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Isl1 is upstream of sonic hedgehog in a pathway required for cardiac morphogenesis
Developmental Biology 295 (2006) 756 – 763 www.elsevier.com/locate/ydbio
Isl1 is upstream of sonic hedgehog in a pathway required for cardiac morphogenesis Lizhu Lin a,b,1 , Lei Bu a,b,c,1 , Chen-Leng Cai a,b , Xiaoxue Zhang a,b , Sylvia Evans a,b,⁎ a
c
Skaggs School of Pharmacy, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA b Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street Charlestown, MA 02129, USA Received for publication 3 January 2006; revised 30 March 2006; accepted 31 March 2006 Available online 7 April 2006
Abstract The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in both foregut endoderm and cardiogenic mesoderm and is required for earliest stages of heart development. Here, we report that isl1 is also required upstream of Shh. We find that, in isl1 null mice, Sonic hedgehog (Shh) is downregulated in foregut endoderm. Shh signals through the unique activating receptor smoothened (Smo). To investigate the role of hedgehog signaling in the isl1 domain, we ablated smo utilizing isl1-cre. Isl1-cre;smo mutants exhibit cardiovascular defects similar to those observed in Shh null mice, defining a spatial requirement for hedgehog signaling within isl1 expression domains for aortic arch and outflow tract formation. Semaphorin signaling through neuropilin receptors npn1 and npn2 is required for aortic arch and outflow tract formation. We find that expression of npn2 is downregulated in isl1-cre;smo mutants, suggesting an isl1/Shh/npn pathway required to affect morphogenesis at the anterior pole of the heart. © 2006 Elsevier Inc. All rights reserved. Keywords: Isl1; Cardiac morphogenesis; Smoothened, Neuropilin2; Outflow tract; Aortic arch artery; Anterior pole of the heart
Introduction Ablation of the LIM homeodomain transcription factor islet1 (isl1) results in embryonic growth arrest at E9.5 and lethality by E10.5 (Cai et al., 2003; Pfaff et al., 1996). Isl1 expression marks an actively proliferating population of cardiac progenitors which will contribute a majority of myocardial cells to the developing heart, including most or all cells in the outflow tract and right ventricle, a majority of cells in the atria, and some cells within the left ventricle (Cai et al., 2003). Recent lineage studies demonstrate two myocardial lineages which contribute to embryonic heart, a primary lineage which differentiates firs, and a secondary lineage which differentiates later, with isl1 marking the latter (Cai et al., 2003; Meilhac et al., 2004). The anterior or secondary heart field which will contribute to the ⁎ Corresponding author. Skaggs School of Pharmacy, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Fax: +1 858 534 4810. E-mail address: [email protected] (S. Evans). 1 These authors contributed equally to this manuscript.
0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.03.053
outflow tract and right ventricle is a subset of the isl1 expressing population (Kelly, 2005; Waldo et al., 2005). Isl1 is required within this population for proliferation, survival and migration into the forming heart (Cai et al., 2003). Isl1 mutant hearts are lacking outflow tract, right ventricle, and have a severe reduction in atrial tissue. Early embryonic lethality of isl1 mutants has precluded an examination of the potential role of isl1 in later events of cardiac morphogenesis. A number of studies have demonstrated that aberrant proliferation in the secondary heart field can result in aortic arch artery and outflow tract anomalies (Hutson and Kirby, 2003; Xu et al., 2005). The prominent role of isl1 in the secondary heart field suggests that isl1 may be required to affect these later aspects of cardiac morphogenesis. Isl1 is expressed in ventral neural tube, foregut endoderm and cardiogenic mesoderm (Cai et al., 2003; Pfaff et al., 1996). Shh is expressed in the notochord, floorplate, and ventral foregut endoderm (Zhang et al., 2001). During our analysis of isl1 null mice, we observed that the ligand Sonic hedgehog (Shh) was selectively downregulated in foregut endoderm, but not in notochord or floorplate. Analysis of a global knockout of Shh has demonstrated a critical role in
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aortic arch artery and outflow tract morphogenesis (Garg et al., 2001; Meyers and Martin, 1999; Washington Smoak et al., 2005). Global knockout of smo results in severe embryonic patterning defects and early embryonic lethality by E10.5 (Zhang et al., 2001). Hearts of smo mutants exhibit aberrant looping and are severely malformed. To investigate whether downregulation of Shh signaling in the isl1 domain would have consequences for heart formation, we ablated the unique activating hedgehog receptor smoothened (Smo) utilizing an isl1-cre mouse line generated in our laboratory. Our results showed that ablation of Smo in the Isl1 field results in aortic arch artery and outflow tract defects. Our data suggest that Shh signaling in the ventral foregut endoderm is required for cardiac morphogenesis at the anterior pole of the heart. Materials and methods Mice Floxed Smoothened (Smo f/f) mice and Smoothened +/− (Smo+/−) mice were obtained from Andy McMahon at Harvard Medical School. Isl1-Cre mice were created in our laboratory (Park et al., in press; Yang et al., 2006). To flox out smoothened in the Isl1-Cre field, Isl1-Cre mice were crossed with Smo+/− mice. Isl1-Cre+/−, Smo+/− males were then crossed with Smo f/f females to obtain Isl1-Cre+/−, Smo−/f for analysis. Primers for mouse genotyping were as follows: Isl1-1650-5′ (5′-actatttgccacctagccacagca-3′) and Cre-3′ (5′-tccctgaacatgtccatcaggttc-3′) were used to genotype the isl1-Cre allele (300 bp); SmoFlox5′ (5′-cgggttccaaagtttgcaaagt-3′) and SmoFlox3′ (5′-catggccaaacagccaactcag-3′) were used to genotype the floxed Smoothened allele (250 bp); Neo5′ (5′-cgcttcctcgtgctttacggtatc-3′) and Smo3′ (5′-gatgtattcgtgaagcaacag-3′) were used to genotype the null smoothened allele (900 bp).
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Whole-mount RNA in situ hybridization, histological analyses, and corrosion casts Whole-mount RNA in situ hybridization was carried out as previously described (Wilkinson, 1992). Sources of specific RNA in situ probes are as follows: Fgf8 (Gail Martin), Wnt11 (Andy McMahon), Tbx1 (Antonio Baldini), Npn2 (EST. Genebank Accession No: BU554259), Shh (Deepak Srrivastava), and PlexinA2 (Jon Epstein). For histological analyses, embryos were fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in paraffin, 8 μm sections prepared on a microtome, and stained with hematoxylin–eosin according to standard protocols. Corrosion casts were prepared and processed according to protocols in Baston's No. 17 plastic replica and corrosion Kit (Polisciences Inc, #07349).
Proliferation and apoptosis analyses Mouse embryos were saturated with 20% sucrose, frozen in OCT and 8 μm sections prepared on a cryotome. Sections were fixed in 2% paraformaldehyde for 10 min at room temperature, blocked and stained with rabbit anti-phosphohistone H3 antibody (#06-570, 1:100, Upstate Biochem) for proliferation analyses or stained with rabbit anti-cleaved Caspase-3 (#9661, 1:100, Cell Signaling Technology) for apoptosis analyses. Secondary antibody Alexa Fluo® 488 was from Molecular Probes (#A11078, 1:250).
Results Endodermal expression of Shh is downregulated in isl1 mutants Hearts of isl1 null mice are severely truncated, and isl1 expressing cells exhibit defects in proliferation, cell survival and migration. To investigate potential downstream pathways which might be affected in isl1 mutants, we examined a number of growth factor signaling pathways. As isl1 and
Fig. 1. Shh expression is downregulated in isl1 mutants. Whole-mount RNA in situ hybridization (A–D) and section analyses (E–J) with a riboprobe for Shh mRNA in isl1 null embryos (C, D, H, I, J) and somite-matched littermate controls (A, B, E, F, G) demonstrated selective downregulation of Shh in foregut endoderm in isl1 mutants. Sections are shown anterior to posterior. FP = floorplate of neural tube; N = notochord; PE = pharyngeal/foregut endoderm.
758 L. Lin et al. / Developmental Biology 295 (2006) 756–763 Fig. 2. Morphological, histological, and corrosion cast analyses of cardiovascular phenotype in isl1-cre;smo mutants. Whole-mount views of hearts at E11.5 (A, B), E12.5 (C, D), and E18.5 (G, H) in isl1-cre;smo mutants (B, D, H) and littermate controls (A, C, G) demonstrated outflow tract abnormalities evident at E11.5 and E12.5, when outflow tracts in mutant hearts appeared shorter than those of littermate controls (outlined by black dots in panels A–D). At E18.5, persistent truncus arteriosus (PTA) was evident in isl1-cre;smo mutants (H), in contrast to control littermates which exhibited well separated pulmonary artery (PA) and aorta (Ao). Section analysis of E12.5 hearts (E, F) demonstrated PTA in isl1-cre;smo mutants, atrial septal defects (ASD) (F), and ventricular septal defects (not shown). Section analysis at E18.5 (I, J) demonstrated PTA, ventricular septal defects (J), and atrial septal defects (the latter not shown) in isl1-cre;smo mutants. Corrosion casting of great vessels was performed (K, L) as described in Materials and methods and demonstrated persistent truncus arteriosus in isl1-cre;smo mutants (L). In the mutant shown, right-sided aortic arch was also observed. The latter phenotype was observed in 75% of mutants examined. Ink injection was performed in E10.0 embryos to examine aortic arch artery patterning (M, N). Isl1-cre;smo mutants were missing the sixth aortic arch artery. Ao = aorta; OFT = outflow tract; LA = left atria; LV = left ventricle; PA = pulmonary artery; PTA = persistent truncus arteriosus; RA = right atria; RV = right ventricle.
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sonic hedgehog are coexpressed in pharyngeal and foregut endoderm, we examined expression of Shh in isl1 mutants by whole-mount RNA in situ hybridization followed by section analyses. Results demonstrated that Shh expression in pharyngeal and foregut endoderm is selectively downregulated in isl1 mutants (Fig. 1). This downregulation is not owing to disappearance of ventral pharyngeal endodermal cells in the isl1 mutant as Isl1 is coexpressed with Shh in ventral pharyngeal endoderm and isl1 expressing cells are still evident in the mutant (Cai et al., 2003). In isl1 null mice, isl1 transcripts are produced, but are non-functional (Pfaff et al., 1996). Ablation of smo by isl1-cre results in aortic arch artery and outflow tract defects To investigate potential consequences of downregulation of hedgehog signaling within endoderm or adjacent cardiogenic mesoderm, we ablated the hedgehog receptor smoothened (smo) utilizing an isl1-cre mouse line generated in our laboratory by knocking cre into the endogenous isl1 locus (Park et al., in press; Yang et al., 2006). Lineage studies have
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demonstrated that this cre line recapitulates expression of the endogenous isl1 gene, with excision occurring at the cardiac crescent stage. Isl1-cre;smo mutants died perinatally, with morphological examination revealing cardiovascular defects comparable to those observed with global knockout of Shh (Garg et al., 2001; Meyers and Martin, 1999; Washington Smoak et al., 2005). Shortened outflow tracts were observed in isl1-cre;smo mutants relative to those of control littermates (Figs. 2A–D). Measurements performed utilizing ImageJ software demonstrated approximately 17.12 ± 0.15%, 18.05 ± 0.19%, and 21.88 ± 0.21% reductions in length of the outflow tract of mutants relative to control littermates at E10.5, E11.5, and E12.5 respectively. Isl1-cre;smo mutants exhibited persistent truncus arteriosus (PTA), ventricular septal defects, atrial septal defects, and anomalies of the aortic arches, including right-sided aortic arch (Figs. 2K, L). Of 20 isl1-cre;smo mutants examined at E18.5, 19 exhibited PTA and 1 exhibited transposition of the great arteries (TGA). Ink injections performed in E10.0 embryos revealed an absence of the left sixth aortic arch in Isl1-cre;smo mutants, although the right sixth arch was normal (Figs. 2M, N and data not shown). This abnormality in aortic arch patterning is
Fig. 3. Whole-mount RNA in situ hybridization analysis of potential downstream effector targets in isl1-cre;smo mutants and control littermates. Expression of Fgf8 (A, B), Wnt11 (C, D), Tbx1 (E, F), Shh (G, H), and Npn2 (I–L) was examined in isl1-cre;smo mutants (B, D, F, H, J, L) and littermate controls (A, C, E, G, I, K). Tbx1 was downregulated in mesodermal core of arches (E, F arrows), and Npn2 was downregulated in the arches (I, J arrowheads) and severely downregulated in the outflow tract (I, J arrows) in isl1-cre;smo mutants. Section analysis also demonstrated decreased Npn2 expression in the arches and in outflow tract of isl1-cre;smo mutants relative to control littermates (K, L arrows), as indicated by arrows.
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similar to that previously observed in global Shh knockout mice (Washington Smoak et al., 2005). Downstream effector targets of smoothened in isl1-cre mutants Mutation of a number of genes results in similar cardiovascular defects to those observed in isl1-cre;smo mutants (Gruber and Epstein, 2004). We investigated several of these genes to determine whether they were downstream of smo signaling in the isl1 domain. Fgf8 and Wnt11 are required for cardiac outflow tract formation (Abu-Issa et al., 2002; Frank et al., 2002; Zhou et al., submitted for publication). Expression of Fgf8 and Wnt11 was unaffected in isl1-cre;smo mutants (Figs. 3A–D). Tbx1 is required for outflow tract and aortic arch artery formation (Vitelli and Baldini, 2003) and was downregulated in pharyngeal mesoderm of isl1-cre;smo mutants, as previously described for Shh null mice (Garg et al., 2001) (Figs. 3E, F). To investigate whether ablation of smo within the isl1 domain affected expression of Shh in pharyngeal endoderm, we analyzed Shh expression in isl1-cre;smo mutants. Results demonstrated that expression of Shh was not affected in isl1cre;smo mutants (Figs. 3G, H). VEGF and semaphorin signaling through neuropilin receptors neuropilin1 (npn1) and neuropilin2 (npn2) is required for multiple aspects of cardiovascular development, including aortic arch artery, outflow tract, and atrial development (Gu et al., 2003). Expression of npn1 was unaffected in isl1-cre;smo mutants (data not shown). Expression of npn2, however, was strongly downregulated in the cardiac outflow tract (Figs. 3I–
L). Npn2 is a marker for pre-migratory and migratory cranial neural crest cells (Yu and Moens, 2005), and expression within pharyngeal arches is decreased in isl1-cre;smo mutants, suggesting a decrease in or aberrant migration of neural crest cells in pharyngeal arches. Proliferation and apoptosis in isl1-cre;smo mutants Previous analysis of global Shh knockout mice has demonstrated extensive apoptosis in cardiovascular tissues (Washington Smoak et al., 2005). We examined apoptosis and proliferation in isl1-cre;smo mutants. We could find no statistically significant differences in proliferation at E10 by immunostaining with anti-phospho-histone H3 (PHH3, Figs. 4A, B). Immunostaining with antibody to cleaved Caspase-3 (C-Casp3) demonstrated increased apoptosis in outflow tract myocardium of isl1-cre;smo mutants relative to controls, as well as in other domains adjacent to or overlapping with isl1 expression. Apoptosis was observed at a frequency of 0.015 ± 0.002 in outflow tract myocardium of isl1-cre;smo mutants, whereas no apoptosis was observed in outflow tract myocardium of littermate controls (P < 0.05). No increased apoptosis was observed in ventral foregut endoderm where isl1 is also expressed (Figs. 4C, D). Cardiac neural crest cells in isl1-cre;smo mutants Cardiac outflow tract formation requires collaboration and crosstalk between a number of cell lineages, including
Fig. 4. Proliferation and apoptosis assays in isl1-cre;smo mutants and control littermates. Proliferation was assessed by antibody staining for phosphorylated histone H3 (PHH3) (A, B). No significant differences were observed in proliferation. Apoptosis was assessed by antibody staining for cleaved Caspase-3 (C-Casp3), in isl1cre;smo mutants and somite-matched littermate controls (C, D). Increased apoptosis was observed in the outflow tract of isl1-cre;smo mutants relative to wildtype controls (arrows).
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Fig. 5. Cardiac neural crest cell migration in isl1-cre;smo mutants and littermate controls. Whole-mount RNA in situ analysis was performed at isl1-cre;smo mutants and littermate controls utilizing a marker specific for cardiac neural crest cells, PlexinA2. Results of these analyses demonstrated that cardiac neural crest cells were still present in the outflow tract of isl1-cre;smo mutants (arrows) by E10.5 but did not penetrate as far into the proximal outflow tract as in control littermates (arrowheads). Indicated segments of photographs in panels A and B are shown at higher magnification in panels C and D, respectively. OFT = outflow tract.
pharyngeal endoderm, ectoderm, cardiogenic mesoderm of the secondary heart field, and cardiac neural crest (Gruber and Epstein, 2004; Hutson and Kirby, 2003). To investigate whether cardiac neural crest cells were affected in Isl1-cre;smo mutants, we examined expression of PlexinA2, a specific marker for cardiac neural crest cells (Brown et al., 2001). Results of this analysis suggested that cardiac neural crest cells were still present in the pharyngeal arches and in the outflow tract of isl1cre;smo mutants but did not penetrate the outflow tract to the same extent as observed in stage-matched wildtype littermate controls (Fig. 5). Discussion Our results have shed light on a genetic pathway required for expression of the secreted ligand Shh in pharyngeal endoderm, a critical expression domain for cardiovascular development. We have found that the LIM homeodomain transcription factor isl1 is required for Shh expression in pharyngeal and foregut endoderm. Shh null mice exhibit multiple cardiovascular defects, including aberrant aortic arch artery formation (Garg et al., 2001; Meyers and Martin, 1999; Washington Smoak et al., 2005) and failure of septation of the outflow tract, ventricles, and atria (Washington Smoak et al., 2005). We have found that ablation of smo in the isl1 domain results in a similar spectrum of cardiovascular defects. Utilization of isl1-cre has defined a temporal and spatial requirement for Shh signaling through smoothened for these aspects of cardiogenesis. At E7.5, Shh is expressed in the node and notochord, two organizing centers of the embryo (Echelard et al., 1993). Isl1, however, is not expressed in these domains, and therefore cardiovascular phenotypes observed in isl1-cre;smo mutants are independent
of smo signaling in these domains. Isl1-cre activity mirrors expression of the endogenous gene and is first evident at E7.5 (Cai et al., 2003; Park et al., in press; Yang et al., 2006). Consistent with a requirement for smo signaling in the isl1 expression domain, active smoothened signaling, as indicated by a patched-lacZ indicator (Goodrich et al., 1997), is observed in pharyngeal endoderm and in cardiogenic mesoderm, both domains of isl1 expression (Washington Smoak et al., 2005; our unpublished observations). Observed defects in atrial septation in isl1-cre;smo mutants may reflect a requirement for smo signaling within isl1expressing cardiogenic mesoderm, which will give rise to regions of the atria, including the muscular septum, in which patched-lacZ expression is observed (Cai et al., 2003; Washington Smoak et al., 2005; our unpublished observations). We observe decreased tbx1 expression in pharyngeal mesoderm of isl1-cre;smo mutants, consistent with previous findings which demonstrated decreased tbx1 expression in Shh null mice (Garg et al., 2001; Yamagishi et al., 2003). Response of a tbx1 pharyngeal mesoderm enhancer to Shh requires conserved Fox binding sites, and Foxa2 is downregulated in the mesodermal core of Shh mutants, suggesting that Shh mediates regulation of tbx1 via Foxa2 (Hu et al., 2004; Yamagishi et al., 2003). Tbx1 is required for aortic arch and outflow tract morphogenesis (Baldini, 2004; Vitelli and Baldini, 2003), and its downregulation in the mesodermal core of pharyngeal arches is likely to contribute to aortic arch anomalies in isl1-cre;smo mutants. Expression of Fgf8 and Wnt11 was not downregulated in isl1-cre;smo mutants, suggesting that these genes are either in an independent pathway or upstream of smoothened. Our studies identified neuropilin2 (npn2) as a potential effector gene downstream of smoothened signaling. In isl1cre;smo mutants, npn2 is strongly downregulated in the
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outflow tract, in a domain where active hedgehog signaling is observed by patched-lacZ staining of wildtype mice (Washington Smoak et al., 2005; our unpublished observations). Neuropilins are receptors for class 3 secreted semaphorin Cs and VEGF (Chen et al., 2005; Washington Smoak et al., 2005). Previous studies have demonstrated a functionally redundant requirement for npn1 and npn2 in outflow tract septation (Gu et al., 2003). Mice which are doubly homozygous null for npn2, and an npn1 receptor mutation which can no longer interact with semaphorin Cs, exhibit persistent truncus arteriosus and ventricular septal defects. Similar outflow tract phenotypes are observed in semaphorin 3C null mice (Feiner et al., 2001), suggesting that sema3C may be the ligand required with npn1 and npn2 to affect outflow tract septation. These observations suggest npn2 as a potential modifier gene for cardiovascular outflow tract malformations in humans. In this light, it is interesting to note that another ligand for npn1 and npn2, VEGF, is a modifier gene for diGeorge syndrome (Lawson et al., 2002; Stalmans et al., 2003) and has also been demonstrated to be downstream of hedgehog signaling (Lawson et al., 2002). Although cardiac neural crest cells are present in isl1-cre; smo mutants, their migration and/or targeting within the outflow tract was aberrant. Similar abnormal targeting of cardiac neural crest cells was observed in Shh mutants (Washington Smoak et al., 2005). Our data indicate that migration or targeting of cardiac neural crest cells may be dependent on smoothened signaling within isl1 descendents. Apparent reduction of neural crest cells within the pharyngeal arches as marked by Npn2 expression and within the outflow tract as marked by PlexinA2 expression may be attributed to aberrant migration or to loss of cells consequent to increased apoptosis. We observed increased apoptosis in the arches and in the outflow tract of isl1-cre;smo mutants. These results are consistent with previous data demonstrating increased apoptosis in Shh mutants (Jeong et al., 2004; Washington Smoak et al., 2005). Outflow tracts in isl1-cre;smo mutants were shorter than observed in wildtype somite-matched littermates, and yet we did not observe significant differences in proliferation at early stages examined. Increased apoptosis, however, was observed. The shortened outflow tract may result from increased apoptosis, aberrant migration of cells from the secondary heart field into the outflow tract, or both. This issue will be further addressed in the future by in vivo imaging studies. Future studies will be aimed at gaining further mechanistic understanding into this critical pathway for cardiac morphogenesis to understand whether regulation of shh expression by Isl1 is direct or indirect and how Shh in turn regulates neuropilin2 expression. Acknowledgments We would like to acknowledge Andy McMahon for providing us with floxed smoothened mice and Smoothened+/− mice and thank Jon Epstein, Andy McMahon, Antonio Baldini, Deepak Srivastava, and Gail Martin for providing us with cDNAs to make riboprobes. Thanks to Jennifer Fu, Cristina
Garcia-Frigola, and Yunqing Shi for expert technical assistance and mouse husbandry. Thanks Dr. Ju Chen for helpful discussion. This work was supported by NIH RO1 HL74066 and R01 HL70867 (SE) and AHA 0525141Y and 0430385N (Lizhu Lin and Chen-Leng Cai). References Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K., Meyers, E.N., 2002. Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 129, 4613–4625. Baldini, A., 2004. DiGeorge syndrome: an update. Curr. Opin. Cardiol. 19, 201–204. Brown, C.B., Feiner, L., Lu, M.M., Li, J., Ma, X., Webber, A.L., Jia, L., Raper, J.A., Epstein, J.A., 2001. PlexinA2 and semaphorin signaling during cardiac neural crest development. Development 128, 3071–3080. Cai, C.L., Liang, X., Shi, Y., Chu, P.H., Pfaff, S.L., Chen, J., Evans, S., 2003. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877–889. Chen, C., Li, M., Chai, H., Yang, H., Fisher, W.E., Yao, Q., 2005. Roles of neuropilins in neuronal development, angiogenesis, and cancers. World J. Surg. 29, 271–275. Echelard, Y., Epstein, D.J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J.A., McMahon, A.P., 1993. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417–1430. Feiner, L., Webber, A.L., Brown, C.B., Lu, M.M., Jia, L., Feinstein, P., Mombaerts, P., Epstein, J.A., Raper, J.A., 2001. Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. Development 128, 3061–3070. Frank, D.U., Fotheringham, L.K., Brewer, J.A., Muglia, L.J., Tristani-Firouzi, M., Capecchi, M.R., Moon, A.M., 2002. An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome. Development 129, 4591–4603. Garg, V., Yamagishi, C., Hu, T., Kathiriya, I.S., Yamagishi, H., Srivastava, D., 2001. Tbx1, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev. Biol. 235, 62–73. Goodrich, L.V., Milenkovic, L., Higgins, K.M., Scott, M.P., 1997. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113. Gruber, P.J., Epstein, J.A., 2004. Development gone awry: congenital heart disease. Circ. Res. 94, 273–283. Gu, C., Rodriguez, E.R., Reimert, D.V., Shu, T., Fritzsch, B., Richards, L.J., Kolodkin, A.L., Ginty, D.D., 2003. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev. Cell 5, 45–57. Hu, T., Yamagishi, H., Maeda, J., McAnally, J., Yamagishi, C., Srivastava, D., 2004. Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors. Development 131, 5491–5502. Hutson, M.R., Kirby, M.L., 2003. Neural crest and cardiovascular development: a 20-year perspective. Birth Defects. Res. C. Embryo. Today 69, 2–13. Jeong, J., Mao, J., Tenzen, T., Kottmann, A.H., McMahon, A.P., 2004. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes Dev. 18, 937–951. Kelly, R.G., 2005. Molecular inroads into the anterior heart field. Trends Cardiovasc. Med. 15, 51–56. Lawson, N.D., Vogel, A.M., Weinstein, B.M., 2002. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev. Cell 3, 127–136. Meilhac, S.M., Esner, M., Kelly, R.G., Nicolas, J.F., Buckingham, M.E., 2004. The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6, 685–698. Meyers, E.N., Martin, G.R., 1999. Differences in left–right axis pathways in mouse and chick: functions of FGF8 and SHH. Science 285, 403–406. Park, E.J., Talbot, A., Ogden L.A., Evans, S., Cai, C., Tygesen, J.,
L. Lin et al. / Developmental Biology 295 (2006) 756–763 Frank, D.U., Moon, A.M., in press. Requisite, tissue-specific roles for FGF8 in OFT formation and septation. Development. Pfaff, S.L., Mendelsohn, M., Stewart, C.L., Edlund, T., Jessell, T.M., 1996. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84, 309–320. Stalmans, I., Lambrechts, D., De Smet, F., Jansen, S., Wang, J., Maity, S., Kneer, P., von der Ohe, M., Swillen, A., Maes, C., Gewillig, M., Molin, D.G., Hellings, P., Boetel, T., Haardt, M., Compernolle, V., Dewerchin, M., Plaisance, S., Vlietinck, R., Emanuel, B., Gittenberger-de Groot, A.C., Scambler, P., Morrow, B., Driscol, D.A., Moons, L., Esguerra, C.V., Carmeliet, G., Behn-Krappa, A., Devriendt, K., Collen, D., Conway, S.J., Carmeliet, P., 2003. VEGF: a modifier of the del22q11 (DiGeorge) syndrome? Nat. Med. 9, 173–182. Vitelli, F., Baldini, A., 2003. Generating and modifying DiGeorge syndromelike phenotypes in model organisms: is there a common genetic pathway? Trends Genet. 19, 588–593. Waldo, K.L., Hutson, M.R., Ward, C.C., Zdanowicz, M., Stadt, H.A., Kumiski, D., Abu-Issa, R., Kirby, M.L., 2005. Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart. Dev. Biol. 281, 78–90. Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., Meyers, E.N., 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.
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Wilkinson, D.G. (Ed.), 1992. In Situ Hybridization: A Practical Approach. Oxford Univ. Press, New York. Xu, H., Cerrato, F., Baldini, A., 2005. Timed mutation and cell-fate mapping reveal reiterated roles of Tbx1 during embryogenesis, and a crucial function during segmentation of the pharyngeal system via regulation of endoderm expansion. Development 132, 4387–4395. Yamagishi, H., Maeda, J., Hu, T., McAnally, J., Conway, S.J., Kume, T., Meyers, E.N., Yamagishi, C., Srivastava, D., 2003. Tbx1 is regulated by tissue-specific forkhead proteins through a common Sonic hedgehogresponsive enhancer. Genes Dev. 17, 269–281. Yang, L., Cai, C.L., Lin, L., Qyang, Y., Chung, C., Monteiro, R.M., Mummery, C.L., Fishman, G.I., Cogen, A., Evans, S., 2006. Isl1-Cre reveals a common Bmp pathway in heart and limb development. Development 133, 1575–1585. Yu, H.H., Moens, C.B., 2005. Semaphorin signaling guides cranial neural crest cell migration in zebrafish. Dev. Biol. 280, 373–385. Zhang, X.M., Ramalho-Santos, M., McMahon, A.P., 2001. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node. Cell 106, 781–792. Zhou, W., Lin, L., Majumdar, A., Li, X., Zhang, X., Liu, W., Etheridge, L., Shi, Y., Martin, J., de Ven W.V., Kaartinen, V., Wynshaw-Boris, A., McMahon, A., Rosenfeld, MG., Evans, S., submitted for publication. Transcriptional regulation by non-canonical Wnt signaling orchestrates a signaling cascade controlling organ morphogenesis. Dev. Cell.
Isl1 is upstream of sonic hedgehog in a pathway required for cardiac morphogenesis Lizhu Lin a,b,1 , Lei Bu a,b,c,1 , Chen-Leng Cai a,b , Xiaoxue Zhang a,b , Sylvia Evans a,b,⁎ a
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Skaggs School of Pharmacy, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA b Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street Charlestown, MA 02129, USA Received for publication 3 January 2006; revised 30 March 2006; accepted 31 March 2006 Available online 7 April 2006
Abstract The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in both foregut endoderm and cardiogenic mesoderm and is required for earliest stages of heart development. Here, we report that isl1 is also required upstream of Shh. We find that, in isl1 null mice, Sonic hedgehog (Shh) is downregulated in foregut endoderm. Shh signals through the unique activating receptor smoothened (Smo). To investigate the role of hedgehog signaling in the isl1 domain, we ablated smo utilizing isl1-cre. Isl1-cre;smo mutants exhibit cardiovascular defects similar to those observed in Shh null mice, defining a spatial requirement for hedgehog signaling within isl1 expression domains for aortic arch and outflow tract formation. Semaphorin signaling through neuropilin receptors npn1 and npn2 is required for aortic arch and outflow tract formation. We find that expression of npn2 is downregulated in isl1-cre;smo mutants, suggesting an isl1/Shh/npn pathway required to affect morphogenesis at the anterior pole of the heart. © 2006 Elsevier Inc. All rights reserved. Keywords: Isl1; Cardiac morphogenesis; Smoothened, Neuropilin2; Outflow tract; Aortic arch artery; Anterior pole of the heart
Introduction Ablation of the LIM homeodomain transcription factor islet1 (isl1) results in embryonic growth arrest at E9.5 and lethality by E10.5 (Cai et al., 2003; Pfaff et al., 1996). Isl1 expression marks an actively proliferating population of cardiac progenitors which will contribute a majority of myocardial cells to the developing heart, including most or all cells in the outflow tract and right ventricle, a majority of cells in the atria, and some cells within the left ventricle (Cai et al., 2003). Recent lineage studies demonstrate two myocardial lineages which contribute to embryonic heart, a primary lineage which differentiates firs, and a secondary lineage which differentiates later, with isl1 marking the latter (Cai et al., 2003; Meilhac et al., 2004). The anterior or secondary heart field which will contribute to the ⁎ Corresponding author. Skaggs School of Pharmacy, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Fax: +1 858 534 4810. E-mail address: [email protected] (S. Evans). 1 These authors contributed equally to this manuscript.
0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.03.053
outflow tract and right ventricle is a subset of the isl1 expressing population (Kelly, 2005; Waldo et al., 2005). Isl1 is required within this population for proliferation, survival and migration into the forming heart (Cai et al., 2003). Isl1 mutant hearts are lacking outflow tract, right ventricle, and have a severe reduction in atrial tissue. Early embryonic lethality of isl1 mutants has precluded an examination of the potential role of isl1 in later events of cardiac morphogenesis. A number of studies have demonstrated that aberrant proliferation in the secondary heart field can result in aortic arch artery and outflow tract anomalies (Hutson and Kirby, 2003; Xu et al., 2005). The prominent role of isl1 in the secondary heart field suggests that isl1 may be required to affect these later aspects of cardiac morphogenesis. Isl1 is expressed in ventral neural tube, foregut endoderm and cardiogenic mesoderm (Cai et al., 2003; Pfaff et al., 1996). Shh is expressed in the notochord, floorplate, and ventral foregut endoderm (Zhang et al., 2001). During our analysis of isl1 null mice, we observed that the ligand Sonic hedgehog (Shh) was selectively downregulated in foregut endoderm, but not in notochord or floorplate. Analysis of a global knockout of Shh has demonstrated a critical role in
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aortic arch artery and outflow tract morphogenesis (Garg et al., 2001; Meyers and Martin, 1999; Washington Smoak et al., 2005). Global knockout of smo results in severe embryonic patterning defects and early embryonic lethality by E10.5 (Zhang et al., 2001). Hearts of smo mutants exhibit aberrant looping and are severely malformed. To investigate whether downregulation of Shh signaling in the isl1 domain would have consequences for heart formation, we ablated the unique activating hedgehog receptor smoothened (Smo) utilizing an isl1-cre mouse line generated in our laboratory. Our results showed that ablation of Smo in the Isl1 field results in aortic arch artery and outflow tract defects. Our data suggest that Shh signaling in the ventral foregut endoderm is required for cardiac morphogenesis at the anterior pole of the heart. Materials and methods Mice Floxed Smoothened (Smo f/f) mice and Smoothened +/− (Smo+/−) mice were obtained from Andy McMahon at Harvard Medical School. Isl1-Cre mice were created in our laboratory (Park et al., in press; Yang et al., 2006). To flox out smoothened in the Isl1-Cre field, Isl1-Cre mice were crossed with Smo+/− mice. Isl1-Cre+/−, Smo+/− males were then crossed with Smo f/f females to obtain Isl1-Cre+/−, Smo−/f for analysis. Primers for mouse genotyping were as follows: Isl1-1650-5′ (5′-actatttgccacctagccacagca-3′) and Cre-3′ (5′-tccctgaacatgtccatcaggttc-3′) were used to genotype the isl1-Cre allele (300 bp); SmoFlox5′ (5′-cgggttccaaagtttgcaaagt-3′) and SmoFlox3′ (5′-catggccaaacagccaactcag-3′) were used to genotype the floxed Smoothened allele (250 bp); Neo5′ (5′-cgcttcctcgtgctttacggtatc-3′) and Smo3′ (5′-gatgtattcgtgaagcaacag-3′) were used to genotype the null smoothened allele (900 bp).
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Whole-mount RNA in situ hybridization, histological analyses, and corrosion casts Whole-mount RNA in situ hybridization was carried out as previously described (Wilkinson, 1992). Sources of specific RNA in situ probes are as follows: Fgf8 (Gail Martin), Wnt11 (Andy McMahon), Tbx1 (Antonio Baldini), Npn2 (EST. Genebank Accession No: BU554259), Shh (Deepak Srrivastava), and PlexinA2 (Jon Epstein). For histological analyses, embryos were fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in paraffin, 8 μm sections prepared on a microtome, and stained with hematoxylin–eosin according to standard protocols. Corrosion casts were prepared and processed according to protocols in Baston's No. 17 plastic replica and corrosion Kit (Polisciences Inc, #07349).
Proliferation and apoptosis analyses Mouse embryos were saturated with 20% sucrose, frozen in OCT and 8 μm sections prepared on a cryotome. Sections were fixed in 2% paraformaldehyde for 10 min at room temperature, blocked and stained with rabbit anti-phosphohistone H3 antibody (#06-570, 1:100, Upstate Biochem) for proliferation analyses or stained with rabbit anti-cleaved Caspase-3 (#9661, 1:100, Cell Signaling Technology) for apoptosis analyses. Secondary antibody Alexa Fluo® 488 was from Molecular Probes (#A11078, 1:250).
Results Endodermal expression of Shh is downregulated in isl1 mutants Hearts of isl1 null mice are severely truncated, and isl1 expressing cells exhibit defects in proliferation, cell survival and migration. To investigate potential downstream pathways which might be affected in isl1 mutants, we examined a number of growth factor signaling pathways. As isl1 and
Fig. 1. Shh expression is downregulated in isl1 mutants. Whole-mount RNA in situ hybridization (A–D) and section analyses (E–J) with a riboprobe for Shh mRNA in isl1 null embryos (C, D, H, I, J) and somite-matched littermate controls (A, B, E, F, G) demonstrated selective downregulation of Shh in foregut endoderm in isl1 mutants. Sections are shown anterior to posterior. FP = floorplate of neural tube; N = notochord; PE = pharyngeal/foregut endoderm.
758 L. Lin et al. / Developmental Biology 295 (2006) 756–763 Fig. 2. Morphological, histological, and corrosion cast analyses of cardiovascular phenotype in isl1-cre;smo mutants. Whole-mount views of hearts at E11.5 (A, B), E12.5 (C, D), and E18.5 (G, H) in isl1-cre;smo mutants (B, D, H) and littermate controls (A, C, G) demonstrated outflow tract abnormalities evident at E11.5 and E12.5, when outflow tracts in mutant hearts appeared shorter than those of littermate controls (outlined by black dots in panels A–D). At E18.5, persistent truncus arteriosus (PTA) was evident in isl1-cre;smo mutants (H), in contrast to control littermates which exhibited well separated pulmonary artery (PA) and aorta (Ao). Section analysis of E12.5 hearts (E, F) demonstrated PTA in isl1-cre;smo mutants, atrial septal defects (ASD) (F), and ventricular septal defects (not shown). Section analysis at E18.5 (I, J) demonstrated PTA, ventricular septal defects (J), and atrial septal defects (the latter not shown) in isl1-cre;smo mutants. Corrosion casting of great vessels was performed (K, L) as described in Materials and methods and demonstrated persistent truncus arteriosus in isl1-cre;smo mutants (L). In the mutant shown, right-sided aortic arch was also observed. The latter phenotype was observed in 75% of mutants examined. Ink injection was performed in E10.0 embryos to examine aortic arch artery patterning (M, N). Isl1-cre;smo mutants were missing the sixth aortic arch artery. Ao = aorta; OFT = outflow tract; LA = left atria; LV = left ventricle; PA = pulmonary artery; PTA = persistent truncus arteriosus; RA = right atria; RV = right ventricle.
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sonic hedgehog are coexpressed in pharyngeal and foregut endoderm, we examined expression of Shh in isl1 mutants by whole-mount RNA in situ hybridization followed by section analyses. Results demonstrated that Shh expression in pharyngeal and foregut endoderm is selectively downregulated in isl1 mutants (Fig. 1). This downregulation is not owing to disappearance of ventral pharyngeal endodermal cells in the isl1 mutant as Isl1 is coexpressed with Shh in ventral pharyngeal endoderm and isl1 expressing cells are still evident in the mutant (Cai et al., 2003). In isl1 null mice, isl1 transcripts are produced, but are non-functional (Pfaff et al., 1996). Ablation of smo by isl1-cre results in aortic arch artery and outflow tract defects To investigate potential consequences of downregulation of hedgehog signaling within endoderm or adjacent cardiogenic mesoderm, we ablated the hedgehog receptor smoothened (smo) utilizing an isl1-cre mouse line generated in our laboratory by knocking cre into the endogenous isl1 locus (Park et al., in press; Yang et al., 2006). Lineage studies have
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demonstrated that this cre line recapitulates expression of the endogenous isl1 gene, with excision occurring at the cardiac crescent stage. Isl1-cre;smo mutants died perinatally, with morphological examination revealing cardiovascular defects comparable to those observed with global knockout of Shh (Garg et al., 2001; Meyers and Martin, 1999; Washington Smoak et al., 2005). Shortened outflow tracts were observed in isl1-cre;smo mutants relative to those of control littermates (Figs. 2A–D). Measurements performed utilizing ImageJ software demonstrated approximately 17.12 ± 0.15%, 18.05 ± 0.19%, and 21.88 ± 0.21% reductions in length of the outflow tract of mutants relative to control littermates at E10.5, E11.5, and E12.5 respectively. Isl1-cre;smo mutants exhibited persistent truncus arteriosus (PTA), ventricular septal defects, atrial septal defects, and anomalies of the aortic arches, including right-sided aortic arch (Figs. 2K, L). Of 20 isl1-cre;smo mutants examined at E18.5, 19 exhibited PTA and 1 exhibited transposition of the great arteries (TGA). Ink injections performed in E10.0 embryos revealed an absence of the left sixth aortic arch in Isl1-cre;smo mutants, although the right sixth arch was normal (Figs. 2M, N and data not shown). This abnormality in aortic arch patterning is
Fig. 3. Whole-mount RNA in situ hybridization analysis of potential downstream effector targets in isl1-cre;smo mutants and control littermates. Expression of Fgf8 (A, B), Wnt11 (C, D), Tbx1 (E, F), Shh (G, H), and Npn2 (I–L) was examined in isl1-cre;smo mutants (B, D, F, H, J, L) and littermate controls (A, C, E, G, I, K). Tbx1 was downregulated in mesodermal core of arches (E, F arrows), and Npn2 was downregulated in the arches (I, J arrowheads) and severely downregulated in the outflow tract (I, J arrows) in isl1-cre;smo mutants. Section analysis also demonstrated decreased Npn2 expression in the arches and in outflow tract of isl1-cre;smo mutants relative to control littermates (K, L arrows), as indicated by arrows.
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similar to that previously observed in global Shh knockout mice (Washington Smoak et al., 2005). Downstream effector targets of smoothened in isl1-cre mutants Mutation of a number of genes results in similar cardiovascular defects to those observed in isl1-cre;smo mutants (Gruber and Epstein, 2004). We investigated several of these genes to determine whether they were downstream of smo signaling in the isl1 domain. Fgf8 and Wnt11 are required for cardiac outflow tract formation (Abu-Issa et al., 2002; Frank et al., 2002; Zhou et al., submitted for publication). Expression of Fgf8 and Wnt11 was unaffected in isl1-cre;smo mutants (Figs. 3A–D). Tbx1 is required for outflow tract and aortic arch artery formation (Vitelli and Baldini, 2003) and was downregulated in pharyngeal mesoderm of isl1-cre;smo mutants, as previously described for Shh null mice (Garg et al., 2001) (Figs. 3E, F). To investigate whether ablation of smo within the isl1 domain affected expression of Shh in pharyngeal endoderm, we analyzed Shh expression in isl1-cre;smo mutants. Results demonstrated that expression of Shh was not affected in isl1cre;smo mutants (Figs. 3G, H). VEGF and semaphorin signaling through neuropilin receptors neuropilin1 (npn1) and neuropilin2 (npn2) is required for multiple aspects of cardiovascular development, including aortic arch artery, outflow tract, and atrial development (Gu et al., 2003). Expression of npn1 was unaffected in isl1-cre;smo mutants (data not shown). Expression of npn2, however, was strongly downregulated in the cardiac outflow tract (Figs. 3I–
L). Npn2 is a marker for pre-migratory and migratory cranial neural crest cells (Yu and Moens, 2005), and expression within pharyngeal arches is decreased in isl1-cre;smo mutants, suggesting a decrease in or aberrant migration of neural crest cells in pharyngeal arches. Proliferation and apoptosis in isl1-cre;smo mutants Previous analysis of global Shh knockout mice has demonstrated extensive apoptosis in cardiovascular tissues (Washington Smoak et al., 2005). We examined apoptosis and proliferation in isl1-cre;smo mutants. We could find no statistically significant differences in proliferation at E10 by immunostaining with anti-phospho-histone H3 (PHH3, Figs. 4A, B). Immunostaining with antibody to cleaved Caspase-3 (C-Casp3) demonstrated increased apoptosis in outflow tract myocardium of isl1-cre;smo mutants relative to controls, as well as in other domains adjacent to or overlapping with isl1 expression. Apoptosis was observed at a frequency of 0.015 ± 0.002 in outflow tract myocardium of isl1-cre;smo mutants, whereas no apoptosis was observed in outflow tract myocardium of littermate controls (P < 0.05). No increased apoptosis was observed in ventral foregut endoderm where isl1 is also expressed (Figs. 4C, D). Cardiac neural crest cells in isl1-cre;smo mutants Cardiac outflow tract formation requires collaboration and crosstalk between a number of cell lineages, including
Fig. 4. Proliferation and apoptosis assays in isl1-cre;smo mutants and control littermates. Proliferation was assessed by antibody staining for phosphorylated histone H3 (PHH3) (A, B). No significant differences were observed in proliferation. Apoptosis was assessed by antibody staining for cleaved Caspase-3 (C-Casp3), in isl1cre;smo mutants and somite-matched littermate controls (C, D). Increased apoptosis was observed in the outflow tract of isl1-cre;smo mutants relative to wildtype controls (arrows).
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Fig. 5. Cardiac neural crest cell migration in isl1-cre;smo mutants and littermate controls. Whole-mount RNA in situ analysis was performed at isl1-cre;smo mutants and littermate controls utilizing a marker specific for cardiac neural crest cells, PlexinA2. Results of these analyses demonstrated that cardiac neural crest cells were still present in the outflow tract of isl1-cre;smo mutants (arrows) by E10.5 but did not penetrate as far into the proximal outflow tract as in control littermates (arrowheads). Indicated segments of photographs in panels A and B are shown at higher magnification in panels C and D, respectively. OFT = outflow tract.
pharyngeal endoderm, ectoderm, cardiogenic mesoderm of the secondary heart field, and cardiac neural crest (Gruber and Epstein, 2004; Hutson and Kirby, 2003). To investigate whether cardiac neural crest cells were affected in Isl1-cre;smo mutants, we examined expression of PlexinA2, a specific marker for cardiac neural crest cells (Brown et al., 2001). Results of this analysis suggested that cardiac neural crest cells were still present in the pharyngeal arches and in the outflow tract of isl1cre;smo mutants but did not penetrate the outflow tract to the same extent as observed in stage-matched wildtype littermate controls (Fig. 5). Discussion Our results have shed light on a genetic pathway required for expression of the secreted ligand Shh in pharyngeal endoderm, a critical expression domain for cardiovascular development. We have found that the LIM homeodomain transcription factor isl1 is required for Shh expression in pharyngeal and foregut endoderm. Shh null mice exhibit multiple cardiovascular defects, including aberrant aortic arch artery formation (Garg et al., 2001; Meyers and Martin, 1999; Washington Smoak et al., 2005) and failure of septation of the outflow tract, ventricles, and atria (Washington Smoak et al., 2005). We have found that ablation of smo in the isl1 domain results in a similar spectrum of cardiovascular defects. Utilization of isl1-cre has defined a temporal and spatial requirement for Shh signaling through smoothened for these aspects of cardiogenesis. At E7.5, Shh is expressed in the node and notochord, two organizing centers of the embryo (Echelard et al., 1993). Isl1, however, is not expressed in these domains, and therefore cardiovascular phenotypes observed in isl1-cre;smo mutants are independent
of smo signaling in these domains. Isl1-cre activity mirrors expression of the endogenous gene and is first evident at E7.5 (Cai et al., 2003; Park et al., in press; Yang et al., 2006). Consistent with a requirement for smo signaling in the isl1 expression domain, active smoothened signaling, as indicated by a patched-lacZ indicator (Goodrich et al., 1997), is observed in pharyngeal endoderm and in cardiogenic mesoderm, both domains of isl1 expression (Washington Smoak et al., 2005; our unpublished observations). Observed defects in atrial septation in isl1-cre;smo mutants may reflect a requirement for smo signaling within isl1expressing cardiogenic mesoderm, which will give rise to regions of the atria, including the muscular septum, in which patched-lacZ expression is observed (Cai et al., 2003; Washington Smoak et al., 2005; our unpublished observations). We observe decreased tbx1 expression in pharyngeal mesoderm of isl1-cre;smo mutants, consistent with previous findings which demonstrated decreased tbx1 expression in Shh null mice (Garg et al., 2001; Yamagishi et al., 2003). Response of a tbx1 pharyngeal mesoderm enhancer to Shh requires conserved Fox binding sites, and Foxa2 is downregulated in the mesodermal core of Shh mutants, suggesting that Shh mediates regulation of tbx1 via Foxa2 (Hu et al., 2004; Yamagishi et al., 2003). Tbx1 is required for aortic arch and outflow tract morphogenesis (Baldini, 2004; Vitelli and Baldini, 2003), and its downregulation in the mesodermal core of pharyngeal arches is likely to contribute to aortic arch anomalies in isl1-cre;smo mutants. Expression of Fgf8 and Wnt11 was not downregulated in isl1-cre;smo mutants, suggesting that these genes are either in an independent pathway or upstream of smoothened. Our studies identified neuropilin2 (npn2) as a potential effector gene downstream of smoothened signaling. In isl1cre;smo mutants, npn2 is strongly downregulated in the
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outflow tract, in a domain where active hedgehog signaling is observed by patched-lacZ staining of wildtype mice (Washington Smoak et al., 2005; our unpublished observations). Neuropilins are receptors for class 3 secreted semaphorin Cs and VEGF (Chen et al., 2005; Washington Smoak et al., 2005). Previous studies have demonstrated a functionally redundant requirement for npn1 and npn2 in outflow tract septation (Gu et al., 2003). Mice which are doubly homozygous null for npn2, and an npn1 receptor mutation which can no longer interact with semaphorin Cs, exhibit persistent truncus arteriosus and ventricular septal defects. Similar outflow tract phenotypes are observed in semaphorin 3C null mice (Feiner et al., 2001), suggesting that sema3C may be the ligand required with npn1 and npn2 to affect outflow tract septation. These observations suggest npn2 as a potential modifier gene for cardiovascular outflow tract malformations in humans. In this light, it is interesting to note that another ligand for npn1 and npn2, VEGF, is a modifier gene for diGeorge syndrome (Lawson et al., 2002; Stalmans et al., 2003) and has also been demonstrated to be downstream of hedgehog signaling (Lawson et al., 2002). Although cardiac neural crest cells are present in isl1-cre; smo mutants, their migration and/or targeting within the outflow tract was aberrant. Similar abnormal targeting of cardiac neural crest cells was observed in Shh mutants (Washington Smoak et al., 2005). Our data indicate that migration or targeting of cardiac neural crest cells may be dependent on smoothened signaling within isl1 descendents. Apparent reduction of neural crest cells within the pharyngeal arches as marked by Npn2 expression and within the outflow tract as marked by PlexinA2 expression may be attributed to aberrant migration or to loss of cells consequent to increased apoptosis. We observed increased apoptosis in the arches and in the outflow tract of isl1-cre;smo mutants. These results are consistent with previous data demonstrating increased apoptosis in Shh mutants (Jeong et al., 2004; Washington Smoak et al., 2005). Outflow tracts in isl1-cre;smo mutants were shorter than observed in wildtype somite-matched littermates, and yet we did not observe significant differences in proliferation at early stages examined. Increased apoptosis, however, was observed. The shortened outflow tract may result from increased apoptosis, aberrant migration of cells from the secondary heart field into the outflow tract, or both. This issue will be further addressed in the future by in vivo imaging studies. Future studies will be aimed at gaining further mechanistic understanding into this critical pathway for cardiac morphogenesis to understand whether regulation of shh expression by Isl1 is direct or indirect and how Shh in turn regulates neuropilin2 expression. Acknowledgments We would like to acknowledge Andy McMahon for providing us with floxed smoothened mice and Smoothened+/− mice and thank Jon Epstein, Andy McMahon, Antonio Baldini, Deepak Srivastava, and Gail Martin for providing us with cDNAs to make riboprobes. Thanks to Jennifer Fu, Cristina
Garcia-Frigola, and Yunqing Shi for expert technical assistance and mouse husbandry. Thanks Dr. Ju Chen for helpful discussion. This work was supported by NIH RO1 HL74066 and R01 HL70867 (SE) and AHA 0525141Y and 0430385N (Lizhu Lin and Chen-Leng Cai). References Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K., Meyers, E.N., 2002. Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 129, 4613–4625. Baldini, A., 2004. DiGeorge syndrome: an update. Curr. Opin. Cardiol. 19, 201–204. Brown, C.B., Feiner, L., Lu, M.M., Li, J., Ma, X., Webber, A.L., Jia, L., Raper, J.A., Epstein, J.A., 2001. PlexinA2 and semaphorin signaling during cardiac neural crest development. Development 128, 3071–3080. Cai, C.L., Liang, X., Shi, Y., Chu, P.H., Pfaff, S.L., Chen, J., Evans, S., 2003. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877–889. Chen, C., Li, M., Chai, H., Yang, H., Fisher, W.E., Yao, Q., 2005. Roles of neuropilins in neuronal development, angiogenesis, and cancers. World J. Surg. 29, 271–275. Echelard, Y., Epstein, D.J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J.A., McMahon, A.P., 1993. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417–1430. Feiner, L., Webber, A.L., Brown, C.B., Lu, M.M., Jia, L., Feinstein, P., Mombaerts, P., Epstein, J.A., Raper, J.A., 2001. Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. Development 128, 3061–3070. Frank, D.U., Fotheringham, L.K., Brewer, J.A., Muglia, L.J., Tristani-Firouzi, M., Capecchi, M.R., Moon, A.M., 2002. An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome. Development 129, 4591–4603. Garg, V., Yamagishi, C., Hu, T., Kathiriya, I.S., Yamagishi, H., Srivastava, D., 2001. Tbx1, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev. Biol. 235, 62–73. Goodrich, L.V., Milenkovic, L., Higgins, K.M., Scott, M.P., 1997. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113. Gruber, P.J., Epstein, J.A., 2004. Development gone awry: congenital heart disease. Circ. Res. 94, 273–283. Gu, C., Rodriguez, E.R., Reimert, D.V., Shu, T., Fritzsch, B., Richards, L.J., Kolodkin, A.L., Ginty, D.D., 2003. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev. Cell 5, 45–57. Hu, T., Yamagishi, H., Maeda, J., McAnally, J., Yamagishi, C., Srivastava, D., 2004. Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors. Development 131, 5491–5502. Hutson, M.R., Kirby, M.L., 2003. Neural crest and cardiovascular development: a 20-year perspective. Birth Defects. Res. C. Embryo. Today 69, 2–13. Jeong, J., Mao, J., Tenzen, T., Kottmann, A.H., McMahon, A.P., 2004. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes Dev. 18, 937–951. Kelly, R.G., 2005. Molecular inroads into the anterior heart field. Trends Cardiovasc. Med. 15, 51–56. Lawson, N.D., Vogel, A.M., Weinstein, B.M., 2002. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev. Cell 3, 127–136. Meilhac, S.M., Esner, M., Kelly, R.G., Nicolas, J.F., Buckingham, M.E., 2004. The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6, 685–698. Meyers, E.N., Martin, G.R., 1999. Differences in left–right axis pathways in mouse and chick: functions of FGF8 and SHH. Science 285, 403–406. Park, E.J., Talbot, A., Ogden L.A., Evans, S., Cai, C., Tygesen, J.,
L. Lin et al. / Developmental Biology 295 (2006) 756–763 Frank, D.U., Moon, A.M., in press. Requisite, tissue-specific roles for FGF8 in OFT formation and septation. Development. Pfaff, S.L., Mendelsohn, M., Stewart, C.L., Edlund, T., Jessell, T.M., 1996. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84, 309–320. Stalmans, I., Lambrechts, D., De Smet, F., Jansen, S., Wang, J., Maity, S., Kneer, P., von der Ohe, M., Swillen, A., Maes, C., Gewillig, M., Molin, D.G., Hellings, P., Boetel, T., Haardt, M., Compernolle, V., Dewerchin, M., Plaisance, S., Vlietinck, R., Emanuel, B., Gittenberger-de Groot, A.C., Scambler, P., Morrow, B., Driscol, D.A., Moons, L., Esguerra, C.V., Carmeliet, G., Behn-Krappa, A., Devriendt, K., Collen, D., Conway, S.J., Carmeliet, P., 2003. VEGF: a modifier of the del22q11 (DiGeorge) syndrome? Nat. Med. 9, 173–182. Vitelli, F., Baldini, A., 2003. Generating and modifying DiGeorge syndromelike phenotypes in model organisms: is there a common genetic pathway? Trends Genet. 19, 588–593. Waldo, K.L., Hutson, M.R., Ward, C.C., Zdanowicz, M., Stadt, H.A., Kumiski, D., Abu-Issa, R., Kirby, M.L., 2005. Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart. Dev. Biol. 281, 78–90. Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., Meyers, E.N., 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.
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Wilkinson, D.G. (Ed.), 1992. In Situ Hybridization: A Practical Approach. Oxford Univ. Press, New York. Xu, H., Cerrato, F., Baldini, A., 2005. Timed mutation and cell-fate mapping reveal reiterated roles of Tbx1 during embryogenesis, and a crucial function during segmentation of the pharyngeal system via regulation of endoderm expansion. Development 132, 4387–4395. Yamagishi, H., Maeda, J., Hu, T., McAnally, J., Conway, S.J., Kume, T., Meyers, E.N., Yamagishi, C., Srivastava, D., 2003. Tbx1 is regulated by tissue-specific forkhead proteins through a common Sonic hedgehogresponsive enhancer. Genes Dev. 17, 269–281. Yang, L., Cai, C.L., Lin, L., Qyang, Y., Chung, C., Monteiro, R.M., Mummery, C.L., Fishman, G.I., Cogen, A., Evans, S., 2006. Isl1-Cre reveals a common Bmp pathway in heart and limb development. Development 133, 1575–1585. Yu, H.H., Moens, C.B., 2005. Semaphorin signaling guides cranial neural crest cell migration in zebrafish. Dev. Biol. 280, 373–385. Zhang, X.M., Ramalho-Santos, M., McMahon, A.P., 2001. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node. Cell 106, 781–792. Zhou, W., Lin, L., Majumdar, A., Li, X., Zhang, X., Liu, W., Etheridge, L., Shi, Y., Martin, J., de Ven W.V., Kaartinen, V., Wynshaw-Boris, A., McMahon, A., Rosenfeld, MG., Evans, S., submitted for publication. Transcriptional regulation by non-canonical Wnt signaling orchestrates a signaling cascade controlling organ morphogenesis. Dev. Cell.