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. 2012 Jan 1;21(1):26-31.
doi: 10.1093/hmg/ddr434. Epub 2011 Sep 22.

Cardiovascular defects in a mouse model of HOXA1 syndrome

Nadja Makki  1 , Mario R Capecchi
Affiliations

Cardiovascular defects in a mouse model of HOXA1 syndrome

Nadja Makki et al. Hum Mol Genet. .

Abstract

Congenital heart disease is one of the most common human birth defects, yet many genes and pathways regulating heart development remain unknown. A recent study in humans revealed that mutations in a single Hox gene, HOXA1 (Athabascan Brainstem Dysgenesis Syndrome, Bosley-Salih-Alorainy Syndrome), can cause severe cardiovascular malformations, some of which are lethal without surgical intervention. Since the discovery of the human syndromes, there have been no reports of any Hox mouse mutants with cardiac defects, hampering studies to explore the developmental causes of the human disease. In this study, we identify severe cardiovascular malformations in a Hox mouse model, which mimic the congenital heart defects in HOXA1 syndrome patients. Hoxa1 null mice show defects such as interrupted aortic arch, aberrant subclavian artery and Tetralogy of Fallot, demonstrating that Hoxa1 is required for patterning of the great arteries and outflow tract of the heart. We show that during early embryogenesis, Hoxa1 is expressed in precursors of cardiac neural crest cells (NCCs), which populate the heart. We further demonstrate that Hoxa1 acts upstream of several genes, important for neural crest specification. Thus, our data allow us to suggest a model in which Hoxa1 regulates heart development through its influence on cardiac NCCs, providing insight into the mechanisms underlying the human disease.

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Figures

Figure 1.
Figure 1.
Hoxa1 alleles used in this study. (A) Hoxa1GFP was generated by inserting EGFP followed by polyadenylation signals into a unique site located in the first exon of Hoxa1 resulting in a frame shift in the Hoxa1 genomic sequence 3′ of the cassette (grey box). This generates a fusion protein, lacking the homeodomain, which is encoded in exon 2. Neo was removed leaving a residual loxP site. The Hoxa1IC allele was generated by inserting an IRES-Cre-NEO cassette in the 3′UTR of Hoxa1 and subsequent removal of Neo, leaving a residual Frt site. Solid boxes represent coding regions and white boxes UTRs. (B) PCR or southern genotyping identifies the different Hoxa1 alleles.
Figure 2.
Figure 2.
Hoxa1 null mice exhibit severe cardiovascular abnormalities that mimic the defects in HoxA1 syndrome patients. (A) Schematic of the heart, great arteries and cerebral arteries. Structures in which defects were found in Hoxa1 mutants are highlighted in red. (B–L) Cardiovascular defects in mutants include abnormalities of the great arteries and cardiac OFT. (B and B′) Anatomy and histology of great arteries in an E18.5 WT embryo. Great artery defects in mutants include IAAB (C, C′), aberrant retro-esophageal right subclavian artery (ASC, D, D′), a combination of IAAB and ASC (E, E′) and RAA (F). Defects in the OFT include BAV and VSD. The aortic valve is tricuspid in WT (G) but bicuspid in mutants (H). The ventricular septum is continuous in WT embryos at E18.5 (I), while a large VSD is present in the mutant (J). Compared with WT (I, K), this embryo also shows an overriding aorta, hypertrophy of the ventricle (J, black lines) and pulmonary stenosis (L), which are the hallmarks of Tetralogy of Fallot (TOF). Ao, aorta; BT, brachiocephalic trunk; DA, ductus arteriosus; E, esophagus; LA, left atrium; LCC, left common carotid artery; LSA, left subclavian artery; LV, left ventricle; LVOT, left ventricular outflow tract; PA, pulmonary artery; RA, right atrium; RCC, right common carotid artery; RSA, right subclavian artery; RV, right ventricle; Tr, trachea. Scale bars: anatomy panels, 0.5 mm; histology panels, 100 μm.
Figure 3.
Figure 3.
Hoxa1 null mice exhibit cerebrovascular and glandular defects. (A) In wild-type embryos, the common carotid artery branches into the external and internal carotid arteries (ICA/ECA) in a stereotyped pattern, while mutants show branching abnormalities (B). Compared with wild-type (C and C′) mutants exhibit thymic (Ty) hypoplasia (D and D′). (E and E′) The parathyroid glands (Ptr) are located adjacent to the thyroid (Tyr) in wild-type embryos but are absent in mutants (F and F′). TC, thyroid cartilage. Scale bars: anatomy panels, 0.5 mm; histology panels, 100 μm.
Figure 4.
Figure 4.
Hoxa1 is expressed in cardiac neural crest progenitors in the posterior hindbrain and is required for their development. (A–D) RNA in situ analysis of Hoxa1 expression in WT embryos. Strong Hoxa1 expression in the posterior hindbrain (brackets), where cardiac NCCs arise, is seen at the six-somite (A, dorsal view) and the eight-somite stage (B, lateral view). Hoxa1 is expressed in this region as early as E7.75 (headfold stage, Hf) (C) and by E9.0 it is absent from the hindbrain and is only present in the posterior neural tube and foregut (D, arrow heads). (E–J) Hoxa1 lineage contributes extensively to the OFT of the heart. (E) X-gal staining for Hoxa1 lineage (blue) in the OFT and atria (A) of a dissected E12.5 heart. (F and G) Transverse sections through the heart of WT embryos comparing Hoxa1 lineage (F) to Wnt1 lineage (G), which represents the NCC lineage, reveals that most if not all cardiac NCCs are derived from Hoxa1-expressing cells. (H–J) Temporal series showing that whereas there is almost no Hoxa1 lineage in the heart at E9.0 (H), at E10.0 the lineage has populated the cardiac region (I), giving rise to the majority of cells in the OFT, as seen at E10.5 (J). (K–M) Neural crest markers are down-regulated in the posterior hindbrain of Hoxa1 mutants. The expression of Hnf1b (K, K′), Foxd3 (L, L′) and Zic1 (M, M′) is strongly reduced in the posterior hindbrain (r6–r8; brackets) of Hoxa1 null embryos. Foxd3 expression in r4 is also absent in mutants (L, L′; arrow heads). (N) Model for the role of Hoxa1 in cardiac neural crest progenitors based on this (black) and other studies (blue) (5,21,28,29). We suggest that Hoxa1 acts upstream of Hnf1b, Zic1 and Foxd3 in neural crest development, potentially in complex with Pbx1 and upstream of Pax3 to control NCC specification. Note that dashed lines and arrows do not necessarily indicate direct gene regulation. e, eye; h, heart. Scale bars: top panels 200 μm, bottom panel 100 μm.
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