Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart

Abstract

Hearts of mice lacking Isl1, a LIM homeodomain transcription factor, are completely missing the outflow tract, right ventricle, and much of the atria. isl1 expression and lineage tracing of isl1-expressing progenitors demonstrate that Isl1 is a marker for a distinct population of undifferentiated cardiac progenitors that give rise to the cardiac segments missing in isl1 mutants. Isl1 function is required for these progenitors to contribute to the heart. In isl1 mutants, isl1-expressing progenitors are progressively reduced in number, and FGF and BMP growth factors are downregulated. Our studies define two sets of cardiogenic precursors, one of which expresses and requires Isl1 and the other of which does not. Our results have implications for the development of specific cardiac lineages, left-right asymmetry, cardiac evolution, and isolation of cardiac progenitor cells.

Figure 1. Whole-Mount mRNA In Situ, Histological, and Scanning EM Analyses of Wild-Type Littermates and isl1 Homozygous Mutants

Wild-type littermates are indicated by +/+ and homozygous null isl1 mice are indicated by −/−. (A–H) Embryos of ED9.5 (20–21 somite pairs) were whole-mount stained with digoxigenin-labeled riboprobes for MLC2a (A and C) or MLC2v (E and G). Left, frontal, and right views of each embryo are shown in respective panels. Corresponding sections are also shown, progressively from anterior to posterior (B, D, F, and H). Results of this analysis demonstrated aberrant cardiac morphology in −/− mice and overall reduction in cardiac tissue. (I–P). Specific probes utilized are indicated below each panel, and specific regions of interest are indicated by white arrows. (I and M) Front view of ED 9.5 (23 somite pairs) embryo hybridized with a probe for tbx5 mRNA. (J and N) Front view of ED8.5 (11 somite pairs) embryo hybridized with a probe for EHand. (K and O) Right side view of ED9.5 (23 somite pairs) embryo hybridized with a probe for fgf10. (L and P) Right side view of ED9.5 (23 somite pairs) embryo hybridized with a probe for wnt11. (Q–T) Scanning electron microscopy analysis was consistent with these findings, as shown. Frontal views of embryos of ED8.75 (12 somite pairs) (Q and S) and ED9.5 (22 somite pairs) (R and T) are shown. Note that in isl1 mutants at 12 somite pairs (S), heart primordia closely resemble those of wild-type embryos of 5–6 somite pairs (Kaufman, 1999). At the 22 somite pairs stage, isl1 mutant hearts (T), when compared to those of wild-type littermates (R), appear to be lacking outflow tract and right ventricular segments, consistent with marker analysis. Atrial (A) and ventricular (V) segments of mutant heart shown in (T) have been labeled according to results of marker analysis. Abbreviations: A, atria; LA, left atria; LV, left ventricle; OT, outflow tract; RA, right atria; RV, right ventricle; SV, sinus venosus; and V, ventricle.

Figure 2. Double and Single Whole-Mount In Situ Analyses of Embryos Stained for isl1 and MLC2a mRNAs

(A–E) Embryos were doubly stained for isl1 mRNA (green) and MLC2a mRNA (red), or singly stained for Isl1 or MLC2a, as indicated to the left of each panel. (A) ED7; (B) ED7.5; (C) ED8.0 (2 somite pairs); (D) ED8.25 (5 somite pairs); (E) ED8.5 (8 somite pairs). (F–I) Sections of embryos doubly stained for isl1 mRNA and MLC2a mRNA. (F and G) isl1 mRNA was detected with BCIP, which resulted in light green, and MLC2a mRNA was detected with fast red, which resulted in red. (H and I) isl1 mRNA was detected with magenta phos-tet red to give purple, and MLC2a was detected with BCIP/ferricyanide/ferrocyanide to give dark blue (MLC2a). For technical details, see Experimental Procedures. Stages of sectioned embryos correspond to those shown in whole mount. (F) ED 7.5; (G) ED8.0 (2 somite pairs); (H) ED8.25 (5 somite pairs); (I) ED8.5, (8 somite pairs). isl1 and MLC2a mRNAs are nonoverlapping, with isl1 mRNA being expressed in foregut endoderm (short arrow) and in splanchnic mesoderm (thin long arrow) and MLC2a mRNA being expressed in differentiated myocardium (wide long arrow). (J–L) Staining for isl1 mRNA was performed on embryos of ED10 (26 somite pairs), shown in whole mount from the right (J) and from the left (K), and corresponding sections from anterior, mid, and posterior levels shown in (L). Isl1 mRNA continues to be expressed in foregut endoderm (short arrow) and in splanchnic mesoderm (thin long arrow) but is not observed in myocardium. Abbreviations: A, atria; LV, left ventricle; OT, outflow tract; RV, right ventricle.

Figure 3. Lineage Analysis of isl1-Expressing Cells during Early Heart Development

CMV β-actin-lacZ indicator mice (Zinyk et al., 1998) were crossed to isl1-cre mice (Srinivas et al., 2001) to visualize the fate of isl1-expressing cells by X-gal staining. Progeny were harvested at different embryonic stages and stained with X-gal. Whole embryos are shown progressively in left, frontal, and right views. Corresponding sections are shown, progressing from anterior to posterior at (A and B) ED8.5 (10 somite pairs); (C and D) ED9.0 (13 somite pairs); and (E and F) ED10.25 (27 somite pairs). The arrow in (F) indicates X-gal-positive cells within the endocardium of the outflow tract. Abbreviations: A, atria; OT, outflow tract; LV, left ventricle; RV, right ventricle; SV, sinus venosus.

Figure 4. Detection of isl1-Expressing Cells by Whole-Mount In Situ Analysis of isl1 mRNA in Wild-Type and isl1 Homozygous Mutants

Wild-type littermates are indicated by +/+, and homozygous null isl1 mice are indicated by −/−. Whole-mount views are shown from left and right, with corresponding sections below, progressing from anterior to posterior. (A, D, G, and J) ED8.5 (8 somite pairs); (B, E, H, and K) ED9.0 (15 somite pairs); (C, F, I, and L) ED9.75 (25 somite pairs). The arrows in each figure indicate regions where, in the wild-type, a greater number of cells is present than in the mutants. Stars in (I) and (L) draw attention to the absence of isl1 expression in motor neurons in isl1 mutants, reflecting absence of motor neurons (Pfaff et al., 1996).

Figure 5. BrdU Labeling, TUNEL Analysis, and Lineage Labeling in the isl1 Mutant Background

Wild-type littermates are indicated by +/+, and homozygous null isl1 mice are indicated by −/−. (A–D) Sections from ED8.75 (12 somite pairs) BrdU-labeled embryos shown at low (A and C) and higher (B and D) magnification. Arrows indicate splanchic mesoderm, where proliferation indices were most strongly adversely affected in mutant embryos; unnotched arrowheads indicate foregut endoderm, which also exhibited lower proliferative activity in isl1 mutants relative to control littermates; notched arrowheads indicate endocardium, where proliferation was unaffected in isl1 mutants relative to control embryos. (E–N) TUNEL analysis performed on sections from embryos of ED8.75 (12 somite pairs) (E–J) and ED9.5 (22 somite pairs) (K–N). (E–G, K, and L) DAPI staining to visualize nuclei. (H–J, M, and N) Fluorescein-labeled dUTP was utilized to visualize nicked DNA. (O–R) Lineage analysis of isl1-expressing cells in an isl1 mutant background. Isl1^+/−; ROSA26-lacZ indicator doubly heterozygous mice were crossed to isl1-cre mice to obtain mice which were either heterozygous mutant or homozygous mutant for isl1 and carried both the isl1-cre and ROSA26-lacZ indicator alleles.

Figure 6. Whole-Mount In Situ Analyses to Detect BMP and FGF mRNAs in Wild-Type Littermates and isl1 Homozygous Mutants

Embryos of at least two stages were hybridized to probes for BMPs and FGFs. Wild-type littermates are indicated by +/+, and homozygous null isl1 mice are indicated by −/−. The mRNAs detected are shown below each relevant group of embryos. From left to right, the stages shown are (A and E) ED8.75 (11 somite pairs), ED9.5 (23 somite pairs); (B and F) ED8.5 (9 somite pairs), ED9.0 (16 somite pairs); (C and G) ED8.75 (12 somite pairs); (D and H) ED9.0 (15 somite pairs), ED9.5 (23 somite pairs); (I and L) ED9.0 (16 somite pairs); (J and M) ED8.5 (9 somite pairs), ED9.5 (21 somite pairs); (K and N) ED8.5 (8 somite pairs), ED9.5 (22 somite pairs). For each mRNA examined, there was a reduction in expression in isl1 mutants, as indicated by arrows (O–V). Section analyses of whole-mount embryos stained for BMP4, BMP7, fgf8, and fgf10. Notched arrows indicate pharyngeal endoderm, triangular arrowheads indicate splanchnic mesoderm, and solid arrows indicate outflow tract myocardium. (O and S) BMP4 mRNA expression at ED8.5 (left) and ED9.0 (right); (P and T) BMP7 mRNA expression at ED9.0; (Q and U) fgf8 mRNA expression at ED9.0; (R and V) fgf10 mRNA expression at ED8.5 (left) and ED9.5 (right).

Figure 7. Working Model of Early Heart Development

isl1-expressing cells are shown in green; MLC2a-positive cells derived from non-isl1-expressing progenitors are shown in red; MLC2a-positive cells derived from isl1-expressing progenitors are shown in purple. (A) Embryo of 3–4 somite pairs; (B) embryo of 6–8 somite pairs; (C) embryo of 11–13 somite pairs. From left to right, embryos are shown in frontal, then lateral views, followed by two cross-section views. Lines drawn through the frontal views indicate the anterior-posterior position of the corresponding sections (a–f). Arrows indicate the direction of migration of isl1-expressing progenitors. (D) Flow chart diagramming the role of Isl1 in heart formation.