Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency

Abstract

The CXCL12-CXCR4 pathway has crucial roles in stem cell homing and maintenance, neuronal guidance, cancer progression, inflammation, remote-conditioning, cell migration and development. Recently, work in chick suggested that signalling via CXCR4 in neural crest cells (NCCs) has a role in the 22q11.2 deletion syndrome (22q11.2DS), a disorder where haploinsufficiency of the transcription factor TBX1 is responsible for the major structural defects. We tested this idea in mouse models. Our analysis of genes with altered expression in Tbx1 mutant mouse models showed down-regulation of Cxcl12 in pharyngeal surface ectoderm and rostral mesoderm, both tissues with the potential to signal to migrating NCCs. Conditional mutagenesis of Tbx1 in the pharyngeal surface ectoderm is associated with hypo/aplasia of the 4th pharyngeal arch artery (PAA) and interruption of the aortic arch type B (IAA-B), the cardiovascular defect most typical of 22q11.2DS. We therefore analysed constitutive mouse mutants of the ligand (CXCL12) and receptor (CXCR4) components of the pathway, in addition to ectodermal conditionals of Cxcl12 and NCC conditionals of Cxcr4. However, none of these typical 22q11.2DS features were detected in constitutively or conditionally mutant embryos. Instead, duplicated carotid arteries were observed, a phenotype recapitulated in Tie-2Cre (endothelial) conditional knock outs of Cxcr4. Previous studies have demonstrated genetic interaction between signalling pathways and Tbx1 haploinsufficiency e.g. FGF, WNT, SMAD-dependent. We therefore tested for possible epistasis between Tbx1 and the CXCL12 signalling axis by examining Tbx1 and Cxcl12 double heterozygotes as well as Tbx1/Cxcl12/Cxcr4 triple heterozygotes, but failed to identify any exacerbation of the Tbx1 haploinsufficient arch artery phenotype. We conclude that CXCL12 signalling via NCC/CXCR4 has no major role in the genesis of the Tbx1 loss of function phenotype. Instead, the pathway has a distinct effect on remodelling of head vessels and interventricular septation mediated via CXCL12 signalling from the pharyngeal surface ectoderm and second heart field to endothelial cells.

Fig 1. Expression of CXCR4/CXCL12 axis downstream of Tbx1 and in pharyngeal NCCs.

(A-D) Cxcl12 expression in Tbx1 mutant embryos at E9.0 (in situ hybridisations): wholemounts (A and B) and transverse sections at the level of PA2 (C and D). Cxcl12 expression is reduced in the pharyngeal surface ectoderm (PSE, arrow) and underlying mesenchyme (m). (E,F) CXCR4 is expressed in migratory NCCs at E9.5. NCCs were detected in sagittal sections of wild type embryos using Ap2α antibody. CXCR4 is highly expressed in the neural tube (NT) and at a lower level in migrating NCCs (boxed regions in E and F, shown enlarged in E’ and F’). (G, H) CXCR4 is expressed in few lineage-traced NCCs in PA3 (Pax3-Cre;R26R^eYFP embryos) at E9.5 (boxed region in panels G and H, shown enlarged in G’ and H’) whilst high levels of CXCR4 can be observed in delaminating NCCs (arrowheads). CXCR4 expression was also observed in lineage-negative cells within PA3 (G(iii)). (I- M) NCC migration into the PAAs is normal in Cxcl12 null embryos, as assessed by wholemount staining with Ap2α antibody (I, J, maximum z projections of confocal stacks) and Sox10 expression (L, M, in situ hybridisations). OV, otic vesicle; NCCs,neural crest cells; PA3, pharyngeal arch 3;PSE, pharyngeal surface ectoderm. Scale bars represent 200μ in panels C-H and 100μ in panel I.

Fig 2. Mutation of Cxcl12 causes cardiovascular defects but does not synergise with Tbx1 heterozygosity.

(A-I) Aortic arch anomalies in Cxcl12 mutants at E15.5 including duplicated LCC (white arrows in B, C and G), ectopic vessels originating from the aortic arch (black arrowhead in panel B), right-sided aortic arch (RAA, Panel D) and RSA apparently absent (B)- however note thin vessel branching off and running parallel with and dorsal to the RCC (blue arrowheads in F and G, serial sections)- or unbranched (C). The LSA was thin and abnormally positioned (panel B) or not visible (C), originating from a more distal part of the aorta in Cxcl12^-/-embryos compared to controls (black arrows in panels H and I). (J, K) India ink injections of E10.5 embryos showed PAA3-6 were normal in both Cxcl12 mutants (n = 8) and controls (n = 9). (L-N) Outflow alignment defects in Cxcl12 mutants at E15.5 e.g. VSD and over-riding aorta (arrow in panel M), and DORV (panel N shows aorta opening into the right ventricle. (O-Q) Development of the intersomitic arteries (ISAs) and vertebral artery (VA) is affected in Cxcl12 mutants. In situ hybridisations (sagittal sections) show expression of Cxcr4 in the VA at E12.5 and absence of the VA in Cxcl12 ^-/- embryos (O). Confocal analysis of wholemount PECAM (green) and SM22α-stained (red) E11.5 embryos shows malformation of the VA (arrows) in the Cxcl12 mutant (Q,) compared to control (P) and hyperplasia of ISAs (indicated by arrowheads; boxed regions are shown enlarged in P’ and Q’- z projections created from shorter confocal substacks show the ISAs more clearly), and failure to regress of some anterior ISAs (asterisks). (R-U) Tbx1 and Cxcl12/Cxcr4 do not synergise in producing aortic arch phenotypes. Retro-oesophageal RSA was observed in a minority of mutants with a Tbx1 mutant allele (e.g. R and S), whilst the majority of Tbx^+/-;Cxcl12^+/- and Tbx^+/-;Cxcl12^+/-;Cxcr4^+/- mutants showed normal vessel patterning (T). mVSDs were observed at low frequency in double and triple heterozygotes (U). Ao, aorta; AoA, aortic arch; DORV, double outlet right ventricle; LCC, left common carotid; LSA, left subclavian; LV, left ventricle; mVSD, membraneous ventricular septal defect; PAA, pharyngeal arch arteries;PT, pulmonary trunk; RCC, right common carotid; RSA, right subclavian; RV, right ventricle; Tr, trachea. Scale bars represent 200μ in panels in E-I and O, and 500μ in panels L-N and P-Q.

Fig 3. Cardiovascular defects in Cxcr4 and Cxcl12 conditional mutants.

(A-F) Knock-out of Cxcr4 in NCC and endothelial lineages. Aortic arches and vasculature were normal in Pax3-Cre;Cxcr4^fl/- mutants (B) but mVSDs were present in 23.5% of embryos examined (asterisk in E indicates VSD with over-riding aorta). Tie2-Cre; Cxcr4^fl/- mice phenocopy both aortic arch and intracardiac defects (C, F) e.g. unbranched RSA, absent or mis-positioned LSA, and an ectopic vessel arising from the aortic arch (arrow in C); asterisk (F) indicates VSD with over-riding aorta. (G-L) Knock-out of Cxcl12 in ectoderm and second heart field. Ap2α-Cre;Cxcl12^fl/- mutants displayed a subset of aortic arch defects only i.e. duplicated LCC in a single case (arrow, H) and ectopic vessels arising from the aortic arch in approximately 40% of embryos examined (arrowhead, I). Aortic arches and vasculature were normal in AHF-Mef2cCre;Cxcl12^fl/- embryos (J) whereas membranous VSDs were found in 36.4% of embryos examined (arrow in L). Ao, aorta; AoA, aortic arch; DORV, double outlet right ventricle; LCC, left common carotid; LSA, left subclavian; LV, left ventricle; mVSD, membranous ventricular septal defect; Oe, oesophagus; PT, pulmonary trunk; RCC, right common carotid; RSA, right subclavian; RV, right ventricle; Tr, trachea; VA, vertebral artery. Scale bars represent 500μ.