SMOC1 Is Essential for Ocular and Limb Development in Humans and Mice

SNP Genotyping, and Fine Mapping with Short Tandem Repeat Markers

Whole-genome SNP genotyping, with the use of GeneChip Human Mapping 50K Array XbaI (Affymetrix, Santa Clara, CA), and fine mapping of possible candidate regions, with the use of additional microsatellite markers, were performed as previously described.^12,15 The list of primers used for fine mapping is presented in Table S2.

Linkage Analysis

Multipoint linkage analyses using aligned SNPs were performed with ALLEGRO software.¹⁶ Two-point linkage analyses of candidate regions were performed with the LINKAGE package MLINK (FASTLINK software, version 5.1). In each program, an autosomal-recessive model of inheritance with complete penetrance and a disease-allele frequency of 0.001 were applied.

Mutation Analysis of Candidate Genes

All coding exons and exon-intron boundaries of RAD51L1 (MIM 602948), ACTN1 (MIM 102575), ERH (MIM 601191), SRSF5 (MIM 600914), DCAF5 (MIM 603812), COX16, EXD2, GALNTL1, SLC39A9, KIAA0247, MED6 (MIM 602984), TTC9 (MIM 610488), MAP3K9 (MIM 600136), and SMOC1 (transcript variant 1, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001034852.1","term_id":"78190497","term_text":"NM_001034852.1"}}NM_001034852.1) were analyzed in the probands of families A, C, and X. The transcript variant 2 of SMOC1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_022137.4","term_id":"78190496","term_text":"NM_022137.4"}}NM_022137.4) is 3 bp shorter than the variant 1, leading to an in-frame amino acid deletion at position 431. PCR was cycled 35 times at 94°C for 30 s, at 60°C for 30 s, and at 72°C for 30–90 s in a total volume of 20 μl containing 30 ng genomic DNA as a template, 0.5 μM forward and reverse primers, 200 μM each deoxyribonucleotide triphosphate (dNTP), 1 × ExTaq buffer, and 0.25 U ExTaq (Takara). All primers were designed with Primer3 software. Detailed information of primers is available upon request. PCR products were purified with ExoSAP (USB) and sequenced with BigDye Terminator 3.1 (Applied Biosystems) on a 3100 Genetic Analyzer. Sequences of patients were compared to reference genome sequences in the UCSC Genome Browser (February 2009 assembly) with Seqscape software, version 2.1 (Applied Biosystems).

Animals

Smoc1 mutant mice, created with the use of the Sleeping Beauty transposon system, have been previously described.¹⁷ Line PV384 was provided by the RIKEN BioResource Center through the National BioResource Project of MEXT, Japan. Three independent mouse lines (no. 1 to no. 3), each with a single insertion in intron 1 of Smoc1, were bred as heterozygotes. Lines 1 and 3 were backcrossed for at least four generations to a C57BL/6J background. Line 2 was maintained with a mixed background of C57BL/6J and ICR. We mainly analyzed line 1, but we confirmed similar phenotypes in lines 2 and 3. Animals were housed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Yokohama City University, School of Medicine. PCR genotyping of mice was performed with the use of genomic DNA from yolk-sac, ear, or tail biopsies. The following primers were used: PV384-WF, 5′-AAAGGCTGGGAATTGTTTGA-3′; PV384-WR, 5′-TGCAGCTGAAACTGTCTCTCC-3′; PV384-MF, 5′-TGTCCTAACTGACTTGCCAAA-3′. The PV384-WF/PV384-WR primers amplified a 441 bp wild-type (WT) product, and the PV384-MF/PV384-WR primers amplified a 218 bp mutant product.

Southern Hybridization

Genomic DNA was extracted from livers or tail biopsies of PV384 heterozygous (Smoc1^Tp/+) mice via standard protocols. The gene-trap insertions were analyzed by Southern hybridization with the use of 10 μg of SacI-, NdeI-, BglII-, and EcoRІ-digested DNA. The probe (451 bp), which hybridized to the internal ribosome entry site (IRES) in the gene-trap vector, was synthesized with the DIG PCR Probe Synthesis Kit (Roche) with the use of the following primers: 5′-CTAACGTTACTGGCCGAAGC-3′ and 5′- CCCAGATCAGATCCCATACAA-3′. Hybridization, washing, and detection of probes were performed according to the manufacturer's protocol. Images were captured with the FluorChem system (Alpha Innotech).

Cloning of Gene-Trap Insertion Sites

After identification of aberrant DNA fragments by Southern hybridization, NdeI-, SacI-, and EcoRІ-digested DNA from PV384 mice was fractionated by electrophoresis, and appropriately sized fragments containing Ol1 (other locus 1), Ol2, and Ol3 were isolated with a QIAEXII Gel Extraction Kit (QIAGEN). The isolated DNA was self-ligated by Ligation High ver.2 (Toyobo), precipitated with ethanol, and dissolved in 20 μl EB buffer (QIAGEN). Inverse PCR was performed in 25 μl reactions, containing 2 μl ligated DNA, 1 × PCR buffer for KOD FX, 0.4 mM each dNTP, 0.5 μM each primer, and 0.5 U KOD FX DNA polymerase (Toyobo). Primers common to Ol1, Ol2, and Ol3 were as follows: Inv-F, 5′- ATCGCCAGTTCTGTATGAACGGTCTGGTCTT-3′; Inv-R, 5′-CCCTCTTTACGTGCCAGCCATCTTAGAGATAC-3′. Confirmatory PCR of gene-trap insertion sites for Ol1, Ol2, and Ol3 loci was performed with the use of the following primers: Ol1-F, 5′-GAGTGGTATTCATTGGATTCTGCTGAT-3′; Ol2-F, 5′-AAATCCAGCTGGCCAACAGACTAAG-3′; Ol3-F, 5′-TTGCCGGGTAGACTCTATCAAGAACCA-3′; TBAL-R, 5′-CTTGTGTCATGCACAAAGTAGATGTCC-3′. Primer sets of Ol1-F/TBAL-R, Ol2-F/TBAL-R, and Ol3-F/TBAL-R could amplify 175 bp, 607 bp, and 767 bp products, respectively. These PCR primer pairs were also used for genotyping of mice harboring a single insertion at the Smoc1 locus.

Confirmation of Promoter- and Poly(A)-Trapped Transcripts

Whole embryos at embryonic day 10.5 (E10.5) and E11.5 were stored in RNAlater solution (QIAGEN). Total RNA was extracted from WT, Smoc1^Tp/+, and Smoc1^Tp/Tp embryos with the use of RNeasy Plus Mini (QIAGEN). One microgram total RNA was subjected to reverse transcription with the use of a PrimeScript 1^st Strand Synthesis Kit with random hexamers (Takara). A control reaction with no reverse transcriptase was included in each experiment. PCR was performed in 20 μl reactions, containing 1 μl cDNA, 1 × PCR Buffer for KOD FX, 0.4 mM each dNTP, 0.3 μM each primer, and 0.4 U KOD FX (Toyobo). Primers used are listed below: Smoc1-F, 5′-GTCCCCACCTCCCCAAGTGCTTTGA-3′; LacZ-R, 5′-TGCCAAAAGACGGCAATATGGTGGAAA-3′; GFP-F, 5′-TCACATGGTCCTGCTGGAGTTCGTGAC-3′; Smoc1-R, 5′-ACACTTGCTCTGGCCAGCATCTTTGCAT-3′. Primer sets of Smoc1-F/Smoc1-R, Smoc1-F/LacZ-R, and GFP-F/Smoc1-R could amplify native Smoc1 (366 bp), promoter-trapped transcripts (Tp-LacZ, 500 bp) and poly(A)-trapped transcripts (Tp-GFP, 308 bp), respectively. The PCR conditions were 98°C for 10 s, 68°C for 1 min, for 30 cycles. Primers for ACTB¹⁸ were used as an internal control. PCR for ACTB was cycled 20 times at 94°C for 20 s, 60°C for 20 s, and 72°C for 30 s in a total volume of 10 μl containing 0.5 μl cDNA, 0.4 μM each primer, 0.2 mM each dNTP, 1 × ExTaq buffer, and 0.5 U ExTaq HS (Takara). All PCR products were electrophoresed on 2% agarose gels.

In Situ Hybridization

Embryos were collected between E9.5 and E13.5. Whole-mount in situ hybridization was carried out as previously described.^19,20 Two fragments of Smoc1 cDNA were obtained as probes by RT-PCR, with the use of total RNA extracted from livers of E16.5 mouse embryos, and subcloned into pCR4-TOPO (Invitrogen). Primer sequences were as follows: probe 1-F, 5′-GTCTGCTCACGCCCCACT-3′; probe 1-R, 5′-CCTGAACCATGTCTGTGGTG-3′; probe P-F, 5′-CAGGAACAGGAAAGGGAAGA-3′; probe P-R, 5′-AAGGGAAAACCACACAGCAC-3′. PCR products were 1023 bp and 1578 bp, corresponding to nucleotide positions 275–1297 and 1849–3426 of the mouse Smoc1 cDNA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001146217.1","term_id":"226246621","term_text":"NM_001146217.1"}}NM_001146217.1), respectively. The cDNA fragment amplified with probe P-F and probe P-R primers was identical to the probe used in a previous report.²¹ Digoxigenin-labeled sense and antisense riboprobes were synthesized with the use of a digoxigenin RNA labeling kit (Roche). These two different antisense probes demonstrated identical staining patterns, and the control sense probes showed no staining. The expression pattern was confirmed with more than three embryos. In addition, the following probes were used: Bmp2 (gift from Y. Takahashi),²² Sox9 (gift from A. Yamada),²² Bmp7 (gift from E.J. Robertson), and Msx2 (gift from Dr. R.E. Maxson, Jr). The numbers of embryos examined were as follows (numerical quantity for WT, Smoc1^Tp/+, and Smoc1^Tp/Tp, respectively, shown in parentheses): Msx2 (2, 1, 3) at E11.5; Bmp2 (3, 0, 3), Bmp7 (3, 0, 3), Msx2 (3, 0, 3), and Sox9 (2, 1, 3) at E12.5; Bmp2 (1, 2, 3), Bmp7 (2, 1, 3), Msx2 (1, 2, 3), and Sox9 (1, 3, 4) at E13.5. Stained embryos were cleared in glycerol to enable images to be produced with a VHX-1000 digital microscope (Keyence).

Histology

Heads of embryos and newborns were fixed overnight in 4% paraformaldehyde in PBS at 4°C. These embryos were then washed in PBS. Frozen samples were serially sectioned at 16 μm (E14.5) and 20 μm (P0). The numbers of eyes examined (WT, Smoc1^Tp/+, Smoc1^Tp/Tp) were as follows: coronally sectioned at E14.5 (8, 10, 12), coronally sectioned at P0 (8, 10, 6), horizontally sectioned at P0 (2, 2, 4). For evaluation of ventral atrophy of the retina, only the coronally sectioned eyes were used. TB staining was performed according to standard protocols. Forelimbs of mice were fixed in 4% paraformaldehyde in PBS, decalcified in 10% EDTA, and embedded in paraffin. Forelimbs were serially sectioned at 4 μm and stained with hematoxylin and eosin.

Evaluation of Optic Nerve Diameter

The palatine and orbital bones were carefully removed to expose the optic chiasm and optic nerve. During the dissection process, 4% paraformaldehyde in PBS was frequently applied onto the gaps between the bone and optic nerve. Xylene cyanol was applied to enhance the outline of optic nerves at poastnatal day 0 (P0). Photographs of optic nerves were taken with a VHX-1000 digital microscope, and the diameter was measured for right and left optic nerves with the bundled software included with the VHX-1000 instrument.

Skeletal Staining

For skeletal preparations, mice were fixed in 99.5% ethanol after removal of the skin and viscera. Cartilage tissues were stained with 0.015% alcian blue and 20% acetic acid in 75% ethanol for three days at 37°C. After dehydration with 99.5% ethanol for three days, bones were stained with 0.002% alizarin red in 1% KOH. Then skeletons were cleared in 1% KOH for several weeks. For P14 mice, soft tissues were dissolved in 2% KOH before alizarin red staining.

Nile Blue Staining

For the study of apoptosis of hindlimbs at E13.5 and E14.5, Nile blue (NB) staining was performed on the basis of a previously described protocol,²³ except that staining was performed at 37°C (not room temperature). Apoptosis was determined by NB-stained (deceased) cells. After rinsing in Tyrode solution, hindlimbs of control (WT and heterozygous littermates) and homozygous mice were evaluated. Photographs of dorsal aspects were taken with a VHX-1000 digital microscope. Experiments were repeated three times, and reproducible representative results are presented.

Smoc1 Expression in the Developing Eye and Limb in Mice

For the examination of Smoc1 expression in the developing eye and limb, whole-mount in situ hybridization of mouse embryos was performed. Smoc1 was expressed in the forebrain, midbrain, hindbrain, pharyngeal arch, somites, and forelimb buds at E9.5 (Figure 2A). At E10.5, Smoc1 expression was observed in the optic stalk (Figure 2B), and at E11.5, expression was localized to the closure site of the optic cup (Figure 2C). Expression of Smoc1 in developing limbs between E10.5 and E11.5 was observed in both dorsal and ventral regions, with a broader pattern of expression in dorsal regions, but expression was not detected in the most anterior, posterior, and distal parts of limb buds (Figures 2D and 2E). Expression coinciding with chondrogenic condensation was observed at E12.5 (Figure 2F), and expression then became restricted to future synovial joint regions at E13.5 (Figure 2G). This dynamic expression suggests that Smoc1 plays a critical role in ocular and limb development.

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Figure 2

Smoc1 Expression in Mouse Embryos

Lateral views of embryos (A–C) and a ventral view of the left part of the head (B′, lateral view is shown at the top).

(A) At E9.5, Smoc1 was expressed in the forebrain, midbrain, hindbrain, pharyngeal arch, somites, and forelimb buds (magenta arrowhead), but not in the optic vesicle (Ov, blue arrow).

(B and B′) Expression in the optic stalk became evident at E10.5 (magenta asterisks), but was not evident in the optic cup (Oc, blue arrow).

(C) Expression was restricted to the closure site of the optic cup (dashed circle) at E11.5.

(D–G) Dorsal and (D′–G′) posterior view of the right hindlimbs (dorsal view is shown at the top in D′–G′). The anterior side is indicated by an A. (D and D′) At E10.5, Smoc1 was more widely expressed in the dorsal part of the limb bud than in the ventral part. Smoc1 expression is undetected in the most anterior, posterior, and distal parts of the limb bud. (E and E′) At E11.5, ventral expression was broader than that in the previous stage. (F and F′) At E12.5, expression was detected in areas consistent with chondrogenic condensation. (G and G′) At E13.5, Smoc1 expression became restricted to future joint regions. Scale bar represents 500 μm.

Ocular and Limb Anomalies in Smoc1 Null Mice

To investigate the pathological basis of MLA due to the loss of SMOC1 function, we obtained Smoc1 mutant mice, PV384.¹⁷ PV384 mice possess gene-trap insertions in the Smoc1 locus and in three other loci. After PV384 mice were bred with C57BL/6J or ICR mice, we obtained three independent lines (no. 1 to no. 3), each with a sole insertion in intron 1 of Smoc1 (Figure S1). We mainly analyzed line 1, but we confirmed similar phenotypes in lines 2 and 3. Heterozygous mutant mice (Smoc1^Tp/+) were healthy and fertile. Homozygous mice (Smoc1^TP/TP) were null mutants, as they showed no native transcript of Smoc1 (Figure S1E). Homozygous mice were viable at P0; however, they did not survive beyond the first 3 wks of life (Figure 3B). Their growth was retarded in comparison to WT and heterozygous littermates at P0 and P14 (Figures 3A and 3C). Developmental defects in eyes and optic nerves were evident at E14.5. Homozygous mice had relatively small eyes, and histological examinations revealed aplasia or hypoplasia of optic nerves (in 10 of 12 optic nerves), atrophy of the anteroventral part of the retina (in 11 of 12 eyes), and extension of the retinal pigmented epithelium (RPE) to the optic nerve (in 10 of 12 eyes) (Figures 3D–3I). These abnormalities were also observed at P0 (aplasia or hypoplasia of optic nerves [in 7 of 10 optic nerves], retinal atrophy [in 6 of 6 eyes], and RPE extension [in 3 of 6 eyes with identifiable optic nerves]) (Figures 3J–3M). WT or heterozygous littermates did not show any such abnormalities, except that a few eyes of heterozygous mice showed extension of the RPE at E14.5, but not at P0 (in 2 of 10 and 0 of 12 eyes, respectively). Toluidine blue (TB) staining showed ganglion cell layers that were thinned and irregular to varying degrees in homozygous mice, suggesting a reduced number of retinal ganglion cells (Figures 3J–3K′). Thus, Smoc1 is required for axon sprouting, elongation, or maintenance of retinal ganglion cells.²⁴ Hypoplasia of optic nerves was further quantitatively confirmed by macroscopic examination: the average diameter of optic nerves of homozygous mice was significantly smaller than that of WT and heterozygous littermates at P0 and P14 (Figures 3L–3Q). These data clearly demonstrate that loss of Smoc1 in mice affects development of the body, retina, and optic nerves, in a manner similar to that seen in MLA patients.^3,4

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Figure 3

Growth and Ocular Phenotypes of Smoc1 Null Mice

(A) Representative Smoc1^Tp/Tp mouse, showing a small body in comparison to Smoc1^Tp/+ and WT littermates.

(B) Genotypes of living pups during the first 3 wk of life.

(C) Body weight of pups of each genotype at P0 (left panel) and P14 (right panel).

(D and E) Relatively small eyes were evident in Smoc1^Tp/Tp mice in comparison to WT mice.

(F–K′) Coronal sections of eyes at E14.5 (F–I) and P0 (J–K′) with TB staining (H, I, and J–K′). (F-I) Atrophy of the anteroventral part of the retina (G, magenta arrowheads, dorsal view shown at the top), hypoplastic optic nerve, and extension of the RPE to the optic nerve (I, magenta arrow) in Smoc1^Tp/Tp mice at E14.5. (J and K) Hypoplastic optic nerve and RPE extension in Smoc1^Tp/Tp mice at P0 (K, magenta arrow). Note that sections in which optic nerves appeared most thick are presented in (H–K). (J′–K′) In higher-magnification views of (J and K), a thinned and irregular ganglion cell layer (white brackets) was observed in Smoc1^Tp/Tp mice. Abbreviations are as follows: le, lens; re, retina; rpe, retinal pigmented epithelium.

(L–P) Ventral views of the brain showing optic nerves at P0 (L and M) and P14 (N–P), showing various degrees of optic nerve hypoplasia.

(Q) Optic nerve diameter. Optic nerves were significantly hypoplastic in Smoc1^Tp/Tp mice in comparison to WT and Smoc1^Tp/+ littermates.

The numbers of pups (B and C) or eyes (Q) corresponding to each genotype are indicated within bars. Error bars indicate standard deviation: ^∗p < 0.01, compared with WT. ^∗∗p < 0.01, compared with WT and Smoc1^Tp/+. Scale bars represent 1 mm (D, E, and L–P), 200 μm (F–I), 500 μm (J and K), and 100 μm (J′ and K′).

Newborn homozygous mice could be readily identified by their hindlimb syndactyly and pes valgus, whereas no abnormalities were observed in WT and heterozygous pups (Figure 4 and Table 1). Interestingly, the severity of syndactyly varied between mouse lines: line 1 exclusively showed soft tissue syndactyly, whereas line 2 frequently showed four digits (Figures 4F and 4J). Skeletal preparations with alcian blue and alizarin red revealed that the foot with four digits had four phalanx and five metatarsals with fusion to each other (Figure 4K). Thus the Smoc1 null mutation resulted in a spectrum of phenotypes, from soft tissue syndactyly to four fused digits, probably due to different genetic backgrounds. Bowed tibiae and hypoplastic fibulae were also consistently observed in homozygous mice (Figures 4H and 4L). The articulation between tibia/fibula and calcanea of homozygous mice appeared malpositioned (Figures 4G and 4K), which might contribute to pes valgus. At P14, soft tissue syndactyly was also evident in most forelimbs of homozygous mice (Figures 4M–4O). Moreover, hindlimbs of homozygous mice showed synostosis between the 4^th and 5^th metatarsals (Figure 4T), which is observed in both the hands and the feet of MLA patients. Thus, many limb anomalies of MLA patients were recapitulated in Smoc1 null mice (Table S1).

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Figure 4

Limb Phenotypes of Smoc1 Null Mice

Limbs of WT (A–D, M, and P) and Smoc1^Tp/Tp mice (E–L, N–O′, and Q–T) at P0 (A–L) and P14 (M–T). Digit identities are indicated by the numbers 1 (thumb, anterior) and 5 (little finger, posterior). Skeletal staining with alcian blue and alizarin red is presented (C, D, G, H, K, L, S, and T). Smoc1^Tp/Tp mice showed pes valgus (E and I), soft tissue syndactyly (F and G), and four digits with metatarsal fusion (J and K). Malposition of the articulation between the tibia/fibula and the calcanea (G and K, magenta arrowheads), bowed tibia (magenta arrows), and hypoplastic fibula (arrowheads) of Smoc1^Tp/Tp mice (H and L) were observed. 2/3 soft tissue syndactyly (N) and 2/3 webbing (O) were evident in forelimbs of Smoc1^Tp/Tp mice. (O′) A transverse section taken at the level indicated by the dashed line in (O) showed 2/3 webbing. 2/3 syndactyly (Q), 2/3/4 syndactyly (R), synostosis between the 2^nd and 3^rd proximal phalanx and metatarsals (S), and synostosis between the 4^th and 5^th metatarsals (T, arrow), observed in the hindlimbs of Smoc1^Tp/Tp mice. Scale bars represent 1 mm (A–O and P–T) or 500 μm (O′).

We would like to thank the patients and their families for their participation in this study. We thank Yoshiko Takahashi (Nara Institute of Science and Technology) and Atsushi Yamada (Showa University) for providing the Bmp2 and Sox9 probes; Elizabeth J. Robertson (University of Oxford) and Makoto Ishibashi (Kyoto University) for the Bmp7 probe; Robert E. Maxson, Jr. (University of Southern California Keck School of Medicine) for the Msx2 probe; Tomonori Hirose, Kazunori Akimoto, and Kazunori Sasaki (Yokohama City University) for providing useful information about mouse breeding, taking photos on a stereo microscope, and mRNA quantification; and Kohei Shiota and Sumiko Kimura (Kyoto University) for helpful comments about NB staining and limb anomalies. This work was supported by research grants from the Ministry of Health, Labour and Welfare (T. Furuichi, N. Miyake, N. Matsumoto, and H.S.) and the Japan Science and Technology Agency (N. Matsumoto), a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (T. Furuichi and N. Matsumoto), and a Grant-in-Aid for Young Scientist from the Japan Society for the Promotion of Science (K.N., H.D., N. Miyake, and H.S.). This work has been carried out at the Advanced Medical Research Center of Yokohama City University.