Isl1Cre reveals a common Bmp pathway in heart and limb development
Lei Yang
0
Chen-Leng Cai
0
Lizhu Lin
0
Yibing Qyang
0
Christine Chung
0
Rui M. Monteiro
0
Christine L. Mummery
0
Glenn I. Fishman
0
Anna Cogen
0
Sylvia Evans
)
0
0
MATERIALS AND METHODS Generation of mutant mice Floxed Bmpr1a/Alk3 mice were kindly provided by Richard Behringer (The University of Texas, MD Anderson Cancer Center). Isl1Cre/
A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 (Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor, Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo.
INTRODUCTION
The frequency of human congenital diseases presenting with heart
and limb anomalies is at minimum 1 out of 5000 live births (Wilson,
1998). A cardiomelic developmental field has been postulated based
on a number of observations, including a significant positive
association of limb defects with heart defects in more than 100
Mendelian disorders (Wilson, 1998). Additionally, fetal autopsy
revealed that 57% of limb defects were coincident with heart defects,
and that heart and limb anomalies coexisted in 81% of partial
aneuploidies (Barr et al., 1994). Also of note, ten out of twelve well
characterized teratogenic syndromes in humans display coincident
heart and limb anomalies (Gorlin and Gorlin, 1990).
Identification of the transcription factor TBX5 as a disease gene
in prototypical heart-limb syndrome I, Holt-Oram syndrome, has
resulted in identification of a common genetic pathway affecting
both heart and limb (Basson et al., 1997; Basson et al., 1999). A
number of heart-limb syndromes, including Holt-Oram syndrome,
are characterized by cardiac arrhythmias (Bell, 1951; Ruiz de la
Fuente and Prieto, 1980; Silengo et al., 1990; Sinkovec et al., 2005;
Temtamy and McKusick, 1978). In the case of Holt-Oram
syndrome, a series of elegant experiments have demonstrated that
the gap junction protein connexin40 is a direct downstream target of
Tbx5 both in the heart and in the limb, accounting for conduction
system anomalies in the heart and growth defects in the limb
(Basson et al., 1999). Another t-box transcription factor, TBX3, is
expressed in developing heart and limb, and is mutated in
ulnarmammary syndrome (UMS) (Davenport et al., 2003). Limb
deformities in UMS patients have been associated with cardiac
defects, including ventricular septal defects, in a subset of patients
(Schinzel et al., 1987) (Craig Basson, personal communication).
Our lab has recently identified a subset of undifferentiated cardiac
progenitors which is marked by expression of a LIM-homeodomain
protein, islet 1 (Isl1) (Cai et al., 2003). Isl1 expression is
extinguished as the progenitors migrate into the forming heart.
Intriguingly, while performing fate mapping studies with an
Isl1Cre/+ mouse line generated by a knockin into the endogenous
Isl1 locus (see Materials and methods), we observed a similar
paradigm for hindlimb progenitors. Isl1 mRNA is highly expressed
in lateral mesoderm at the site where the hindlimb bud originates.
Fate mapping with Isl1Cre/+ and an R26R-lacZ reporter (Soriano,
1999), revealed that Isl1-expressing progenitors migrate into the
hindlimb bud to contribute a substantial proportion of mesodermal
cells to the limb bud, in a posterior to anterior gradient. Our results
reveal that Isl1 marks both heart and hindlimb progenitors,
suggesting potential common genetic pathways downstream of Isl1,
which could be involved in heart-limb syndromes.
To investigate common pathways in heart and limb, we have
examined the requirement for Bmp signaling utilizing Isl1Cre/+ to
ablate the Type1 Bmp receptor, Bmpr1a in early progenitors.
Ablation of the receptor mitigates issues of ligand redundancy
during heart and limb formation (Dudley and Robertson, 1997;
Katagiri et al., 1998; Lyons et al., 1995; Schneider et al., 2003).
Results of our analysis reveal novel requirements for Bmp signaling,
and common downstream targets for Bmp signaling in heart and
limb, one of which is a limb disease gene also likely to play a critical
role in heart development, Tbx3.
produce doubly heterozygous Isl1Cre/+;Bmpr1a floxed/null mice. The (...truncated)