spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 3 May 2006
doi: 10.1242/dev.02395

Development 133, 2263-2273 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Capellini, T. D.
Right arrow Articles by Selleri, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Capellini, T. D.
Right arrow Articles by Selleri, L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression

Terence D. Capellini1,2,*, Giuseppina Di Giacomo1,*, Valentina Salsi3, Andrea Brendolan1, Elisabetta Ferretti1, Deepak Srivastava4, Vincenzo Zappavigna1,3 and Licia Selleri1,{dagger}

1 Department of Cell and Developmental Biology, Cornell University Weill Medical School, New York, NY 10021, USA.
2 New York Consortium in Evolutionary Primatology, The Graduate School and University Center, The City University of New York, NY 10016, USA.
3 Department of Animal Biology, University of Modena-Reggio Emilia, Modena 41100, Italy.
4 Gladstone Institute of Cardiovascular Disease, Department of Pediatrics, University of California, San Francisco, CA 94158, USA.


Figure 1
View larger version (68K):

[in a new window]
  		Fig. 1. Abnormal morphology of Pbx1–/–;Pbx2+/– and Pbx1–/–;Pbx2–/– embryos. (A) At E10.25 (and E10.5), Pbx1–/–;Pbx2+/– embryos exhibit abnormal morphology and appear delayed. Their forelimbs are PD shortened and AP dysmorphic, while their hindlimbs appear relatively normal compared with wild type. Pbx1–/–;Pbx2–/– embryos are severely delayed compared with wild type, exhibiting striking morphological abnormalities, including the lack of limbs. Enlargements of all wild-type and mutant limbs are shown on the right-hand side. In addition, both wild-type and Pbx1–/–;Pbx2+/– hindlimbs are shown at two gestational days in enlargements on the far right, whereas Pbx1–/–;Pbx2–/– limbs are shown at E11 to demonstrate rudimentary forelimb bud formation (lower magnification). (B) At E13.5, Pbx1–/–;Pbx2+/– embryos are smaller than wild type with abnormal morphology. Forelimbs display shorter zeugopodia (short red arrow) and dysmorphic autopodia (short black arrow) compared with wild type (long red and black arrows, respectively); hindlimbs exhibit rudimentary autopodia (short red arrow) compared with wild type (long red arrow). Proximal (left); anterior (top). FL, forelimb; HL, hindlimb.

 

Figure 2
View larger version (53K):

[in a new window]
  		Fig. 2. Limb skeletal phenotypes of Pbx1–/–;Pbx2+/– (mutant) embryos. (A-D) At E12.5, Sox9 expression indicates that mutant forelimb (B) mesenchymal condensations for digits 1 (empty red arrowhead) and 5 (empty black arrowhead) are rudimentary compared with wild type (A). In mutant hindlimbs (D), digit (red empty arrowhead) and fibula condensations (empty black arrowhead) are absent compared with wild type (C) (black arrow). (E-H) At E12.5, unlike wild type (E) (red arrow), mutant scapular (F) anlagen is reduced (red arrowhead) and the humerus is dysmorphic with a hypertrophic anlagen reminiscent of a deltoid tuberosity (F) (black arrowheads). In mutant (H), the pelvis is virtually absent, except for one element (empty black arrowhead) that may be a rudimentary ischium (G) (compare with wild type, long black arrow). (I-N) At E13.5, unlike wild type (I, red arrow), mutant scapula (K; inset) is dysmorphic (red arrowhead) and the humeral head is fused to it and duplicated medially (double black arrowhead), more severely than in Pbx1–/–;Pbx2+/+ mutants (J) (arrowheads). In mutant hindlimbs (N), only a rudimentary anlagen reminiscent of an ischium (open black arrowhead) remains fused to the dysmorphic femur (red arrow). This phenotype is more severe than in Pbx1–/–;Pbx2+/+ mutants (M) (arrowheads). The mutant forelimb (K) zeugopod (ra and ul) appears shortened (short black arrows), while the mutant hindlimb (N) zeugopod (ti and fi) lacks the fibula (open red arrowhead) when compared with wild type (I and L, respectively) (L, red arrow). The double mutant forelimb (K) autopod exhibits three central digits, with digits 1 (open red arrowhead) and 5 (open black arrowhead) absent or rudimentary. The mutant hindlimb (N) autopod lacks all digits (dashed red rectangle) except one rudimentary ray (open black arrowhead; inset) compared with wild type (L). fe, femur; fi, fibula; hu, humerus; pg, pelvic girdle; ra, radius; sc, scapula; ti, tibia; ul, ulna.

 

Figure 3
View larger version (47K):

[in a new window]
  		Fig. 3. Pbx1 and Pbx2 colocalize in lateral plate-intermediate mesoderm and early limb fields. (A,E,I) At E8.5, Pbx1 (A) and Pbx2 (E) colocalize (I) in lateral plate-intermediate mesoderm (red arrowheads). (B,F,J) At E9, Pbx1 (B) and Pbx2 (F) colocalize (J) in the early limb fields (red arrowheads). (C,G,K) At E9.25, although overlapping proximally (K), Pbx1 (C) and Pbx2 (G) forelimb expression domains become mostly complementary, being proximal and distal, respectively (red arrowheads). (D,H,L) At E9.25, Pbx1 (D) and Pbx2 (H) hindlimb expression domains remain overlapping (L) (red arrowheads). Proximal is leftwards; anterior is towards the top. som, somites.

 

Figure 4
View larger version (48K):

[in a new window]
  		Fig. 4. Pbx1 and Pbx2 mesenchymal expression in later limb development is dynamic and, in part, complementary. (A,B) At E9.5-10.75, Pbx1 is expressed in proximal forelimbs and hindlimbs, mostly anteriorly, whereas Pbx2 is expressed distally and throughout the limb. At E11-11.75, Pbx1 is still expressed proximally in limbs, but localized anteriorly and posteriorly in the mesenchyme proximal to the autopod. Similarly, Pbx2 expression becomes restricted to this domain, albeit more broadly. (C) Diagrams depicting dynamic expression of Pbx1, Pbx2 and overlapping domains of Pbx1/Pbx2 at different gestational days. These schemes represent summaries of the expression patterns present at the gestational time-points indicated by the somite range at the top of each panel. Proximal is leftwards, anterior towards the top. som, somites.

 

Figure 5
View larger version (86K):

[in a new window]
  		Fig. 5. Mesenchymal gene expression in Pbx1–/–;Pbx2+/– (mutant) hindlimbs. (A) Expression of Alx4, Hand2 and Gli3 is relatively unperturbed in mutant hindlimbs, before and/or at Shh onset, indicating that until E10.5 the mesenchyme is patterned similar to wild type. (B) Shh expression is absent throughout mutant hindlimb development at E10.5 and E11.5. At E11.5, Shh absence (empty purple arrowhead) in mutant hindlimbs is associated with a similar and expected alteration in Fgf8 expression (empty blue arrowheads), compared with wild-type hindlimbs (normal Shh expression indicated by solid purple arrowhead and Fgf8 by blue arrowheads). (C) Although reduced, expression of Ptch1 and Gli1 is present (E10.75), suggesting minimal and transient Shh activity. Meis2 is unperturbed in mutant hindlimbs (E11.0), suggesting that proximal limbs remain relatively intact. (D) Grm is extended posterodistally in mutant hindlimbs (E10.75), indicating Shh absence in the ZPA (Scherz et al., 2004Go). Hand2 expression is reduced (E10.75) and Gli3 expression is diffused throughout mutant hindlimbs (E11), resembling expression patterns in mice that lack Hoxa/Hoxd (Kmita et al., 2005Go) and suggesting an AP patterning defect in the absence of sustained Shh activity. Proximal is leftwards; anterior is towards the top.

 

Figure 6
View larger version (102K):

[in a new window]
  		Fig. 6. Hox gene expression is altered in Pbx1–/–;Pbx2+/– (mutant) limbs. (A) Hox gene expression (E10.25-10.5) is absent or reduced in somite-matched mutant hindlimb posterior domains before, or at, Shh onset, despite their relatively normal morphology. Open black arrowheads indicate lack of posterior expression (Hoxa9, Hoxa11, Hoxd9). Open red arrowhead indicates reduced posterior expression (Hoxd11). White arrowheads indicate absence of expression (Hoxa13, Hoxd12, Hoxd13). Diagrams depict summaries of expression data across two time-points (E10.25 and E10.5): Hox gene expression domains are represented by the same colors displayed in their respective in situ hybridization panels. Dashed domains in mutant indicate reduced Hoxd11 (pink) expression, whereas missing domains reflect complete absence of expression (Hoxa13, Hoxd12, Hoxd13). Blue domain in Hoxd diagram represents Hoxd10 expression. (B) Distal 5' Hoxa (Hoxa11 and Hoxa13) expression (E11.5) remains relatively unperturbed in mutant hindlimbs, while Hoxd (Hoxd11 and Hoxd13) expression is severely reduced anteriorly (open black arrowheads). Open red arrowheads indicate perturbed proximal posterior expression (Hoxa11 and Hoxd11). (C) 5' Hoxa (Hoxa11 and Hoxa13) expression (E11.5) remains relatively unperturbed in mutant forelimbs, whereas 5' Hoxd (Hoxd11 and Hoxd13) expression is reduced anteriorly (open black arrowheads). Proximal is leftwards; anterior is towards the top.

 

Figure 7
View larger version (32K):

[in a new window]
  		Fig. 7. 5' HoxD proteins are bound to and directly activate transcription from the Shh limb enhancer (ShhE). (A) ChIP analyses on E10.5 mouse fore- and hindlimbs, using specific anti-Hoxd10 ({alpha}Hoxd10), anti-Hoxd13 ({alpha}Hoxd13) and anti-Pbx ({alpha}Pbx) antisera, demonstrate their direct in vivo binding to the mouse ShhE. A 424 bp fragment of the ShhE was amplified by PCR. No PCR amplification of a 373 bp negative-control region (Co) located 1400 bp upstream of this enhancer was detected using E10.5 limb chromatin. Representative reactions of all PCR amplifications, carried out in triplicate, are shown. (B) ChIP on pooled E13.5 limbs using the {alpha}Hoxd13 antiserum revealed no amplification of the ShhE. (C) At E10.25 and E11, Pbx1 (blue) and Shh (purple) mRNA transcripts do not colocalize in wild-type and Pbx2–/– (KO) hindlimbs. (D) HoxD proteins can activate transcription from the ShhE (eightfold for Hoxd9, fourfold for Hoxd10 and threefold for Hoxd13) in P19 embryonal carcinoma cells. Luciferase activity, in arbitrary units (R.L.U.), was assayed from extracts of transiently transfected P19 cells. Co-transfection assays were performed in the presence (+) of the indicated expression vectors encoding Pbx2, Hoxd9, Hoxd10 and Hoxd13, and with a luciferase reporter construct (pT81mShhE) containing the ShhE. Bars represent the mean±s.e.m. of at least four independent experiments. (E) Simplified model (top) depicts the overlapping genetic hierarchical roles of Pbx1 and Pbx2 in controlling Hox gene spatial distribution and Hox recruitment to the limb ShhE. In addition, Pbx2, albeit not functionally essential within this network, is also recruited to this enhancer in limb cells. Intensity of the hue of Pbx2 correlates with its functional relevance. Lower left diagram illustrates the requirement of Pbx1 (violet), Pbx2 (rose) and Pbx1/Pbx2 (purple) for controlling the positioning of 5' Hox genes (i.e. Hoxa/Hoxd, orange) to future Shh-expressing domain (green). In Pbx1–/–;Pbx2+/– (mutant) limbs, Hox gene expression remains external to the ZPA, and Shh activation does not occur (red cross). Lower right diagram depicts our refinement of a previous model (Zakany et al., 2004Go) and highlights that Shh loss, owing to early Pbx1/Pbx2 perturbation, leads to partial disruption of 5' Hoxd reverse colinearity (yellow), but not of 5' Hoxa expression (deep orange). Proximal is towards the left; anterior is towards the top. Co, control; FL, forelimb; HL, hindlimb; I, input chromatin; NR, non-specific control antibody; NoAb, no antibody-control resin.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006