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(Circulation Research. 2007;101:971.)
© 2007 American Heart Association, Inc.

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Isl1 Expression at the Venous Pole Identifies a Novel Role for the Second Heart Field in Cardiac Development

Brian S. Snarr, Jessica L. O’Neal, Mastan R. Chintalapudi, Elaine E. Wirrig, Aimee L. Phelps, Steven W. Kubalak, Andy Wessels

From the Department of Cell Biology and Anatomy (B.S.S., J.L.O., M.R.C., E.E.W., A.L.P., S.W.K., A.W.) Medical University of South Carolina, Charleston; and the Department of Pediatrics (S.W.K., A.W.), Division of Pediatric Cardiology, Medical University of South Carolina, Charleston.

Correspondence to Andy Wessels, PhD, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425. E-mail wesselsa{at}musc.edu

	Abstract

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The right ventricle and outflow tract of the developing heart are derived from mesodermal progenitor cells from the second heart field (SHF). SHF cells have been characterized by expression of the transcription factor Islet-1 (Isl1). Although Isl1 expression has also been reported in the venous pole, the specific contribution of the SHF to this part of the heart is unknown. Here we show that Isl1 is strongly expressed in the dorsal mesenchymal protrusion (DMP), a non–endocardially-derived mesenchymal structure involved in atrioventricular septation. We further demonstrate that abnormal development of the SHF-derived DMP is associated with the pathogenesis of atrioventricular septal defects. These results identify a novel role for the SHF.

Key Words: Isl1 • Nkx2.5 • dorsal mesenchymal protrusion • second heart field • venous pole • vestibular spine

	Introduction

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The myocardium of the right ventricle (RV) and outflow tract (OFT) is derived from mesodermal cells in the second heart field (SHF).^1–3 SHF cells have been shown to express the transcription factor Islet1 (Isl1).⁴ Isl1 is necessary for normal RV and OFT development.^4,5 In Isl1-deficient mice, abnormalities are also found in structures at the venous pole.^4,6 Although atrial Isl1 expression at the venous pole has been reported,^4,5 a specific atrial role for Isl1 in development has not been established.

	Materials and Methods

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Tie2-Cre and Rosa26R mice, X-Gal staining, and 3-dimensional reconstruction techniques have been described.⁷ Immunohistochemistry was performed with antibodies recognizing Isl1 (39.4D5;DSHB), Nkx2.5 (H-114; Santa Cruz), sarcomeric actin (A2172;Sigma), and MLC2a. Some experiments were performed on previously collected Ts16 specimens.⁸ Detailed information is available in the supplemental materials (available online at http://circres. ahajournals.org).

	Results and Discussion

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We studied Isl1 protein expression in the developing mouse between embryonic day (ED)11 and ED14.5. At the earliest stage, Isl1 expression was, as previously described,⁵ found in the OFT (Figure 1A and 1C), foregut endoderm (Figure 1C), and splanchnic mesoderm (Figure 1A, 1C, 1D, and 1F). Isl1 expression was also seen in cardiac tissues at the venous pole (Figure 1D and 1F). Three-dimensional reconstructions illustrate that these Isl1 expression domains are contiguous (Figure 1G through 1J), demonstrating the contribution of the SHF to both poles of the heart.

View larger version (91K): [in this window] [in a new window]   Figure 1. Isl1 expression in the dorsal mesenchymal protrusion at ED11.0 reveals a specific contribution of the secondary heart field to the venous pole. A and D, Transverse sections immunostained for Isl1. C and F, Higher magnifications of boxed areas in A and D. B and E, Sister sections to C and F, respectively, stained for MLC2a. Isl1 expression at the arterial pole (A, C) includes the splanchnic mesoderm, OFT myocardium (black arrowheads), endocardium (white arrowheads), and some cushion mesenchymal cells. Isl1 expression at the venous pole (D,F) is seen in the DMP and in a subset of myocytes (white arrows). G through J, 3D-AMIRA reconstructions of Isl1 expression at ED11.0. G through H, Posterior view of the heart showing Isl1 expression in nonmyocardial (blue) and myocardial (purple) SHF cell populations, demonstrating continuity of SHF cells from arterial to venous poles. I through J, Reconstructions showing the relationship between the SHF-derived cells and the mesenchymal AV and OFT cushions. AS indicates aortic sac; AVC, atrioventricular cushions; CAP, mesenchymal cap on primary atrial septum; DA, dorsal aorta; DMP, dorsal mesenchymal protrusion; FG, foregut; iAVC, inferior atrioventricular cushion; LA, left atrium; LV, left ventricle; NT, neural tube; OFT, outflow tract; OFTC, outflow tract cushions; RA, right atrium; RV, right ventricle; sAVC, superior atrioventricular cushion; Sp-meso, splanchnic mesoderm.

Isl1-expression at the venous pole was observed in distinct subsets of atrial myocytes (Figure 1D and 1F) and, most notably, in a discrete set of mesenchymal cells that was found to be associated with the dorsal mesocardium (Figure 1D through 1J). This particular mesenchyme was first mentioned by His, who referred to it as the "spina vestibule",^9,10 describing it as "ein besonderes bindegewebiges Gebilde" (a special[ized] connective tissue), and later by others using a variety of different names.¹¹ In recent years, several studies have rekindled interest in the role of this mesenchyme in cardiovascular development.^7,10–12 While studying its role in the development of the human heart, we came to the conclusion that the term "dorsal mesenchymal protrusion (DMP)", most accurately described the anatomical features of this intracardiac extension of the splanchnic mesenchyme into the atrial cavities.¹¹ Thus, as we continued to gain more insight into the matter we opted to use this terminology.^7,12 We acknowledge, however, that "atrial spine", "vestibular spine", and "spina vestibuli" are also commonly, and legitimately, used in other articles to describe this mesenchymal population.

We previously showed that the DMP is a non–endocardially-derived mesenchymal structure that forms an integral component of the AV mesenchymal complex, thereby playing an important role in cardiac septation.^7,10,13 Historically, septal defects in the AV canal region have been associated with abnormal development of the AV cushions, hence the term "endocardial cushion defects", a term still frequently used.¹⁴ Recent studies suggest that impaired development of the DMP is involved in the pathogenesis of atrial and AV septal defects,^7,12,13 particularly in Downs Syndrome patients and in the Trisomy 16 (Ts16) mouse model for this condition.^15,16 The identification of this structure as an Isl1-expressing SHF derivative prompted us to revisit its involvement in AV canal defects of the Ts16 mouse.¹⁶ Examination of ED12 Ts16 mouse embryos with AV canal defects confirmed that the DMP was severely underdeveloped. Virtually no Isl1-positive cells were found in the region where it normally develops (Figure 2D, asterisk). Although we still know little about the molecular mechanisms that lead to normal DMP development, we believe that these results provide the first evidence for a specific role for the SHF in AV septation.

View larger version (124K): [in this window] [in a new window]   Figure 2. Impaired development of the SHF-derived DMP is involved in the etiology of AV canal defects. (A-D) sections of ED11.0 wild-type (A, C) and ED12.0 Ts16 (B, D) specimens stained for MLC2a (A, B) and Isl1 (C, D). In the WT heart, the Isl1-expressing DMP extends into the heart (A, C) flanking the developing pulmonary vein to the right (white arrows). In the Ts16 specimen, this Isl1-positive mesenchyme is absent. Note the normal expression of Isl1 in other components of the Ts16 heart (black arrows in D). For abbreviations see Figure 1.

After completion of septation, the DMP largely becomes myocardial, forming the inferior muscular rim at the base of the atrial septum.^7,13 To clarify whether muscularization of the DMP results from ingrowth of flanking myocardium or from a mesenchymal-to-myocardial transition, we examined whether Isl1-expressing SHF cells are undergoing myocardial differentiation, using Nkx2.5, a transcription factor commonly associated with this event.

In the developing mouse heart, Nkx2.5 is widely expressed in the atrial and ventricular myocardium (Figure 3A through 3C). Most, if not all, cells in the DMP at ED11.0 express Isl1 (Figure 3E). Nkx2.5 expression, however, is limited to the flanking atrial myocardium (Figure 3F). By ED13.0, as the DMP is undergoing its myocardial transition (Figure 3G), many cells still express Isl1 (Figure 3H and 3J). A significant number of cells now also express Nkx2.5 (Figure 3I through 3J). After completion of muscularization (ED14.5), few, if any, DMP derivatives still expressed Isl1. The myocardium in these tissues was, however, expressing Nkx2.5 (Figure 3K through 3M). The expression profiles of Isl1 and Nkx2.5 in the DMP throughout muscularization demonstrate that this event occurs by a mesenchymal-to-myocardial differentiation of SHF-derived DMP cells.

View larger version (130K): [in this window] [in a new window]   Figure 3. The Isl1 and Nkx2.5 DMP expression profile demonstrates a mesenchymal-to-myocardial differentiation of SHF-derived DMP cells. A through C, Sections of an ED11.0 heart at the level of the AV junction, stained for sarcomeric actin (A), Isl1 (B), and Nkx2.5 (C). None of these antigens are expressed in the AV cushion mesenchyme, and, in this part of the heart, Isl1 is not seen in any myocardial or mesenchymal structures (B). D through F, High-magnification images of the venous pole at ED11.0, stained for sarcomeric actin (D), Isl1 (E), and Nkx2.5 (F). Note the strong expression of Isl1 and absence of Nkx2.5 from the core of the DMP. G, At ED13.0 the DMP (boxed region) is undergoing muscularization. Immunofluorescent colabeling of sister sections (H-J) with Isl1 (green in H and J) and Nkx2.5 (red in I and J) shows that at this stage many SHF-derived cells coexpress Isl1 and Nkx2.5 (yellow arrows in H through J). Cells that exclusively express Isl1 are identified by white arrows, and cells that exclusively express Nkx2.5 are indicated by black arrows. The DMP at ED14.5 after completion of muscularization (K, L, M). Sister sections were stained for sarcomeric actin (K), Isl1 (L), and Nkx2.5 (M). The muscularized DMP now predominantly expresses Nkx2.5. AS indicates atrial septum; LVV, left venous valve; meso, mesoderm; PAS, primary atrial septum; PuV, pulmonary vein; RVV, right venous valve.

In conclusion, our studies confirm previous notions that the SHF contributes to the cardiac venous pole¹⁷ and shows that within this region, the DMP is an Isl1-expressing derivative of the SHF, playing a crucial role in AV development. This new insight into the significance of the SHF may provide an explanation for the atrial and atrioventricular septation defects observed in Isl1 mutant mice^4,6 and in mice carrying perturbations in other SHF-associated genes.¹⁸ Futhermore, it will form the base for further studies on the development of the DMP and its involvement in the etiology of congenital heart disease.

	Acknowledgments

 
Sources of Funding

This work was supported by NIH Grant C06 RR018823 and C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources, American Heart Association Grant-in-aid 0655530U (to B.S.S., A.W.), NIH-T-32-HL07260 (to B.S.S.), NIH-R01-HL084285 (to A.W., E.E.W.), NIH-R01-HL083116 (to S.K.) and the South Carolina Center of Biomedical Research Excellence (COBRE) NIH-NCRR-P20-RR016434 (to A.W.).

Disclosures

None.

	Footnotes

 
Original received August 17, 2007; revision received October 2, 2007; accepted October 4, 2007.

	References

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 101/10/971    most recent CIRCRESAHA.107.162206v2 CIRCRESAHA.107.162206v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Snarr, B. S. Articles by Wessels, A. Search for Related Content PubMed PubMed Citation Articles by Snarr, B. S. Articles by Wessels, A. Related Collections Animal models of human disease Cardiac development