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. 2011 Jan 15;20(2):271-85.
doi: 10.1093/hmg/ddq462. Epub 2010 Oct 20.

Disruption of PCP signaling causes limb morphogenesis and skeletal defects and may underlie Robinow syndrome and brachydactyly type B

Bing Wang  1 , Tanvi Sinha, Kai Jiao, Rosa Serra, Jianbo Wang
Affiliations

Disruption of PCP signaling causes limb morphogenesis and skeletal defects and may underlie Robinow syndrome and brachydactyly type B

Bing Wang et al. Hum Mol Genet. .

Abstract

Brachydactyly type B (BDB1) and Robinow syndrome (RRS) are two skeletal disorders caused by mutations in ROR2, a co-receptor of Wnt5a. Wnt5a/Ror2 can activate multiple branches of non-canonical Wnt signaling, but it is unclear which branch(es) mediates Wnt5a/Ror2 function in limb skeletal development. Here, we provide evidence implicating the planar cell polarity (PCP) pathway as the downstream component of Wnt5a in the limb. We show that a mutation in the mouse PCP gene Vangl2 causes digit defects resembling the clinical phenotypes in BDB1, including loss of phalanges. Halving the dosage of Wnt5a in Vangl2 mutants enhances the severity and penetrance of the digit defects and causes long bone defects reminiscent of RRS, suggesting that Wnt5a and Vangl2 function in the same pathway and disruption of PCP signaling may underlie both BDB1 and RRS. Consistent with a role for PCP signaling in tissue morphogenesis, mutation of Vangl2 alters the shape and dimensions of early limb buds: the width and thickness are increased, whereas the length is decreased. The digit pre-chondrogenic condensates also become wider, thicker and shorter. Interestingly, altered limb bud dimensions in Vangl2 mutants also affect limb growth by perturbing the signaling network that regulates the balance between Fgf and Bmp signaling. Halving the dosage of Bmp4 partially suppresses the loss of phalanges in Vangl2 mutants, supporting the hypothesis that an aberrant increase in Bmp signaling is the cause of the brachydactyly defect. These findings provide novel insight into the signaling mechanisms of Wnt5a/Ror2 and the pathogenesis in BDB1 and RRS.

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Figures

Figure 1.
Figure 1.
The mammalian PCP gene Vangl2 is required for digit skeletal formation and genetically interacts with Wnt5a during limb skeletal development. (A–C) Forelimbs from E18.5 wild-type embryos or Vangl2Lp/Lp and Wnt5a+/−; Vangl2Lp/Lp mutants were imaged with the ventral side facing up. Note that in Vangl2Lp/Lp and Wnt5a+/−; Vangl2Lp/Lp mutants, digits 3, 4 and 5 had no nails or only rudimentary nails [black arrowheads in (B)]. (D) Skeletal staining with Alcian blue and Alizarin red revealed that in wild-type forelimb, digits 2–5 consisted of the metacarpal (mc) and three phalanges (p1, p2 and p3). (E) In Vangl2Lp/Lp forelimbs, however, only one proximal phalanx (p1) and one distal phalanx resembling the wild-type terminal phalanx p3 formed [referred to as p2/3 in (E)]. (F) In Wnt5a+/−; Vangl2Lp/Lp mutants, reduced dosage of Wnt5a caused more severe defects where digits 4 and 5 had only one phalanx [red arrows in (F)]. (G–I) In the hindlimb of Vangl2Lp/Lp mutants, only digits 2 and 5 displayed loss of the middle phalanges [black arrows in (H)], whereas in Wnt5a+/−; Vangl2Lp/Lp mutants, all digits 2–5 displayed a loss of the middle phalanges. (J–N) In contrast to the digit, the long bones in Vangl2Lp/Lp mutants appeared normal at E18.5 [compare (J) with (K) and (M) with (N)]. In Wnt5a+/−; Vangl2Lp/Lp mutants, however, the radius and ulna in the forelimb were shorter [red arrowhead in (L)], whereas the tibia and fibula in the hindlimb became grossly misshapen and shortened [red arrowhead in (O)].
Figure 2.
Figure 2.
Digit skeletal defects in mouse PCP mutants arise at early stages of endochondral bone formation. (A–D) E12.5 forelimbs from control, Vangl2Lp/Lp, Wnt5a+/−; Vangl2Lp/Lp and Wnt5a−/− mutants were stained with Alcian blue; anterior to the top and posterior to the bottom of each panel. Note the shortening and widening of digit condensates (red brackets) in each mutant. (E–H) Digit 4 from each forelimb was micro-dissected and visualized transversely from its side, dorsal to the left and ventral to the right. Note the thickening of the digit condensates along the dorsal–ventral axis (black brackets) and the increased distance between the distal end of the condensates and the AER (blue brackets). (I–L) E13.5 forelimb from control and mutant embryos stained with Alcian blue. The metacarpal–phalangeal [mp, red arrows in (G)] and inter-phalangeal [p1/p2, blue arrow in (G)] joints have formed in control limbs at this stage, whereas only a single joint is visible in most of the digits in Vangl2Lp/Lp and Wnt5a+/−; Vangl2Lp/Lp mutants [red arrow in (J) and (K)]. (M–P) Measurement of the length, width, thickness and distance to the AER of digit 4 (D4) pre-chondrogenic condensates in E12.5 control, Vangl2Lp/Lp and Wnt5a+/−; Vangl2Lp/Lp forelimb. *P ≤ 0.05 and ***P ≤ 0.005.
Figure 3.
Figure 3.
Early limb bud shape and dimensions are altered in mouse PCP mutants. Forelimb buds from 40 somites (approximately E11) wild-type (A and C) and Wnt5a+/−; Vangl2Lp/Lp mutants were imaged from the dorsal side (A and B) or from the posterior side (C and D). In (A) and (B), two perpendicular double arrow lines (black and red) were drawn to measure the maximum length and width of the limb buds. The white brackets in (C) and (D) represent the presumptive progress zone region, defined as 100 µm from the AER. A blue line was drawn perpendicular to the dorsal–ventral midline to determine the thickness of the progress zone.
Figure 4.
Figure 4.
Reduced cell density and cell number in the distal region of Vangl2Lp/Lp and Wnt5a+/−; Vangl2Lp/Lp forelimb buds. E12.5 forelimbs from wild-type, Vangl2Lp/Lp and Wnt5a+/−; Vangl2Lp/Lp embryos were serially sectioned sagittally. Every third section was stained with Alcian blue to identify pre-chondrogenic condensates [(A), (F) and (K), only the images of digit 4 are shown in each panel]. One of the adjacent sections was subsequently stained with H&E to examine the histology (B, G and L). The images from Alcian blue and H&E staining were then super imposed (C, H and M) in Photoshop, and pre-chondrogenic condensates were outlined in yellow in each panel. The neighboring distal regions were outlined in black boxes and shown in high magnification in (D), (I) and (N), respectively. To examine the cell morphology in the distal region [red boxes in (D), (I) and (N)] in greater detail, another adjacent section was stained with the fluorescent dye calcein and the distal 200 µm regions were imaged with confocal microscopy (E, J and O). White scale bars represented the presumptive progress zone. Red scale bars represented regions immediately proximal to the progress zone.
Figure 5.
Figure 5.
Perturbed signaling network in E11.5 Vangl2 mutant forelimb buds caused defects in cell death and proliferation. Transverse sections of E11.5 control (A) and Vangl2Lp/Lp mutant (B) forelimb buds were stained with antibody against cleaved caspase 3. Apoptotic cells were not found in wild-type distal limb mesenchyme within 200 µm from the AER [white circle in (A) and (B)], but could be detected in this region in Vangl2Lp/Lp mutants [green signals indicated by white arrowheads in (B)]. Nuclei were counterstained in red. White signals were auto-fluorescent blood cells in the limb. Sections from E11.5 control (C) and Vangl2Lp/Lp mutant (D) forelimb buds were also stained with an anti-phospho-histone H3 (pHH3) antibody to assess the cell proliferation rate in the distal limb mesenchyme in a 200 µm diameter circle adjacent to the AER [white circles in (C) and (D)]. Nuclei were counterstained in red. The total number of nuclei within the 200 µm circle was not changed in the Vangl2Lp/Lp mutant at this stage (465 ± 39 in controls versus 479 ± 22 in Vangl2Lp/Lp), but the percentage of pHH3-positive nuclei [green signals in (C) and (D)] was reduced significantly by 30.3% in Vangl2Lp/Lp mutants (6.5 ± 1.7% in controls versus 4.5 ± 1.2% in Vangl2Lp/Lp). (E) Quantitative real-time PCR indicated that in Vangl2Lp/Lp forelimb buds, Grem1 expression was not changed at the 39 somite stage (ss, approximately E11), but was reduced by 34.2% at 45ss (approximately E11.5), whereas Msx2 was increased by 2.1-fold. The levels of Fgf4 and Spry4 were also reduced by 42.9 and 35.3%, respectively, but the expression of Fgf8 was not altered. ***P ≤ 0.005 and *P ≤ 0.05. Transverse sections of E11.5 control (F) and Vangl2Lp/Lp mutant (H) forelimb buds were stained with an anti-phospho-Smad1/5/8 antibody to examine Bmp signaling activity. In wild-type embryos, pSmad1/5/8 staining was only detected in the proximal region of the forelimb [green signals in (F)]. In Vangl2Lp/Lp mutants, however, pSmad1/5/8 staining could be detected in both the proximal and distal limb mesenchyme (H). (G) and (I) are higher magnification views of the distal region in (F) and (H), showing that pSmad1/5/8 staining could be detected in the AER in Vangl2Lp/Lp mutants (I), but not in controls (G). Whole-mount in situ hybridization indicated that by E13.5, although Fgf8 remained to be expressed in the distal region of each digit primordial in the wild-type (J), its expression was completely abolished in Vangl2Lp/Lp mutant forelimbs [red arrows in (J)]. (K) and (L) are higher magnification views of the boxed areas in (J).
Figure 6.
Figure 6.
Reducing Bmp4 dosage by 50% partially rescued the loss of phalanges in Vangl2Lp/Lp forelimb. E18.5 forelimbs from Bmp4LacZ/+; Vangl2Lp/+ (A), Vangl2Lp/Lp (B) and Bmp4LacZ/+; Vangl2Lp/Lp (C) embryos were stained with Alcian blue and Alizarin red. In the control forelimb from Bmp4LacZ/+; Vangl2Lp/+ embryo, 50% reduction in Bmp4 dosage did not alter limb skeletal formation [(A), digits 2–5 consisted of metacarpal (m) and three phalanges (p1, p2 and p3)]. In contrast, reducing Bmp4 dosage in Vangl2Lp/Lp mutants was able to partially rescue the digit defects. In Vangl2Lp/Lp mutants, all digits in the forelimb lost one middle phalanx [(B), green arrows]. In Bmp4LacZ/+; Vangl2Lp/Lp embryos, however, the reduction in Bmp4 dosage allowed a normal number of phalanges to form in digits 2 and 4 [(C), red arrowheads]. When digit 2 from Bmp4LacZ/+; Vangl2Lp/Lp forelimb (E) was compared side by side with that from Vangl2Lp/Lp littermates, we found that the length of m and p1 was similar [compare black lines between (D) and (E)]. In contrast, the total length of the distal p2 and p3 in Bmp4LacZ/+; Vangl2Lp/Lp mutants was significantly longer than the fused p2/3 in Vangl2Lp/Lp mutants [red line between (D) and (E)]. Similarly, in digit 4, the length of m and p1 was not different between Bmp4LacZ/+; Vangl2Lp/Lp and Vangl2Lp/Lp embryos [compare black lines between (F) and (G)], but the total length of the distal p2 and p3 in Bmp4LacZ/+; Vangl2Lp/Lp mutants was significantly longer than the fused p2/3 in Vangl2Lp/Lp mutants [red line between (F) and (G)]. These data suggested that the additional phalanges in Bmp4LacZ/+; Vangl2Lp/Lp mutants arose from longer distal pre-chondrogenic condensate formation, rather than altered segmentation of the digit pre-chondrogenic condensates. (H) Schematic diagram of our proposed model: Wnt5, probably through Ror2, activates PCP-mediated morphogenesis to control limb bud shape and dimensions. Proper limb bud shape and dimensions in turn sustain the signaling network important for limb growth, patterning and skeletal formation.
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