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First published online 11 October 2006
doi: 10.1242/dev.02647

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Essential pro-Bmp roles of crossveinless 2 in mouse organogenesis

¹ Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.
² Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.
³ Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA.

View larger version (42K): [in a new window]   Fig. 1. Generation and external phenotypes of Cv2 knockout mice. (A) Construction of the targeting vector. In the mutant allele, the first methionine of Cv2 was replaced in-frame with that of tau-lacZ. The locations of PCR primers used for selection of recombinant ES cells (blue arrows) and genotyping (white arrowheads), and probes for Southern blot analysis (black and orange boxes) are indicated. Bg, BglII; Sc, SacI; Xb, XbaI. (B,C) Genomic PCR and Southern blot analyses for the homologous recombinant ES cell line. (B) Recombination at the 3' region of the Cv2 genome (resulting in a 3 kb PCR fragment) was observed. (C) Correct integration was further examined by Southern blot using the 5' probe (wild-type, heterozygous and homozygous, from left to right lanes). (D) PCR genotyping of mutant mice. The wild-type allele band (564 bp) and the mutant allele band (412 bp) were amplified by PCR. (E) RT-PCR analysis of Cv2 expression in E12.5 mutant mice. (F,G) External appearances of P0 neonates (F) and E15.5 embryos (G). The control mice are on the left side and the null mutant mice are on the right side. At a low penetrance, exencephaly was also observed (arrows) at E15.5 (about 7% of embryos; n=86).

View larger version (110K): [in a new window]   Fig. 2. Expression of Cv2 analyzed with nlacZ-knocked-in mice. (A,B) At E10.5, lacZ staining was observed in the dorsal midline of the neural tube (arrowhead), migrating neural crest (nc), head mesenchyme, trigeminal ganglion (t), otic vesicle (o), para-aortic region and mesonephros (mn). (B) Transverse section at position shown in A. (C,D) E11.5 embryo. Strong staining appeared in the presumptive vertebral body (arrow) and arch regions (arrowhead). Black arrowhead indicates the dorsal midline of the neural tube. (E-G) At E14.5, the staining was observed in the vertebral body (vb), vertebral arch (va), dorsal root ganglion (drg), part of the neural tube, lung (l), heart (h) and ribs (r). (E) Sagittal section of the trunk region, showing that intervertebral discs (ivd) are lacZ negative. (G) Higher magnification of the neural tube and vertebra. (H-K) Developing kidneys. The condensed nephrogenic mesenchymes were stained with X-gal at E14.5 (H). At P0 (I-K), the staining was observed in the condensed nephrogenic mesenchymes (arrowheads) surrounding the distal tip of the collecting duct, and in the comma-shaped bodies (cb).

View larger version (64K): [in a new window]   Fig. 3. Skeletal defects in the Cv2-/- mice. (A-O) Control littermate and (A'-O') Cv2-/- mutant. (A,A') Skeletons of P0 littermates, (B,B') the atlas, (C,C') the axis, (D,D') the first thoracic vertebra, (E,E') the first lumbar vertebra and (F,F') the first sacral vertebra at P0. (A-F,A'-F') In the mutant, the vertebral arch was partially or completely lost (arrows in B'-F'). (G,G') Ventral views of the thoracic to lumber regions. In the mutant, the 13th ribs were only vestigial (arrows in G'). T13, 13th thoracic vertebrae. (H,H',I,I') Defects of the vertebral arches were also observed in the Cv2-/- mutants at E14.5 (H',I'). Black arrows (H) show the normal vertebral arches. Gray arrows (H') indicated the vestigial lateral components of the vertebral arches. (J,J') In the mutant, the laryngeal cartilages were small (or partially lost) and the bronchial cartilages were completely lost (arrow in A' and bracket in J') at P0. (K,K') Dorsal views of the skull. In the mutant, the unossified region of the metopic suture was widely opened (arrow in K'). f, frontal bone; p, parietal bone. (L,L') Ventral views of the skull base. In the mutant, the basisphenoid (bs) had a cavity in the middle (arrow in L'). bo, basioccipital bone. (M,M') Side views of the skull. In the mutant, the retrotympanic process (rp, white arrow in M) of the squamosal bone was lost. st, squamosal temporaris. (N,N') In the mutant, the scapula was small or had a hole (arrowhead in N'), and the deltoid tuberosity was lost (arrow in N'). (O,O') In the mutant, reduction of the pubic bone body and diastasis of the symphysis were observed (O'). (A-G,A'-G',J-O,J'-O') P0, (H,I,H',I') E14.5.

View larger version (72K): [in a new window]   Fig. 4. Requirement of Cv2 for cartilage differentiation in vivo and in vitro. (A-H) E14.5 and (I-R) E12.5. (A,B) Nuclear Fast Red and Alcian Blue staining of the cross-sections of the trunk region at E14.5. The vertebral arch was lost in the mutant (B). (C,D) Hematoxylin and Eosin staining of the neighboring sections of A and B (higher magnification views of the vertebral arch region indicated by squares in A and B). The condensed cell mass corresponding to the dorsal vertebral arch cartilage (arrowheads) was replaced with the mesenchymal cells in the mutant (D). (E,F) The vertebral body was reduced and had less cartilage matrix in the mutant (F). (G,H) Sagittal sections showed that the rostrocaudal length of the vertebral body was also reduced in the mutant (bracket, compare H with G). ivd, intervertebral disc. (I,J) Immunostaining of the early dorsal sclerotomal marker Zic1 in the presumptive vertebral arch region showed no reduction in the mutant (J) at E12.5. (K,L) In situ hybridization analysis of the early sclerotomal gene Pax1 in the presumptive vertebral body region showed no reduction in the mutant (L). (M) RT-PCR analysis of the trunk tissues also showed that the sclerotomal markers Pax1 and Sox9 were expressed normally in the mutant at E12.5. The expression levels of the Bmp genes were also unchanged in the mutant. (N,O) Hematoxylin and Eosin staining of the presumptive vertebral body (surrounding the notochord) in the control (N) and mutant (O) mice. (P,Q) Ki67 immunostaining. Ki67+ mitotic cells were increased in the vertebral body region in the mutant (Q). Scale bar: 50 µm. (R) Statistical analysis of P and Q. The percentages of Ki67+ cells in the 150 µm x 150 µm square regions surrounding the notochord were presented. The averages ±s.d. (error bars) from three sets of littermates are shown. *P<0.05. (S) RT-PCR analysis showed that Cv2-/- MEF cells had attenuated responses to Bmp4 for the induction of aggrecan 1 and Col2a1.

View larger version (116K): [in a new window]   Fig. 5. Genetic enhancement of Cv2-/- phenotypes observed by deleting one copy of Bmp4. (A-F) Skeletal samples of P0 neonates. Lumbar vertebrae. (A-C) Frontal view. (D-F) Left lateral view. Arrow, ossification of the vertebral body; arrowhead, ossifying part of the vertebral arch. (G-I) External appearance of P0 neonate heads. Arrowhead, missing eye. Cv2+/-;Bmp4+/- (A,D,G), Cv2-/-;Bmp4+/+ (B,E,H) and Cv2-/-;Bmp4+/- (C,F,I) mice.

View larger version (85K): [in a new window]   Fig. 6. Kidney defects in the Cv2 mutant. (A-D) External appearances (A,B) and longitudinal sections (C,D; along the longest axis) of the kidneys at P0. Scale bars: 500 µm. Reduced kidney size was observed in the Cv2-/- mutant (B,D). (E,F) HE staining showed no obvious morphological defects of the renal corpuscle in the Cv2-/- mutant kidney at P0 (F). (G,H) The kidney was smaller in the Cv2-/- mutant (H) at E14.5. (I,J) Whole-mount in situ hybridization of Pax2 expression in the control (I) and mutant (J) kidneys. The Pax2-positive condensed mesenchymes were formed in the mutant, but the number was reduced (K; *P<0.05, t-test). (L,M) WT1-positive condensed mesenchymes and glomerular podocytes (green) and E-cadherin-positive collecting ducts (red) are shown in whole-mount immunostaining. The general architecture of the kidney was largely unaffected in the Cv2-/- mutant (M).

View larger version (50K): [in a new window]   Fig. 7. Kidney defects are enhanced in the Cv2;Kcp compound mutant. (A) Numbers of the glomeruli in the maximal longitudinal section of the kidney. There was a significant difference between the control and Cv2-/- mutant kidneys. In addition, a significant difference was found between the Cv2-/- mutant with both Kcp alleles and that without one or two Kcp alleles (***P<0.001; Tukey test). Error bars show s.d.; n.s., no significant difference. (B-G) External appearances (B-D) and longitudinal sections (E-G; along the longest axis) of the kidneys at P0. Scale bars: 500 µm. (B,E) There was no decrease in size in the Kcp-/- kidney, even on the Cv2+/- background (not shown). (C,D,F,G) The small-kidney phenotype of the Cv2-/- mutant was enhanced on the Kcp+/- and Kcp-/- backgrounds.