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doi: 10.1242/10.1242/dev.00399

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Cell-autonomous involvement of Mab21l1 is essential for lens placode development

¹ Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
² Department of Molecular Embryology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670, Japan
³ RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan
⁴ Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

View larger version (36K): [in a new window]   Fig. 1. Generation of the Mab21l1-null allele. (A) Maps of the Mab21l1 locus, the targeting vector used for mutagenesis and the Mab21l1 targeted allele. White boxes represent the Mab21l1-coding region, the neo cassette and the DTA cassette. Black bars represent DNA fragments used as 5' and 3' probes for Southern blots; the sizes of restriction fragments detected are shown with lines. Arrowheads indicate the location of the primer pairs (primers m1F, m1R and neoR) used for PCR genotyping, with sizes of the PCR products given above. E, EcoRI; B, BamHI; K, KpnI; S, SalI. (B) Southern blots of tail DNA from progeny of heterozygous mice after KpnI and SalI digestion. Blots were probed with the 5' probe, a 3.0 kb KpnI-EcoRI fragment, or the 3' probe, a 1.3 kb BamHI-SpeI fragment. The endogenous KpnI fragment hybridizing with the 5' and 3' probes is 19 kb in length. Insertion of the neo cassette containing SalI sites reduced the KpnI fragments to 8.3 and 7.5 kb. (C) PCR genotyping of genomic DNA. Sizes of PCR products are indicated on the right. (D) Whole-mount in situ hybridization of Mab21l1 in E10.5 heterozygous and homozygous embryos. Note, Mab21l1 expression seen in the heterozygous embryo (left) is absent in the homozygote (right).

View larger version (86K): [in a new window]   Fig. 2. Gross morphological and histological analyses of Mab21l1 mutant adult mice. Adult heterozygous (A) and homozygous (B) mice, the latter lacking eyes. (C) Whole-mount view of adult eyes shows the iris and lens to be absent in a homozygous eye. (D,E) Hematoxylin and Eosin (HE) stained coronal sections of 3-month-old adult eyes. The adult Mab21l1-/- eye (E) is much smaller than that of Mab21l1+/- mice (D). Although a comparable lens structure is not present in the former, an optic nerve and retinal pigmented epithelium are apparent (E). (a-d) Higher magnification views (boxes in D and E). The connection between the lens-like rudiment and the cornea is abnormal in the Mab21l1-/- case (b). Morphologically, retinal lamination and major classes of retinal cells appear in both Mab21l1+/- and Mab21l1-/-. However, outer and inner nuclear layers in the Mab21l1-/- eye (d) are much thinner than in the Mab21l1+/- counterpart (c). Adult heterozygous (F) and homozygous (G) male sex organs. The arrow indicates the small preputial gland in a homozygote. (H) Dissected male preputial glands from adult heterozygote (left) and homozygote (right) embryos. Insets show HE-stained paraffin wax sections. Overall size and intralumenal spaces (inset) in the homozygous preputial gland are reduced. co, cornea; inl, inner nuclear layer; ir, iris; lru, lens-like rudiment; on, optic nerve; onl, outer nuclear layer; pe, retinal pigmented epithelium; P, penis; Pu, pureputial gland; T, testis. Scale bars: 1mm for D,E, insets in H; 200 µm for a,b; 100 µm for c,d.

View larger version (75K): [in a new window]   Fig. 3. Histological and morphological analysis of eye development in Mab21l1-deficient mice. Gross appearance of Mab21l1+/- (A) and Mab21l1-/- (B) eyes. The Mab21l1+/- lens is well developed (A; P1, E16) and pigmented retinal epithelium is almost round, whereas the Mab21l1-/- eye has no lens and the retinal pigmented epithelium is abnormally indented (B; P1, E16, E12.5). No difference is evident at E10.5. HE-stained coronal sections of E17 (C,D), E13 (E,F), E10.5 (G,H) and E9.5 (I,J) embryos. At E17 and E13, no lens is evident in the Mab21l1-/- eye (D,F). Brackets (C,D) indicate the thickness of the retina, arrowheads (D) indicate abnormal corneal swelling and the arrow (D) indicates the optic nerve. (H) At E10.5, a malformed lens placode is evident in the Mab21l1-/- eye (arrowheads). (I,J) At E9.5, little difference is evident between Mab21l1+/- (I) and Mab21l1-/- (J) eyes. The presumptive lens placode is abnormally thin and narrow in Mab21l1-/- embryos (J; brackets) compared with Mab21l1+/- embryos (I; brackets). Scale bars: 200 µm for C,D; 100 µm for E,F; 50 µm for G-J.

View larger version (89K): [in a new window]   Fig. 4. Expression of crystallin genes and retinal differentiation marker genes in Mab21l1+/- and Mab21l1-/- embryos. In situ hybridization of paraffin wax sections at E12.5 (A-D) and E15 (E-H). Expression of: A-crystallin in Mab21l1+/- (A) and Mab21l1-/- (B) embryos; A-crystallin in Mab21l1+/- (C) and Mab21l1-/- embryos (D); Chx10 in Mab21l1+/- (E) and Mab21l1-/- (F) embryos; and Brn3b in Mab21l1+/- (G) and Mab21l1-/- (H) embryos. Arrowheads (B,D) indicate crystallin distribution in an Mab21l1-/- embryo. Scale bars; 100 µm for A-D; 200 µm for E-H.

View larger version (70K): [in a new window]   Fig. 5. Expression of Mab21l1 and Mab21l2 transcripts during eye development. In situ hybridization of coronal frozen (A-F) and paraffin wax (G,H) sections of wild-type eyes. Mab21l1 but not Mab21l2 transcripts are expressed in lens, whereas both are similarly present in retina. lp, lens placode; nr, neural retina; on, optic nerve; ov, optic vesicle; pe, retinal pigmented epithelium; pnr, presumptive neural retina; ppe, presumptive retinal pigmented epithelium; le, lens epithelium; se, surface ectoderm. Scale bars: 50 µm for A,B; 100 µm for C-H.

View larger version (50K): [in a new window]   Fig. 6. Analysis of cell proliferation and apoptotic cell death in Mab21l1+/- and Mab21l1-/- lens placodes. Immunohistochemical detection of BrdU incorporation in Mab21l1+/- and Mab21l1-/- embryos at various somite stages (A-H). Brackets (D,F) indicate reduced incorporation of BrdU in Mab21l1-/- small lens placodes. Red dotted lines indicate a Mab21l1+/- lens vesicle (G) and a Mab21l1-/- malformed lens placode (H). The inset shows a HE-stained serial section of the area within the box (H). TUNEL assay of 35-somite stage Mab21l1+/- (I) and Mab21l1-/- (J) embryos. Numbers of TUNEL-positive cells are slightly increased in the Mab21l1-/- lens pit (arrowheads). Histogram showing the percentage of BrdU-positive cells in Mab21l1+/- and Mab21l1-/- lens anlages (K). The ratio of BrdU-positive cells is reduced in the Mab21l1-/- mutants (t-test; 27 and 32-somite stages, P<0.004; other somite stages, P<0.0001). Vertical bars represent standard errors. lp, lens placode; lv, lens vesicle; ov, optic vesicle; pi, lens pit; se, surface ectoderm. Scale bars: 50 µm.

View larger version (108K): [in a new window]   Fig. 7. Distribution of Mab21l1-/- cells in chimeric embryos. (A) Whole-mount view of an Mab21l1+/+ chimeric embryo. (B-D) Whole-mount views of Mab21l1-/- chimeric embryos. (A'-D') Histological sections of embryos identical to those shown in A-D, respectively. (a-d) Higher magnification views of areas within the boxes in A'-D'. In the Mab21l1+/+ chimeric embryo, ß-galactosidase negative cells are distributed in the retina (A') and lens epithelium (a, arrowheads). No Mab21l1-/- cells (ß-galactosidase negative cells are white) are present in the lens, whereas both wild-type (ß-galactosidase positive cells are blue) and Mab21l1-/- cells are present in retina (B',C',b,c). (C') Despite the high contribution of Mab21l1-/- cells in the retina, the lens is similar in size to that of the chimera in B'. A high contribution of Mab21l1-/- cells leads to absence of the lens (D,D',d). Scale bars: 100 µm for A'-D', 50 µm for a-d.

View larger version (66K): [in a new window]   Fig. 8. Expression of PAX6 and Foxe3 in Mab21l1+/- and Mab21l1-/- embryos. In situ hybridization (A,B,E,F,I,J) or immunohistochemistry (C,D,G,H) of Mab21l1+/- and Mab21l1-/- frozen sections at 28- (A-D) and 35- (E-H) somite stages, and paraffin wax sections at the 40-somite stage (I,J). Expression of: Foxe3 transcripts in (A,E,I) Mab21l1+/- and (B,F,J) Mab21l1-/- embryos; (C,G) PAX6 protein in Mab21l1+/- and (D,H) Mab21l1-/- embryos. Expression of Foxe3 is reduced in Mab21l1-/- presumptive lens placode (B, arrowheads), whereas expression of PAX6 is maintained at the 28-somite stage (D). At the 35-somite stage, Foxe3 is expressed in some cells (F, arrowhead), whereas PAX6 is expressed in most cells in the Mab21l1-/- lens lineage structure (H). By the 40-somite stage, Foxe3 expression has disappeared (J). Scale bars: 50 µm.

View larger version (92K): [in a new window]   Fig. 9. Expression of Mab21l1 in Sey/Sey mutant embryos. In situ hybridization of wild-type (A,C,E) and Sey/Sey (B,D,F) frozen sections at 22-somite (A,B) and 35-somite stages (C-F). Mab21l1 expression is almost totally abolished at the 22-somite stage (B), and greatly reduced at the 35-somite stage in the Sey/Sey mutant embryos (D). However, the Mab21l2 expression level is basically identical between wild-type and Sey/Sey mutant embryos (E,F). Scale bars: 100 µm.