Trigenic neural crest-restricted Smad7 over-expression results in congenital craniofacial and cardiovascular defects

Abstract

Smad7 is a negative regulator of TGFbeta superfamily signaling. Using a three-component triple transgenic system, expression of the inhibitory Smad7 was induced via doxycycline within the NCC lineages at pre- and post-migratory stages. Consistent with its role in negatively regulating both TGFbeta and BMP signaling in vitro, induction of Smad7 within the NCC significantly suppressed phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo, resulting in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion cells. At the cellular level, increased cell death was observed in pharyngeal arches. However, cell proliferation and NCC-derived smooth muscle differentiation were unaltered. NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were not affected, but subsequent colonization of the OFT was significantly reduced. Induction of Smad7 in post-migratory NCC resulted in interventricular septal chamber septation defects, suggesting that TGFbeta superfamily signaling is also essential for cardiac NCC at post-migratory stages to govern normal cardiac development. Taken together, the data illustrate that tightly regulated TGFbeta superfamily signaling plays an essential role during craniofacial and cardiac NCC colonization and cell survival in vivo.

Figure 1. Evaluation of the trigenic Cre/loxP-dependent, tetracycline inducible transgenic system

(A) Schematic illustrating operation of the trigenic system. The R26^rtTA-EGFP knockin mice will only express the rtTA from the Rosa26 locus upon Cre-mediated recombination (Belteki et al., 2005). Wnt1-Cre transgenic mice have been shown to label almost all NCC, including the cardiac and craniofacial NCC lineages (Jiang et al., 2000; Chai et al., 2000). In the tetO-Smad7 transgenic mice, myc-Smad7 full length cDNA (myc tag is blue box 5' of Smad7 cDNA) is under the control of heptamerized tetOn promoter, but Smad7 is not expressed until both the transactivator (rtTA) and inducer (doxycycline) are present within the same cell. Although all three transgenes are individually silent within the compound trigenic mice, myc-tagged Smad7 can be specifically induced within Cre-positive neural crest lineages upon doxycycline feeding. Thus, the spatiotemporal timing of myc-tagged Smad7 expression is determined by the timing of doxycycline addition and positionally by the expression of Cre recombinase. (B) RT-PCR analysis reveals that Smad7 mRNA expression is upregulated ~10 fold in trigenic E10.5 embryos fed doxycycline at E7.5 compared to control embryo expression of endogenous Smad7 (n= 4 embryos of each genotype). Loading was normalized using GAPDH housekeeping control. (C,D) Western analysis verifies that myc-Smad7 is only expressed within duplicate doxycycline-fed duplicate trigenic E10.5 embryos fed doxycycline at E7.5, and is absent from duplicate control embryos (n=3 duplicates). The presence of myc-Smad7 suppressed both pSmad1/5/8 and pSmad2 levels relative to total Smad1 and pSmad2/3 levels when compared to age-matched control littermates, indicating over-expression of Smad7 attenuates both BMP and TGFβ signaling by ~55% (D). Loading was normalized using Tubulin housekeeping control. αTubulin signal was detected after 5 sec, while Myc and Smads was after 30 sec exposures. Abbreviations: tri, trigenic tetO-Smad7/Wnt1-Cre/Rosa^rtTA-EGFP embryos; con, control embryos.

Figure 2. Early Smad7 induction within the neural crest lineage suppresses normal craniofacial and pharyngeal arch development

(A–C) E10.0 control and trigenic (right embryo in A) whole embryos fed doxycycline at E7.5 appear grossly similar except smaller facial processes, but higher magnification reveals that the mandibular component of the E10.0 trigenic mutant 1^st pharyngeal arch is undersized and the oropharyngeal region below the nasal process is enlarged (indicated by * in C) when compared to age-matched control (B). (D–F) E11.5 control and trigenic (right embryo in D) whole embryos. Note trigenic 1^st and 2^nd arches are dramatically underdeveloped and the facial processes are hypoplastic (F) when compared to controls (E), but the size of the trigenic heart appears is unaffected. (G–I) E13.5 control and trigenic (right embryo) whole embryos fed doxycycline at E7.5. Note in isolated heads (severed immediately below lower jaw), both the upper and lower jaws of the trigenic mutant are severely hypoplastic and largely absent (I) when compared to control littermates (H). Abbreviations: 1, first pharyngeal arch; 2, second pharyngeal arch; a, atrium; v, ventricle; lv, left ventricle; e, eye. Bar in A=0.1mm; B,C=0.05mm.

Figure 3. Histological examination of E13.5 Smad7 trigenic phenotypes fed doxycycline at E7.5

(A,B) Control and trigenic littermate cranial regions sectioned sagittally and stained with H&E. Note the absent ventral extremity of the lower jaw and lip, hypoplastic tongue, absent primary palate, and absent upper jaw and lip in trigenic mutant (* in B). However, Meckel's cartilage is still present in the mutant (yellow arrowhead). Whilst the choroid plexus (cp) extending into the trigenic lateral ventricle is present, the choroid plexus differentiating from the roof of the fourth ventricle is absent (arrow in B). (C–H) Low power images of serial sagittal sections through control (C,E,G) and trigenic (D,F,H) cardiothoracic regions at the level of the OFT and high power images of the interventricular septum (G,H). While the control has separate ascending aorta and pulmonary trunk vessels (C,E), the trigenic mutant outflow tract has failed to septate and remains as a single outlet (PTA; E) and the right subclavian is retroesophageally located in the trigenic embryo (arrow in D). Additionally, the trigenic embryo exhibits accompanying interventricular septal defects (H). (I–L) Higher power images of transverse sections reveal that both the trigenic dorsal root ganglia (J) and thymus (L) are histologically normal when compared to control littermates (I,K), but are proportionately smaller in line with the overall reduced size of the trigenics. Abbreviations: t, tounge; nt, neural tube; PTA, Persistent Truncus Arteriosus; VSD, Ventricular Septum Defect; sep, septum; rv, right ventricle; lv, left ventricle; la, left ventricle; Ao, aortic trunk; dAo, descending aorta; p, pulmonary trunk; drg, dorsal root ganglia; thy, thymus; X, vagal X trunk. Bars in I,J=10μm.

Figure 4. NCC develop in Smad7 trigenic mutant embryos fed doxycycline at E7.5

To lineage map both the trigenic and control NCC populations, R26r lacZ reporter mice were crossed with Smad7 trigenic to enable us to visualize NCC migration and colonization of the craniofacial and OFT regions. (A–C) E10 wholemount lacZ staining of trigenic and control littermates, revealed that initial NCC migration was largely unaffected within the trigenic (right embryo in A) cranial, cardiac and trunk regions when compared to control littermates. Robust lacZ expression is evident in trigenic frontonasal prominence, trigeminal nerve ganglia, hypoplastic 1^st, 2^nd and 3^rd arches and within the facial nerve ganglia, and primordium of the 3^rd pharyngeal arch. Similarly, lacZ-marked NCC are present within the cardiac 4/6^th arch region and the DRGs (*) in trigenic mutants. (D–J) LacZ stained E11.5 trigenic and control littermate whole embryos (D); isolated control (E,F) and trigenic (G,H) hearts viewed frontally (E,G) and from the right (F,H); and higher power views of control (I) and trigenic (J) craniofacial regions. Note there is a deficiency of NCC-derived Schwann cells within SNS of trigenic forelimb (* in D), but trunk NCC migration is unaltered. (E–H) Higher power views of isolated hearts clearly show that Smad7 trigenic NCC reach the pharyngeal arches and aortic sac region, that a few mutant NCC can enter the OFT truncal region but that there are no lacZ stained NCC within the trigenic OFT conal region (* in H) when compared to controls (E,F). Similarly, although trigenic NCC do colonize the hypoplastic craniofacial regions, there are reduced numbers of lacZ positive cells, particularly evident within the frontal nasal process and 3^rd, 4^th and 6^th pharyngeal arches (arrow in J). (K,L) Sections through OFT from distal to proximal in E11.5 control (K, K', K”) and trigenic embryos (L, L', L”). Histology confirms a lack of trigenic NCC colonizing the OFT, that there are fewer cells within the conal cushions and that the mutant NCC are not found in ectopic locations within the adjacent myocardial cuff (arrow in L') or overlying endothelium. (M–P) LacZ stained E13.5 control (M,O) and trigenic littermate whole embryos and isolated hearts viewed frontally. Note the absent lacZ NCC within the trigenic upper and lower craniofacial regions (arrow in N) and the absence of lacZ positive NCC within the trigenic OFT (arrow in P). Abbreviations: fl, forelimb; lv, left ventricle; a, atria; oft, outflow tract; ec, endocardial cushions; rv, right ventricle; ra, right atria; e, eye. Bars in I,J=0.2mm.

Figure 5. Elevated cell death of NCC in Smad7 trigenic mutants fed doxycycline at E7.5

(A–C) Ki67 staining at E10.5 on sagittal sections reveals equivalent levels of cell proliferation (n=4) within the trigenic 1^st (B) and 2^nd (not shown) pharyngeal arches when compared to controls (A). (D–F) In contrast, TUNEL staining at E10.5 (n=4) demonstrates a notable increase in cell death in trigenic mutants within the mandibular component of the 1^st arch (arrow in E) compared to controls (D). (G) TUNEL staining (brown) of lacZ-stained E11 R26r trigenic embryo (transverse section of right pharyngeal/OFT region lying on left hand side), demonstrates that apoptotic cells (arrows) are of NCC (blue) origin. Note that trigenic non-NCC derived core arch mesenchyme (indicated by yellow line), cephalic mesenchyme (cm) and body wall (bw) are largely devoid of apoptotic cells. However, as expected the pharyngeal pouch endoderm exhibits robust apoptosis (*). (H–K) α-SMA staining showed that NCC differentiation into smooth muscle within the E10.5 trigenic (I) aortic sac was comparable to controls (H), that α-SMA staining (brown) within the trigenic OFT cushions was reduced (*) and that transient cardiomyocyte expression of α-SMA was unaffected in both control and trigenic mutant hearts. Similarly, NCC-derived smooth muscle colonization of the control (J) and trigenic (K) 6^th aortic arch arteries (arrows) was comparable at E13.5. Abbreviations: as, aortic sac; a, atria; h, heart; PTA, Persistent Truncus Arteriosus.

Figure 6. NCC-restricted Smad7 over-expression results in decreased TGFβ and BMP signaling

(A–H) pSmad2 immunohistochemistry on E10.5 sagittal sections demonstrated that TGFβ signaling is reduced in both craniofacial and cardiovascular tissues colonized by NCC within trigenic (B,D,F) embryos fed doxycycline at E7.5 when compared to control littermates (A,C,E) but that pSmad2 expression is unaffected within control (G) and trigenic (H) ventricles. Note that pSmad2-positive cells (brown staining) are significantly reduced within the trigenic mesenchymal nasal process and the overlying epithelium (* in B), as well as within the 1^st pharyngeal arch mesenchyme and overlying epithelium (* in D). Similarly, there is almost a complete absence of pSmad2-positive NCC around the trigenic aortic sac and within the truncal region of the OFT (F). (I–P) pSmad1/5/8 immunohistochemistry on E10.5 sagittal sections similarly demonstrated that Bmp signaling is attenuated in both craniofacial and cardiovascular tissues colonized by NCC within trigenic (J,L,N) embryos fed doxycycline at E7.5, when compared to control littermates (I,K,M) but that pSmad2 expression (brown staining) is unaffected within control (O) and trigenic (P) ventricles to which NCC do not contribute. Sections were counterstained with 0.05% toluidine blue. Abbreviations: mes, mesenchyme of 1^st arch; as, aortic sac. Bars in B,G,J,O=10μm.

Figure 7. Molecular marker analysis of E10.5 trigenic mutant phenotype

Radioactive in situ hybridization detection of Crabp1 (A,B), Ap2α (C,D) and Fgf8 (E,F) mRNA in control (A,C,E) and trigenic mutants (B,D,F) fed doxycycline at E7.5. (A,B) Transverse sections through the OFT region of trigenic mutants reveal that Crabp1 expression is reduced within the pharyngeal arch region around the foregut (indicated by * in B) and along the NCC migration routes around the dorsal aorta, but not within the thoracic body wall (arrows). (C,D) Sagittal sections of trigenic mutants indicate that Ap2α expression is reduced in the trigenic nasal process (* in D) and that Ap2α is ectopically expressed within the mesenchyme of 1^st arch (arrow) when compared to control littermate (C). (E,F) Sagittal sections of trigenic mutants also reveal that Fgf8 expression is upregulated within the epithelium overlying the nasal process (* in F) and 1^st arch mesenchyme (arrow in F) but that trigenic Fgf8 expression within the neural epithelial layer of the optic cup is comparable with control expression levels. Abbreviations: da, dorsal aorta; oft, outflow tract; e, eye.

Figure 8. Analysis of E14.5 Smad7 trigenic heart phenotypes fed doxycycline at E10

(A,B) Control and trigenic littermate OFT regions sectioned transversely and stained with H&E. Note both the control (A) and trigenic (B) left and right lobes of the thymic rudiment are normally situated and unaffected by myc-Smad7 induction (n=4 trigenic and 5 control fetuses). However, the trigenic OFT truncal endocardial cushions are smaller and the lumen of the outlet of the right ventricle is abnormally dilated (arrow in B). (C,D) Additionally, the trigenic embryo exhibits an isolated VSD (arrow in D) but the control heart is septated normally (C). (E,F) To lineage map both the trigenic and control post-migratory NCC populations, R26rlacZ reporter mice were crossed with Smad7 trigenic mice. Transverse sections through the E13.5 OFT revealed similar patterns of cardiac NCC (blue) within the aorticopulmonary septum (not shown) and valves in both control (E) and trigenic (F) littermates. Note that trigenic cardiac NCC are still present and that the valves are normal. (G,H) α-SMA staining showed that myocardialization occurs normally in both E13.5 control (G) and trigenic (H) hearts. Note active myocardialization of endocardial cushions at base of the OFTs (indicated by *). (I,J) TUNEL staining (brown) revealed that both the patterns and level of apoptosis (indicated by arrows) are grossly similar in E13.5 control (I) and trigenic (J) OFTs. Abbreviations: thy, thymic lobes; rv, right ventricle.