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.