Fbln1 gene disruption by gene trap insertion
Mapping of the gene trap insertion element within the mouse Fbln1 gene () revealed that the start of the element was at position 71145 (GI: 15591330) within intron 14 ( and Supplementary Fig. 1 for the sequence of the 5′ junction region of the insertion within intron 14).
Disruption of the Fbln1 gene by gene trap insertion
To determine the effect of the gene trap insertion on splicing of the Fbln1 transcript, RT-PCR was performed using a primer from exon 13 and a primer from the CD4 component of the gene trap element. DNA sequencing of the resulting amplicon revealed that the gene trap insertion caused an aberrant splicing of exon 14 to the splice acceptor site of the gene trap cassette. The mis-spliced mRNA encoded the first 568 amino acids of the Fbln1 protein in-frame with the CD4 transmembrane containing segment and β-gal element (Supplementary Fig. 2). Additional RT-PCR analysis showed that embryos (E9.5) homozygous for the gene trap insertion lacked transcripts encoding exons located downstream from the insertion element (i.e., exons 15–18) (). These findings indicate that homozygous embryos lack Fbln1C and D transcripts.
To assess the effects of the gene trap insertion on in vivo Fbln1 protein expression, immunoblot and immunohistological analyses were performed. By immunoblot analysis using a Fbln1 polyclonal antibody there was no immunoreactive Fbln1 polypeptide in 8M urea extracts from embryos homozygous for the Fbln1 gene trap insertion (). Heterozygous embryos showed approximately half the level of Fbln1 present in wild-type embryos extracts.
Embryos homozygous for the Fbln1 gene trap insertion have no immunologically detectable Fbln1
Embryonic heart tissue sections from wild-type embryos and embryos homozygous for the Fbln1 gene trap insertion (E10.5) were next evaluated by immunohistochemistry. In wild-type embryos, Fbln1 staining is prominent in the ECM surrounding mesenchymal cells of the AV and OFT cushions (). By contrast, no Fbln1 staining was evident in these tissues () or any other tissues examined from Fbln1 gene trap mutants (data not shown). Using a recombinant amino terminal fragment of mouse Fbln1 (amino acid residues 1–568), we confirmed that the polyclonal antibody used in immunohistochemistry and immunoblotting was reactive with the truncated portion of Fbln1 in the Fbln1-CD4-β-gal fusion protein (data not shown). We performed additional immunohistochemical analysis using a Fbln1 monoclonal antibody directed to an amino terminal epitope contained in the Fbln1-CD4-β-gal fusion protein (Tran et al., 1997
). Pronounced Fbln1 staining was evident in the wild-type tissues (E10.5) (), but extremely low levels were detectable in tissues of embryos homozygous for the gene trap (). Furthermore, the low levels of immunoreactive material appeared as discrete intracellular foci. Based on these findings, it can be concluded that the Fbln1-CD4-β-gal fusion protein becomes quantitatively degraded similar to what has been described in mice homozygous for other genes containing secretory gene trap insertions (Schymeinsky et al., 2002
; Zhou et al., 2004
). Taken together, the findings indicate that embryos homozygous for the Fbln1 gene trap insertion are effectively Fbln1-deficient.
Lethality occurs in embryos homozygous for the Fbln1 gene trap insertion
Genotypic analysis was performed on 117 offspring from heterozygous matings. As a result, 37 wild-type, 80 heterozygous and 0 homozygous offspring were detected (Supplementary Table 1). This non-Mendelian ratio indicated that lethality was occurring in offspring homozygous for the gene trap insertion. To determine the stage of lethality, retrograde genotypic analysis was performed on embryos from timed heterozygous matings. From E9.5-18.5, homozygous embryos were detected at a frequency in accordance with Mendelian expectations (Supplementary Table 1). These findings indicated that homozygous embryos were dying at birth. When litters were examined immediately after birth, null embryos were observed that were hemorrhagic and either dead or experiencing labored breathing.
Fbln1−/− embryos have blood vessel and lung anomalies
Morphological analysis revealed that homozygous embryos exhibit bleeding within the eyes and petechial bleeding in the head and neck (). In addition, fewer superficial blood vessels are apparent in the heads of mutant embryos (). These abnormalities, which were 100% penetrant, could be seen as early as E14.5 and the severity increased with gestational age. In later stage null embryos (E18.5), blood cells could be found in extravascular spaces around the spinal cord. Several homozygous embryos also displayed significant edema on the back of the head overlying the 4th ventricle. Upon histological examination of the eyes of E15.5 Fbln1-deficient embryos, the hyaloid blood vessels, which extend through the hyaloid cavity to the caudal surface of the lens capsule, appear distended and filled with blood cells ().
Mice homozygous for the Fbln1 gene trap insertion display a hemorrhagic phenotype
The lungs of the Fbln1 null embryos (E17.5) appeared smaller than normal. Histological analysis reveals that the lungs of homozygous embryos have a greater cell density as compared to lungs of wild-type or heterozygote embryos of the same gestational age (Supplementary Fig. 3). Most notable is that lungs of Fbln1 nulls have few saccules.
Outflow tract abnormalities in Fbln1−/− embryos
Abnormalities of the remodeling of the OFT were evidenced by the high incidence of double outlet right ventricle (DORV) ( and Supplementary Fig. 4A–F) and overriding aorta in homozygotes (). Persistent truncus arteriosus (PTA), resulting from a failure of OFT septation, was not observed in Fbln1−/− embryos. Together, these observations indicate that Fbln1 is not required for the septation of the aorta and pulmonary artery, but that it is required for the proper rotation of the aorta and pulmonary arteries during remodeling of the OFT.
Fbln1−/− hearts have DORV, VSD and aortic arch artery abnormalities
Penetrance of cardiac defects in embryos homozygous for gene trap disruption of the fibulin-1 gene.
Cardiac septation defects in Fbln1−/− embryos
Ventricular septal defects (VSD) (both muscular and membranous) were seen in 86% of the hearts from Fbln1−/− embryos (, Supplementary Fig. 4E and F and Table 1). The majority of these VSDs (53%) occurred in combination with either DORV or overriding aorta. Atrial septal defects (ASDs) were also observed in 40% (6 out of 15) of Fbln1−/− hearts and included septum primum and septum secundum anomalies Supplementary Fig. 4G. The prevalence of VSD and ASD in Fbln1−/− hearts emphasizes the important role of Fbln1 in proper cardiac septation.
Aortic arch abnormalities in Fbln1−/− embryos
Abnormalities of morphology of the aortic arch arteries were apparent in Fbln1 null embryos. The right subclavian artery (RSA), which is derived from the right 4th-pharyngeal arch artery and branches off the brachiocephalic artery was missing from its normal position in 20% of the Fbln1−/−
mutants (). Moreover, in several of these mutants, there was an anomalous blood vessel that emerged from the aorta, caudal to the left subclavian, that passed behind the esophagus (i.e., retroesophageal RSA). In addition, constrictions of the brachiocephalic artery were also apparent in one Fbln1 null (). These anomalies are consistent with defective remodeling of the pharyngeal arch arteries, a process that requires proper NCC investment into the arch artery sheath layers (Bockman et al., 1989
Myocardial wall defects in Fbln1−/− embryos
In 40% of Fbln1−/− hearts (6 of 15 at E17.5-18.5), the compact layer of the ventricular wall myocardium of both ventricles was thinner compared to that of wild-type hearts (Supplementary Fig. 5).
Pharyngeal gland abnormalities in Fbln1−/− embryos
Examination of the pharyngeal glands of Fbln1-deficient embryos revealed a number of morphological abnormalities. In all Fbln1 null embryos examined (E16.5-18.5, n = 15), the thymus, which is derived from the third pharyngeal pouch, was hypoplastic (). Thymic aplasia or uni-lobe thymus was not observed in null embryos. Fbln1−/− embryos also displayed hypoplasia of the thyroid, which is derived from the 3rd and 4th pharyngeal arches and pouches ().
Hypoplastic thymus and thyroid and under mineralized and reduced sized skull bones in Fbln1−/− embryos
Craniofacial skeletal abnormalities in Fbln1−/− embryos
Analysis of the ossified and cartilaginous tissues of the craniofacial skeleton of Fbln1 mutant embryos (E17.5) revealed decreased ossification of most cranial bones, particularly the frontal, parietal, tympanic ring, nasal and premaxillary bones (). There is also reduction in the size of many skull bones in the nulls including the mandible (micrognathia) () and tympanic ringbones () as well as an overall reduced cranial size as compared to littermate controls. No cleft palate was observed in the Fbln1 nulls. The neural crest-derived elements of the cartilages of the throat (i.e., hyoid and thyroid) also appear reduced in the nulls as compared to wild-type embryos (data not shown).
Fbln1 expression in the neural crest, somites, pharyngeal arches and distal OFT/secondary heart field
As a result of the gene trapping, β-gal is placed under the control of the Fbln1 promoter. X-gal staining was performed to establish the pattern of Fbln1 expression in heterozygous embryos. X-gal staining was apparent in presomitic mesoderm from E8.75 to E9.0 (). X-gal staining was also prominent in somites from E8.75 to E10.5 (). X-gal staining was also apparent along the margins of neural folds in caudal regions of the embryo and dorsal regions of the neural tube (, arrowheads). At E10.5, the dorsal neural tube region was relatively free of X-gal stained cells, however, pronounced X-gal staining was apparent in populations of cells lateral to the hindbrain, around the otic vesicle, in pharyngeal arches 1–3, in anterior and posterior regions of the somites, in the limb buds and in the region cardiac inflow tract ().
Fbln1 expression in the neural crest, somites, secondary heart field and pharyngeal arches
Consistent with the X-gal staining, immunohistological analysis showed pronounced Fbln1 immunolabeling in the ECM of the mesenchyme lateral to the neural tube at the level of the pharyngeal arches in E9.5-10.5 embryos (). By contrast, relatively low levels of Fbln1 immunolabeling were detected in the arch mesenchyme directly adjacent to the aortic sac/distal OFT (, brackets). At E9.5, this area was rich in Pax3-expressing NCCs (, brackets). Prominent Fbln1 labeling was evident in the ECM of the distal OFT, in the region of the SHF (, arrowheads). Pharyngeal arch mesenchyme underlying the neural tube had high levels of Fbln1 and few Pax3-positive cells (, asterisk). Little or no Fbln1 labeling was apparent in pharyngeal arch ectoderm or endoderm at E9.5-10.5.
Fbln1 expression in the head mesenchyme and secondary heart field
Whole-mount in situ RNA hybridization analysis of wild-type E8.3-9.5 embryos showed that Fbln1 mRNA is expressed in presomitic mesoderm and somites, similar to what was observed in X-gal stained embryos heterozygous for the gene trap insertion (). Fbln1 RNA was also detected in rhombomeres 2, 4, 6/7 and in pharyngeal arches 1–4 (). The rhombomere expression of Fbln1 is similar to that of the NCC marker Crabp1 (). Together, the findings from Fbln1 expression analysis indicate that Fbln1 expression is associated with migratory pathways of NCCs derived from rhombomeres 2–7, which contribute to morphogenesis of the OFT and pharyngeal glands. However, it is not known whether Fbln1 is expressed by migrating NCCs or by non-neural crest derived-mesenchymal cells located along the path of migrating NCCs.
Fbln1 in situ RNA hybridization analysis
Fbln1 gene trap nulls have cranial nerve patterning abnormalities
NCCs derived from rhombomeres 2, 4, 6 and 7 also contribute to the formation of the cranial nerves. In situ hybridization analysis using a Sox-10 probe revealed abnormalities of the patterning of cranial nerve NCCs in Fbln1 null embryos (). In particular, interruptions and inappropriately directed streams of Sox-10-positive NCCs that comprise the primordia for cranial nerves IX (glossopharyngeal) and X (vagus) were observed (). The apparent misguidance of NCCs resulted in abnormal mixing of the distal regions of the glossopharyngeal and vagal streams (, brackets), which may be interpreted to have resulted from a caudal misdirection of the glossopharyngeal stream. Abnormal branching of the proximal region of the vagal stream was also observed in some embryos (data not shown).
Fbln1 deficiency leads to anomalies of cranial NCC patterning and cranial nerve morphogenesis as well as increased apoptosis in hindbrain regions
To evaluate the impact of Fbln1 deficiency on cranial nerves, E10.5 embryos were immunostained to detect neurofilament-M. The results reveal abnormalities in cranial nerves IX and X (). Null embryos display an interruption in the proximal portion of cranial nerve IX and a less than normal degree of branching in the distal segment (). Furthermore, there is an abnormal fusion of cranial nerves IX and X in the epibranchial placode region (). Cranial nerve X in Fbln1 null embryos also appeared more compact and less reticulated than in wild-type embryos ().
Fbln1 gene trap nulls have increased apoptosis of NCCs in the hindbrain
TUNEL analysis was performed to assess the consequence of Fbln1 deficiency on NCC survival. Null E10.5 embryos displayed increased levels of TUNEL-positive cells in the hindbrain region as compared to wild-type embryos (). In particular, relatively high levels of TUNEL-positive cells were apparent in the otic vesicle epithelium and around rhombomeres 4, 6 and 7 as compared to wild-type embryos. To determine if the apoptotic cells observed in Fbln1 nulls might correspond to NCCs, immunolabeling was performed using antibodies to the NCC marker, Crabp1. Crabp1-positive cells were observed in null embryos in streams extending from rhombomeres 4, 6 and 7, which corresponded to areas having high levels of TUNEL-positive cells ().
Genetic background influences penetrance of cardiac anomalies in Fbln1 nulls
While the bleeding, lung abnormalities and perinatal lethality in Fbln1
gene trap homozygotes are in agreement with an earlier analysis of mouse Fbln1 deficiency (Kostka et al., 2001
), abnormalities of the OFT, arch arteries, pharyngeal glands, cranial nerves and cephalic skeleton were not previously reported. Differences in genetic background could account for the phenotypic disparities given that the Fbln1 gene trap homozygotes were a mixed C57BL/6 and 129P2/OlaHsd background whereas embryos homozygous for Fbln1 exon 1
deletion were a mixed C57BL/6 and 129/Sv background (Kostka et al., 2001
). When we examined embryos homozygous for the Fbln1 exon 1
deletion we found hypoplastic thymus (100% penetrance) as well as cardiac defects corresponding to those observed in embryos homozygous for the Fbln1 gene trap mutation (e.g., VSD associated with DORV) (Supplementary Fig. 6). However, the penetrance of cardiac defects in the Fbln1 exon 1 mutants was lower than that of the Fbln1 gene trap mutants (). We subsequently found that penetrance of cardiac defects in the Fbln1 gene trap could be affected by the genetic background. When the Fbln1 gene trap line was backcrossed further onto a C57BL6 background the penetrance of cardiac defects was reduced to levels comparable with the Fbln1 exon 1 deletion on a mixed C57BL/6 and 129/Sv background. Fbln1-deficient embryos were also generated by mating the Fbln1 gene trap strain (mixed C57BL/6 and 129P2/OlaHsd background) to the Fbln1 exon 1 deletion strain (C57BL/6 and 129/Sv background). As shown in , the hybrids displayed a level of penetrance higher than the Fbln1 exon 1 deletion homozygotes having the mixed C57BL/6 and 129/Sv background. These findings suggest that the severity of cardiac defects in embryos deficient in Fbln1 is increased by genetic modifiers found within the129P2/OlaHsd background.
Influence of background on penetrance of cardiac defects in embryos homozygous for the Fbln1 gene trap insertion or homozygous for Fbln1 exon 1 deletion.