GATA factors efficiently direct cardiac fate from embryonic stem cells
Harma K. Turbendian
1
Miriam Gordillo
1
Su-Yi Tsai
1
Jia Lu
0
Guoxin Kang
0
Ting-Chun Liu
1
Alice Tang
1
Susanna Liu
1
Glenn I. Fishman
0
Todd Evans
1
0
Leon H. Charney Division of Cardiology, New York University School of Medicine
,
New York, NY 10016
,
USA
1
Department of Surgery, Weill Cornell Medical College
,
New York, NY 10065
,
USA
SUMMARY The GATA4 transcription factor is implicated in promoting cardiogenesis in combination with other factors, including TBX5, MEF2C and BAF60C. However, when expressed in embryonic stem cells (ESCs), GATA4 was shown to promote endoderm, not cardiac mesoderm. The capacity of related GATA factors to promote cardiogenesis is untested. We found that expression of the highly related gene, Gata5, very efficiently promotes cardiomyocyte fate from murine ESCs. Gata5 directs development of beating sheets of cells that express cardiac troponin T and show a full range of action potential morphologies that are responsive to pharmacological stimulation. We discovered that by removing serum from the culture conditions, GATA4 and GATA6 are each also able to efficiently promote cardiogenesis in ESC derivatives, with some distinctions. Thus, GATA factors can function in ESC derivatives upstream of other cardiac transcription factors to direct the efficient generation of cardiomyocytes.
INTRODUCTION
The transcription factors required for cardiogenesis during normal
development include members of the NKX2, MEF2, HAND, SRF,
TBX and GATA transcription factor families. These proteins
regulate each other, functioning through stabilization of cardiogenic
transcriptional networks (Olson, 2006). Forced expression of
combinations of these transcription factors can direct differentiation
of cardiac fate. In particular, Gata4 has been implicated, as this gene
is essential for normal embryonic myocardial growth (Holtzinger
and Evans, 2005; Rojas et al., 2008), and embryos lacking both
Gata4 and the sister gene Gata6 display acardia (Zhao et al., 2008).
Forced expression of Gata4 with Tbx5 and the chromatin modulator
Baf60c (Smarcd3 Mouse Genome Informatics), can reprogram
somatic mesoderm to cardiac fate (Takeuchi and Bruneau, 2009),
and combined expression of Gata4 with Tbx5 and Mef2c was shown
to reprogram fibroblasts to cardiomyocytes (Ieda et al., 2010). Yet
cardiac reprogramming is inefficient, not highly reproducible (Chen
et al., 2012) and has not been adapted to the embryonic stem cell
(ESC) system, which can be used for generating unlimited cardiac
cells (Murry and Keller, 2008). Expression of Gata4 in ESCs drives
development of primitive endoderm, not cardiac cells (Fujikura et
al., 2002). We found previously that expression of Gata4 in a
defined window of ESC-derived embryoid body (EB) development
can enhance production of cardiac cells, but it does so indirectly by
inducing the formation of cardiac-promoting endoderm (Holtzinger
et al., 2010).
The Gata5 gene is under-studied compared with Gata4 or
Gata6, because the Gata5-null embryo does not display a cardiac
phenotype (Molkentin et al., 2000). However, in addition to cardia
*These authors contributed equally to this work
Author for correspondence ()
bifida, some gata5/faust zebrafish embryos showed a relative loss
of cardiomyocytes (Reiter et al., 1999). Subsequent studies in
zebrafish and Xenopus showed that gata5 has an essential but
redundant role (with gata6) in cardiac progenitor specification
(Holtzinger and Evans, 2007; Haworth et al., 2008).
Overexpression of gata5 in combination with smarcd3b generates
ectopic cardiac tissue in developing zebrafish embryos (Lou et al.,
2011). In comparison with fish, mouse embryos apparently better
compensate for loss of Gata5. However, Gata4/5 compound
mutant mice display progressively severe cardiac defects (thinned
ventricular wall, hypoplastic compact myocardium, abnormal
trabecular structures and endocardial cushion defects) secondary
to poor myocardial cell proliferation (Singh et al., 2010). Gata4/5
and Gata5/6 compound heterozygotes present outflow tract defects
that include double outlet right ventricle and ventricular septal
defects (Laforest and Nemer, 2011). Here, we tested the capacity
for Gata5 to program cardiac fate in murine ESC derivatives.
Using an engineered ESC line that allows conditional expression
of Gata5, we found that expression of this single factor efficiently
promotes abundant populations of beating and phenotypically
normal cardiac cells. This led us to investigate more closely why
Gata4 failed in previous studies to direct cardiac fate. We found
that, under defined culture conditions, Gata4 (and the other sister
gene, Gata6) are capable of similar, but not identical,
cardiacpromoting capacities.
MATERIALS AND METHODS
ESC line derivation and culture
The murine ESC line AinV18 (Kyba et al., 2002) was used to derive and
validate lines that allow conditional expression of Gata5 or Gata6,
essentially as described previously (Holtzinger et al., 2010), and wer (...truncated)