QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00192

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marcil, A. Articles by Drouin, J. Search for Related Content PubMed PubMed Citation Articles by Marcil, A. Articles by Drouin, J.

Pitx1 and Pitx2 are required for development of hindlimb buds

¹ Laboratoire de génétique moléculaire, Institut de recherches cliniques de Montréal, 110 avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada
² Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109-0638, USA

View larger version (24K): [in a new window]   Fig. 1. Pitx gene alleles and left-right (LR) asymmetry during hindlimb development. (A) Schematic representation of the mouse Pitx1 and Pitx2 genes and of mutant alleles used in the present study. Numbered boxes represent exons, and in each case, the null alleles were produced by deletion of the homeodomain-encoding exon. In the Pitx2neo allele, arrowheads indicate the position of loxP sites used by CRE recombinase to yield the null allele. (B) Size reduction in femur length observed in Pitx1-/- embryos. Dissected femurs from right and left side of the same skeleton stained at E17.5 for bone (red) and cartilage (blue) are shown for wild-type and knockout (-/-) embryos. (C) All Pitx1-/- embryos examined in the pure 129sv genetic background showed a loss of hindlimb (HL) digit 1 (I) on the right side only, whereas in mixed genetic background (129sv / Balb/c) all five digits were present on both sides. (D) Ventral view of skeletal preparations showing vertebrae and HL of E17.5 mice either heterozygous (+/-) or homozygous (-/-) for the Pitx1 knockout allele. The first sacral vertebra is indicated (S1); pelvic bones are normally (+/-) attached to S1 by the distal end of the ilium. In the majority of Pitx1-/- embryos, this attachment is through the acetabulum because the ilium does not form in Pitx1-/- embryos (Lanctôt et al., 1999b). However, in few rare cases, femur and pelvic bone attachment to the vertebrae is displaced posteriorly, usually to S2, and in some cases (like the one shown here), displacement is even greater (S3) on the right than left side.

View larger version (58K): [in a new window]   Fig. 2. Loss of proximal (femur) and anterior (tibia and first digit) bones in hindlimbs (HL) of mice mutant for both Pitx1 and Pitx2. Skeletal preparations (Alizarin Red, bone; Alcian Blue, cartilage) of E16.5 wild-type (WT) and Pitx1-/-,Pitx2neo/neo embryo showing the pelvic area (top right), the right and left dissected HL with one remaining zeugopod bone and four digits (I,III,IV,V; bottom right), as well as an enlargement of the right HL autopod (bottom left) showing the remaining zeugopod bone contacting the calcaneus (Ca). Based on this, it is concluded that the remaining bone is the fibula. Small cartilaginous remnants (arrowheads) between the pelvic bone and fibula could be the only remain of the femur. This skeleton represents the most extreme phenotype seen in this embryo series.

View larger version (26K): [in a new window]   Fig. 3. Progressive penetrance of hindlimb (HL) phenotype observed in series of Pitx1-/-,Pitx2+/- embryos. All embryos observed fitted the sequence of bone losses illustrated here. All dissected hindlimbs are positioned similar to the wild-type preparation (A). (B) The first digit of the right HL is missing. (C) The right tibia is severely affected. (D) The right tibia and femur did not form and the left digit 1 has disappeared. (E) The left tibia is partially lost. (F) There is only a remnant of the left femur. Fe, femur; T, tibia; Fi, fibula; digit numbers are shown in parentheses.

View larger version (54K): [in a new window]   Fig. 4. Analysis of hindlimb (HL) bud formation in wild-type, Pitx1-/- and Pitx1-/-,Pitx2+/- embryos. Dorsal views of embryos are shown with assessment of developmental stage provided by somite (so) count. (A) The HL-specific transcription factor Tbx4 mRNA was revealed by whole-mount in situ hybridization and found to be downregulated in mutant embryos (E10.5). This staining offered the best contrast to outline the position of somites along the AP axis and these are indicated by numbers for each embryo. Both mutant embryos show smaller right and left HL bud compared with WT, with greater reduction on the right side. (B) In situ hybridization for Fgf8 revealing the AER. (C) In situ hybridization for Fgf10 marking the HL bud mesenchyme (E10.0). (D) In situ hybridization for Meis2 mRNA revealing the proximal segment of the HL bud (E11.5). Similar results were obtained with Meis1 (data not shown). (E) In situ hybridization for Fgf10 in HL field of 25-somite embryos (E9). (F) In situ hybridization for Bmp7 in HL field at onset of bud growth. (G) In situ hybridization for Mxs2. (H) In situ hybridization for Fgf8 (E9.5), revealing early expression of Fgf8 and initiation of HL bud outgrowth. Fgf8 expression is delayed in Pitx1-/-,Pitx2+/- embryos from about 27- to 30-somite stages of development.

View larger version (76K): [in a new window]   Fig. 5. Hindlimb (HL) anteroposterior markers reveal apparent loss of anterior bud mesenchyme. Dorsal views of whole-mount in situ hybridization embryos are shown. (A) The anterior border of Hoxc11 mRNA is revealed at the junction between somites 26 and 27. In mutant embryos, the proportion of Hoxc11-negative anterior mesenchyme relative to Hoxc11-positive mesenchyme is reduced, consistent with the loss of anterior bud mesenchyme revealed in Fig. 5A. (B) The zone of polarizing activity (ZPA) is revealed by hybridization for sonic hedgehog (Shh). Whereas in wild-type and Pitx-/- embryos the ZPA occupies the posterior quadrant of the HL bud, this structure extends all the way up to half the HL buds in Pitx1-/-,Pitx2+/- embryos. (C) Expression of Gli3 in anterior hindlimb buds is present in embryos of the three genotypes. (D) Expression of Fgf4 in AER. The extent of Fgf4 expression appears anteriorized in mutant embryos compared with wild type, again in agreement with the loss of anterior mesenchyme. (E) Expression of the posterior limb bud mesenchyme marker, Hand2 (dHand in figure) is also extended anteriorly. Two examples at different developmental stages are shown with LR differences in the anterior extension of Hand2 expression. (Bottom row) Anterior bud expression of Hand2 is shown in the right HL bud of an E11.5 Pitx1-/- embryo, and in the right HL of a E12.5 Pitx1-/-,Pitx2+/- embryo.

View larger version (46K): [in a new window]   Fig. 6. Further loss of digits in Pitx1-/-,Pitx2neo/— embryos. A few Pitx1-/-, Pitx2neo/— embryos were obtained and found to miss more than one hindlimb (HL) digit. (A,B) Photograph (A) and skeletal preparations (B) of E13.5 wild-type and Pitx1-/-,Pitx2neo/— embryos showing loss of one digit on the left side and of two digits on the right side. Note absence of forelimb (FL) defects. (C) Similar embryos in which cartilaginous condensation of the digits were revealed at E12.5 using whole-mount in situ hybridization for Sox9. The Pitx1-/-,Pitx2neo/— embryo has three digits on left and two digits on right side. The left HL of the wild-type embryo was damaged during preparation. (D) Whole-mount in situ hybridization of AER Fgf8 in the single Pitx1-/-,Pitx2-/- embryo obtained. This embryo (E12.5) was underdeveloped and smaller than the Pitx1+/-,Pitx2-/- embryo shown for comparison. Whereas FL bud development appeared normal in those embryos, very small HL buds were present in the double null embryo, with a small patch of Fgf8 expressing tissue on the left side.

View larger version (80K): [in a new window]   Fig. 7. Early expression of Pitx1 and Pitx2 proteins revealed by whole-mount and section immunohistochemistry. Pitx1 (top) protein is revealed in stomodeum (oral ectoderm) of nine- (A) and 15- (C) somite embryos. Expression in posterior lpm of nine- (A), 11- (B) and 15-somite (C, right side view; D, ventral view) embryos is shown to be bilateral. Pitx2 (bottom) protein is revealed in the head (bilateral) and in left lpm of seven- (A), 11- (B) and 15- (C) somite embryos. In tail bud area, note stronger expression on left side and weaker but significant expression on the right side. (E) Immunohistochemical analysis of Pitx1, Pitx2 and MyoD protein expression in consecutive transverse sections of E10.5 embryos, revealing Pitx1 only in hindlimb (HL) buds. (F) Consecutive sections of E11.5 HL, revealing Pitx1 protein throughout the mesenchyme and Pitx2 protein in muscle cells that colocalize with MyoD-positive cells.

View larger version (32K): [in a new window]   Fig. 8. Role of Pitx genes in limb bud development. (A) Model for role Pitx1 and Pitx2 genes in hindlimb bud formation. The early co-expression of Pitx genes in the mesoderm of the limb bud field appears to be required for growth of bud mesenchyme in response to signals such as Fgf10. At this time, we do not have specific evidence to implicate Fgf10 more than other signals, except for the early expression of Pitx genes. This model is consistent with relatively normal signaling in Pitx mutant embryos. (B) As double null Pitx mutants could not be studied extensively, it cannot be excluded that Pitx genes are required for expression of Fgf10 and that they control the growth capacity of hindlimb bud mesenchyme in this way. (C) Differential control of limb bud outgrowth and specification by Pitx and Tbx genes. Previous work suggested that Tbx5 is a determinant for specification of forelimb identity whereas Pitx1 and the downstream Tbx4 gene both contribute to specification of hindlimb identity. The present work shows that Pitx1 and Pitx2 genes are required for hindlimb bud outgrowth but this function does not appear to require mouse Tbx4 (V. Papaioannou, personal communication). By contrast, Tbx5 appears to fulfill in forelimb buds an outgrowth function similar to that fulfilled by Pitx genes in hindlimb buds (Agarwal et al., 2003) (M. Logan, personal communication).