporal analysis of Isl1-nLacZ embryos illustrated a transient, stage-specific reporter gene activity in mesendodermal and neuroectodermal cells. Particularly, at embryonic day (E)9.5, reporter gene activity (x-gal), was strong in the outflow tract (OFT), and exhibited a weak, spotty pattern in the heart. At E12.5, x-gal was strong in the neural tube (NT); reduced in the OFT; and undetectable in the heart. Isl1 immunohistochemistry (IHC) illustrated similar activity of x-gal with endogenous Isl1 expression. Importantly, x-gal remained permanently undetected in the heart. Isl1 IHC in E12.5 Wnt1-Cre;tdTomato and Wnt1::FlpE;RC::Fela embryos indicated that Isl1 + cells in the OFT and NT colocalized with Wnt1 reporter transgenes. At E18.5 and postnatal day 1 (PN1), Isl1 + cells were exclusively of Wnt1 lineage, indicating that Isl1 + CPCs are CNC- and not SHF-derived. Since CNCs minimally contribute cardiomyocytes in mammals, a presumptive full cardiomyogenic capacity of Isl1 + CPCs potentially contrasts with a CNC origin. To address this controversy, we differentiated iPSC Wnt1 toward the CNC lineage, using a previously established embryoid body (EB) differentiation protocol involving transient BMP antagonism. At ~EB-day 9, tdTomato + /Isl1 + CNCs emerged, which progressively differentiated into spontaneously beating, Nkx2.5 + cardiomyocytes. Conclusions: Our findings clarify that Isl1 + CPCs are of CNC and not SHF origin, and suggest a novel role of the mammalian CNC, as a contributor of long-lived myocardial progenitors that could be targeted for heart-related therapeutic purposes.
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