The del22q11.2 Candidate Gene Tbx1 Controls Regional Outflow Tract Identity and Coronary Artery Patterning

Materials and Methods

Results

Tbx1 is required within the SHF for myocardial progenitor cells to proliferate normally; as a result the distal OFT of Tbx1^−/− embryos is shorter and narrower than in control embryos at E10.5 (Figure 1A through 1D).^20–24 To investigate this further we analyzed Tbx1^−/− embryos carrying the 96-16 transgene, normally expressed in the inferior (or caudal) wall of the OFT at embryonic day (E) 10.5 and subsequently in myocardium at the base of the pulmonary trunk.²⁷ The strong asymmetrical expression domain of 96-16 in the inferior OFT wall is lost in mutant embryos (Figure 1A through 1D); however, β-galactosidase positive cells are distributed around the circumference of the proximal region of the mutant OFT (Figure 1B and 1D). In control hearts, the 96-16 transgene is expressed in a small number of cells in the superior (or cranial) wall of the proximal OFT at E10.5 and in myocardium around the base of the aorta at E14.5 (Figure 1C and 1E).²⁷ At E14.5 low-level and patchy transgene expression was observed around the entire undivided ventricular outlet of Tbx1^−/− hearts (Figure 1F). However, a domain of higher expression in the dorsal/left side of the common outlet likely identifies a residual and malpositioned myocardial domain with subpulmonary identity (Figure 1F).

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Figure 1. 96-16 transgene expression in Tbx1 mutant hearts. A and C, Ventral and right views of an E10.5 heart showing β-galact- osidase expression in the inferior OFT wall (arrow). Note a small number of positive cells in the superior OFT wall (arrowhead). Inset in C, Section through the OFT, superior to the top. B and D, Ventral and right views of an E10.5 Tbx1^−/− heart showing a reduced number of 96-16 positive cells evenly distributed around the OFT. Inset in D, Section through the OFT at the level of the arrowhead. E, Superior view of an E14.5 96-16 heart showing strong expression in myocardium at the base of the pulmonary trunk (PT, arrow) and a narrow ring of expression at the base of the aorta (Ao, arrowhead). F, Superior view of a Tbx1^−/− heart showing a patchy distribution of 96-16 positive cells around the common ventricular outlet with a region of stronger expression in the dorsal/left domain (arrowhead).

The T55 transgene is expressed in the superior OFT wall and base of the aorta in a complementary pattern to that of the 96-16 transgene (Figure 2).²⁸ We observed that the T55 negative (96-16 positive) OFT domain was reduced in Tbx1^−/− hearts at midgestation (Figure 2A and 2B). At later developmental stages transgene expression was observed in the ventral region of Tbx1^−/− hearts, normally comprising T55 negative subpulmonary myocardium (Figure 2C through 2F). A domain of T55 negative myocardium was invariably observed on the left side of the common ventricular outlet (Figure 2D). This result supports our observations with the 96-16 transgene and suggests that a residual myocardial domain with subpulmonary identity is maintained and malpositioned in Tbx1^−/− hearts.

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Figure 2. T55 transgene expression in Tbx1 mutant hearts. A, Ventral view of an E10.5 heart showing β-galactosidase expression in the superior wall of the myocardial OFT (arrow). Inset in A, Section through the OFT, superior to the top. B, Ventral view of an E10.5 Tbx1^−/− heart showing a reduced T55 negative domain (arrowheads in A and B). Inset in B, Section through the OFT, superior to the top. C and E, Ventral and right lateral views of an E14.5 heart showing T55 transgene expression in myocardium at the base of the aorta (arrow). D and F, Ventral and right lateral views of a Tbx1^−/− heart showing a ventrally expanded T55 positive domain (arrow); note that the left side of the common ventricular outlet does not express the transgene (arrowhead in D).

We subsequently examined OFT cushion and outlet valve structure in Tbx1^−/− hearts. At E12.5 2 major and 2 intercalated cushions could be scored in 8/12 mutant hearts (Figure 3A through 3D). The major cushions fail to spiral around one another consistent with lack of rotation of the Tbx1^−/− OFT (supplemental Figure I).²³ In addition, the mutant cushions were hypoplastic compared to controls, the leftward positioned (pulmonary) intercalated cushion being severly affected (4/12) or absent (4/12) in a fraction of mutant hearts (Figure 3C and 3D). Three outlet valve leaflets were observed in 34/39 Tbx1^−/− hearts analyzed at E18.5 by histology and scanning electron microcopy (Figure 3E through 3H); 5 hearts were observed with only 2 valve leaflets. In contrast, 4 outlet valve leaflets have been reported in Tbx1 mutant hearts homozygous for a Tbx1^lacZ allele,²³ suggesting that cushion and valve leaflet development may be subject to variation arising from differences between Tbx1 mutant alleles or genetic backgrounds.

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Figure 3. Outflow tract cushion and valve development in Tbx1^−/− hearts. A–D, Paraffin sections through the OFT of E12.5 Tbx1^+/− (A) and Tbx1^−/− hearts (B–D). Two major (white asterisks) and two intercalated (black asterisks) hypoplastic cushions are observed in the Tbx1^−/− heart shown in B. The leftward positioned intercalated cushion is extremely hypoplastic in the heart shown in C and D (arrow). E and F, Cryosections through the outlet valves of E18.5 Tbx1^+/− (E) and Tbx1^−/− hearts (F). Valve cusps are indicated by asterisks. G and H, Scanning electron micrographs of an E18.5 Tbx1^+/− heart (G) and a Tbx1^−/− common ventricular outlet (H) in a superior view. Three leaflets can be seen in control and mutant outlet valves. Note the comparable diameter of the Tbx1^−/− common outlet and control aorta. Proximal coronary stems (E) and ostia (F and H) are indicated by arrowheads. Scale bars A–D), 100 μm; E and F, 200 μm.

The distribution of coronary arteries in Tbx1^−/− hearts was investigated using a Connexin40-eGFP allele expressed in coronary endothelial cells.²⁹ Whole mount visualization of Cx40-eGFP expression revealed a spectrum of anomalous coronary artery trajectories in Tbx1^−/− hearts at E18.5 (Table; Figure 4E through 4L). In control hearts left and right coronary arteries are observed along the lateral sides of the ventricles, the first of a series of branches being the conal artery from the right (and occasionally the left) and circumflex artery from the left (Figure 4A through 4D). In 28/34 (82%) Tbx1^−/− hearts the proximal left coronary artery courses abnormally across the ventral region of the heart, connecting with the common ventricular outlet close to the right coronary ostium (Figure 4E through 4G and 4K) or merging with the right coronary artery in a single ostium (Figure 4H through 4J and 4L). Additional coronary arteries were observed in Tbx1^−/− hearts including circumflex (Figure 4F, 4H, and 4L) or conal (Figure 4G and 4K) arteries with independent ostia, generally positioned ventrally. A high origin of additional coronary arteries on the common ventricular trunk was observed in a number of mutant hearts (Figure 4H). In 4 Tbx1^−/− hearts the left coronary artery was connected to the left side of the common trunk and in two hearts passed dorsally to the common ventricular outlet (supplemental Figure II). A similar spectrum of defects was observed using a FITC conjugated antismooth muscle actin antibody and the smooth muscle marker DAF-2DA (Figure 4J; supplemental Figure II). In contrast to Tbx1^−/− embryos, coronary arteries do not cross the ventral region of Tbx1^+/− hearts at E18.5 (n=53) or adult (n=5) time points (supplemental Figure III). Furthermore, proximal coronary artery trajectories are normal in Cx40^eGFP/+ (n=17) and Cx40^eGFP/eGFP (n=9) embryos wild-type for Tbx1 (supplemental Figure IV), consistent with detection of Cx40-eGFP expression in coronary endothelial cells only after arterialization.²⁹

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Figure 4. Coronary artery patterning in Tbx1^−/− hearts at E18.5. A–D, Cx40-eGFP expression in a wild-type heart in ventral (A), right (B), left (C), and superior (D) whole-mount views. Cx40-eGFP is expressed in atrial myocardium and coronary artery endothelial cells, identifying right (R) and left (L) coronary arteries and their branches, including conal (C) and circumflex (Cx) arteries. E–I, K, and L, Connexin40-eGFP expression in Tbx1^−/− hearts showing anomalous coronary artery patterning on the ventral surface of the heart. E, Left coronary artery connecting to the common outlet adjacent to the right coronary artery; F, as in E with a separate circumflex artery; G, as in E with a separate conal artery; H, convergence of right and left proximal coronary stems and independent circumflex artery. I and J, Tbx1^−/− hearts with common proximal coronary stems revealed by Cx40-eGFP expression (I) and smooth muscle actin FITC-coupled immuofluorescence (J). K and L, Superior views showing the proximal coronary stems of the hearts in G and H.

Analysis of coronary ostia position revealed that unlike the situation in the wild-type aorta whereby 1 of the 3 valve leaflets is noncoronary, 2 noncoronary leaflets were observed in 16/34 (47%) Tbx1^−/− hearts (Figure 5; Table). 26/34 (75%) mutant hearts lacked a coronary ostium connected to the left side of the common ventricular outlet. In 23/34 investigated hearts both right and left proximal coronary arteries were connected above the right and slightly ventrally positioned valve leaflet (Figure 5B through 5D). The left coronary artery crossed the ventral region of the heart to form either a distinct ostium (14/34 hearts; Figure 5B) or a direct connection with the right coronary stem resulting in a single ostium positioned above the right/ventral valve leaflet (9/34 hearts; Figure 5C, 5G, and 5H). Independent origins of the circumflex artery (Figure 5C) or conal artery (Figure 5D) were observed, occasionally positioned over the left or noncoronary valve leaflets. In 3/5 Tbx1^−/− hearts with a bicuspid valve leaflet the left coronary artery was connected to ostia over the ventrally positioned cusp (data not shown). Endothelial (PECAM, Cx40-eGFP) and smooth muscle (SMA, DAF-2DA) markers were normally associated with coronary arteries in Tbx1^−/− hearts (Figure 5E through 5H).

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Figure 5. Coronary artery patterning in Tbx1^−/− hearts at E18.5. Cryostat sections of Tbx1^+/+ (A and E) and Tbx1^−/− (B–D, F–H) hearts showing coronary arteries revealed by Cx40-eGFP expression (A–F), PECAM staining (D, G, H), and smooth muscle actin FITC-coupled (G and H) or uncoupled (E and F) immunofluorescence. A, In Tbx1^+/+ hearts right (R) and left (L) ostia are positioned over the right and left aortic valve leaflets. B, Right and left ostia positioned over the right/ventral valve leaflet of a Tbx1^−/− heart. C, Common left and right ostium (arrow) and independent circumflex artery. D, Independent conal artery over the right/ventral valve leaflet. E–H, Smooth muscle is associated normally with coronary arteries in Tbx1^−/− hearts. G and H, Unique ostium (arrow) over the rightward valve leaflet giving rise to all coronary arteries. Ao indicates aorta; PT, pulmonary trunk. Scale bars A–F, G, and H, 200 μm.

The etiology of this novel aspect of the Tbx1 mutant phenotype was analyzed by investigation of the distribution of the coronary vascular plexus at E12.5, before arterialization. Coronary ostia formation results from selective maintenance of endothelial connections between the coronary plexus and ascending aorta.^7–11 PECAM staining revealed an abnormal distribution of the coronary plexus in Tbx1^−/− hearts (Figure 6). The plexus is restricted to the dorsal region of the outflow and ventricular myocardium of wild-type hearts, with a coronary free region ventrally at the base of the pulmonary trunk (Figure 6A, 6C, and 6E). In Tbx1^−/− hearts coronary endothelial cells were observed in the ventral region of the common ventricular outlet (Figure 6B, 6D, and 6F), consistent with the reduction and malpositioning of a normally coronary-free subpulmonary myocardial domain.

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Figure 6. PECAM immunostaining in Tbx1 mutant hearts. Whole-mount views of E12.5 hearts after PECAM staining to reveal the coronary artery endothelial plexus in wild-type (A, C, E) and Tbx1^−/− (B, D, F) hearts. A and B, Ventral views; C and D, right lateral views; E and F, left lateral views. The PECAM positive vascular plexus does not cover the ventral pulmonary trunk of wild-type hearts (arrowheads) but invades the ventral region of the Tbx1^−/− ventricular outlet (arrows).

The 96-16 transgene is expressed in the heart as the result of an integration site position effect. Using fluorescent in situ hybridization we mapped the integration site to chromosome 5 A3-B1 (Figure 7A). Semaphorin 3c, mapping to this region, encodes a secreted protein of the semaphorin signaling pathway that is required for OFT division and expressed in OFT myocardium, although not in the entire OFT wall.³⁰ We found that Sema3c transcripts are distributed in a profile similar to that of the 96-16 transgene in the inferior OFT wall at E10.5 and in myocardium around the base of the pulmonary trunk at E12.5 (Figure 7B and 7C; compare with Figure 1A and 1E). We investigated whether Sema3c expression was maintained in Tbx1 mutant hearts at E12.5. In contrast to control hearts (Figure 7D and 7E), Sema3c transcript accumulation was severely reduced or absent in Tbx1^−/− hearts (Figure 7F). Sema3c encodes a molecule involved in chemorepulsion and chemoattraction³² that may potentially contribute to the maintenance of a coronary artery free zone in subpulmonary myocardium. Analysis of Sema3c^−/− hearts revealed that proximal coronary arteries do not course across the ventral region of mutant hearts but connect laterally to the base of the aorta or common ventricular outlet (7/7 hearts; Figure 7G and 7H); 4 valve leaflets were observed in Sema3c^−/− hearts with common arterial trunk (Figure 7I).³⁰ We conclude that, despite restricted expression and Tbx1-dependence, Sema3c is not a critical downstream mediator of the Tbx1^−/− coronary artery phenotype.

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Figure 7. Evaluation of Semaphorin3c as a 96-16 candidate gene. A, Fluorescent in situ hybridization with a lacZ probe to metaphase chromosomes identifies the 96-16 transgene integration site on chromosome 5 A3–B1. B and C, In situ hybridization showing Sema3c transcripts in the E10.5 heart (B) and in myocardium at the base of the pulmonary trunk (PT) but not aorta (Ao) at E12.5 (C), in a similar distribution to the 96-16 transgene. D–F, Ventral views showing Sema3c transcripts in subpulmonary myocardium of E12.5 Tbx1^+/+ and Tbx1^+/− control hearts but not Tbx1^−/− hearts (arrows in D–F). G and H, Superior views of Sema3c^+/− (G) and Sema3c^−/− (H) hearts labeled with anti-SMA-FITC to reveal smooth muscle cells at the base of the great arteries and in proximal coronary arteries. Note that coronary arteries (L, left; R, right) do not cross the ventral region of the Sema3c^−/− heart. I, Cryosection through the common ventricular outlet of a Sema3c^−/− heart labeled with anti–α-actinin antibody (red) to detect myocardium and PECAM (green) to detect endothelial cells, showing 4 valve leaflets (asterisks) and lateralized coronary arteries. Scale bar: 200 μm.

Discussion

Loss of Tbx1 results in hypoplasia of the SHF leading to a short and narrow embryonic OFT and a common ventricular outlet.^20–24 Our analysis of 2 transgenes expressed in complementary subdomains of the embryonic OFT reveals differential Tbx1 sensitivity in that the inferior OFT wall and subpulmonary myocardial domain are particularly Tbx1-dependent. The similar results observed with the 2 transgenic lines analyzed, coupled with the decrease in distal OFT length and diameter, suggest that this phenotype corresponds to a reduction in cell number, although transcriptional changes cannot be excluded. Persistence of a residual region with subpulmonary identity on the left side of the common outlet, together with our analysis of cushion morphology, supports the suggestion of Vitelli and colleagues that there is a failure of OFT rotation in Tbx1^−/− hearts.²³

The inferior OFT wall may be derived from the caudal pharyngeal region, which is severely hypoplastic in Tbx1^−/− embryos. Lineage tracing experiments using a Cre transgene under control of a Tbx1 regulatory element suggested that Tbx1 is not expressed in the entire SHF but in future subpulmonary myocardium.²⁵ Recent analysis of a Tbx1 Cre allele support this conclusion and reveal a lineage derived from Tbx1-expressing cells in the inferior wall of the embryonic OFT.²⁶ The lack of regional markers has precluded analysis of the impact of loss of Tbx1 on different domains of OFT myocardium. Our phenotypic analysis of Tbx1^−/− hearts provides evidence for a mutant phenotype consistent with the lineage studies. Together, these results suggest an explanation for the pulmonary atresia associated with tetralogy of Fallot frequently observed in del22q11.2 syndrome patients. Interestingly, in avian embryos laser ablation of the SHF results in pulmonary hypoplasia or atresia.⁶ In the most severely affected cases 3 outflow valve leaflets were detected, similar to the situation in Tbx1^−/− hearts. Thus experimental and genetic reduction of the SHF converge on a pulmonary hypoplasia phenotype.

A distinguishing feature of the aorta versus pulmonary trunk is the presence of coronary ostia. Coronary arteries form from a plexus of endothelial cells of epicardial origin that selectively invade the base of the aorta.^7–11 Although the molecular and cellular mechanisms underlying this selective invasion remain unknown, multiple cell types are involved in this process, including epicardially-derived, cardiac neural crest, and SHF-derived cells, ablation of which in avians leads to coronary artery patterning defects.^6,10,13 Abnormal coronary artery patterning has been reported in mice mutant for lrdynein, Connexin43, and Perlecan; in each case the defects are associated with specific OFT anomalies including defects associated with laterality, infundibular pouches, or transposition of the great arteries.^14–16 Tbx1^−/− mice provide a model for investigation of coronary artery patterning in common arterial trunk. In the majority of Tbx1^−/− hearts both right and left coronary arteries connect to ostia over the rightward/ventrally positioned valve leaflet and proximal coronary arteries course across the ventral, normally coronary free, region of Tbx1^−/− hearts. At E12.5, before arterialization, the plexus of epicardially derived vessels is abnormally distributed in the ventral region of Tbx1 mutant hearts. Recently it has been shown that 5% of normal C57BL/6 mice have a single coronary ostium³³; however, the elevated incidence of failure of a left ostium in Tbx1^−/− hearts and the normal coronary trajectories observed in control hearts suggest that the coronary patterns observed in the present study result directly or indirectly from loss of Tbx1. We favor the hypothesis that persistence of a residual and malpositioned coronary-refractory myocardial domain results in failure of development of a coronary ostium on the left site of the common outlet. Tbx1 may therefore play an indirect role in coronary patterning by controlling the contribution and positioning of a coronary-refractory subset of cardiac progenitor cells during OFT formation. Abnormal development of this myocardial region is likely to impact on formation of the underlying intercalated cushion resulting in a tricuspid common outlet valve. Alternatively, loss of Tbx1 may impact directly on coronary artery development through abnormal SHF-neural crest interactions or defective arterial pole epicardial or smooth muscle development. Cells of the SHF have recently been shown to give rise to smooth muscle at the base of the great arteries,^26,34–37 suggesting that there may be a direct SHF contribution to coronary artery development. Future experiments will distinguish between these potential mechanisms.

As a first step toward the identification of genes differentially expressed between subaortic and subpulmonary myocardium, localization of the integration site of the 96-16 transgene led to the observation that Sema3c is expressed in the inferior OFT wall and subpulmonary myocardium. Sema3c is a member of the Semaphorin family of signaling molecules which effect axonal growth cone guidance and vascular patterning through plexin and neuropilin coreceptors; Sema3c is required for neural crest influx and endothelial cell function during OFT septation.^30,32,38 Given the Tbx1-dependent expression pattern of Sema3c, and the chemorepulsive/attractive role of this molecule, Sema3c is a candidate mediator of the coronary-free nature of subpulmonary myocardium. However, investigation of coronary artery configuration in Sema3c^−/− hearts shows that coronary arteries are lateralized and do not cross the ventral region of the heart. Thus, despite credentials as a candidate gene, Sema3c is not the critical downstream mediator of the Tbx1^−/− coronary artery phenotype. Semaphorin signaling may nevertheless play a role in coronary artery patterning, possibly by repelling coronary endothelial cells through redundant ligand sources.

Several studies have documented coronary artery trajectories in human hearts with a common ventricular outlet, revealing a wide range of proximal coronary artery defects.^39–41 In general it has been found that the right coronary artery connects with the rightward positioned valve leaflet and the left coronary artery connects dorsally.⁴¹ In contrast, the left coronary artery of Tbx1^−/− mice commonly traverses the ventral part of the heart to connect over the right valve leaflet. Ongoing analysis will determine whether there is a specific coronary artery configuration associated with common arterial trunk in del22q11.2 patients.