Sall genes regulate region-specific morphogenesis in the mouse limb by modulating Hox activities

doi:10.1242/dev.027748

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First published online January 23, 2009
doi: 10.1242/10.1242/dev.027748

Development 136, 585-594 (2009)
Published by The Company of Biologists 2009

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Sall genes regulate region-specific morphogenesis in the mouse limb by modulating Hox activities

Yasuhiko Kawakami¹^,2^,3^,4, Yukako Uchiyama⁵, Concepcion Rodriguez Esteban¹, Toshiaki Inenaga⁵, Naoko Koyano-Nakagawa²^,4^,6, Hiroko Kawakami¹^,2^,3, Merce Marti⁷, Marie Kmita⁸, Paula Monaghan-Nichols⁹, Ryuichi Nishinakamura⁵ and Juan Carlos Izpisua Belmonte¹^,7^,*

¹ Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
² Stem Cell Institute, University of Minnesota, 2001 6th SE, Minneapolis, MN. 55455, USA.
³ Department of Genetics, Cell Biology and Development, 6-160 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA.
⁴ Developmental Biology Center, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455, USA.
⁵ Division of Integrative Cell Biology, Institute of Molecular Embryology and Genetics, Global COE `Cell Fate Regulation Research and Education Unit', Kumamoto University, Kumamoto, Japan 860-0811.
⁶ Department of Neuroscience, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455, USA.
⁷ Center of Regenerative Medicine in Barcelona, Doctor Aiguader, 88, 08003 Barcelona, Spain.
⁸ Laboratory of Genetics and Development, Institut de Recherches Cliniques de Montréal (IRCM), Université de Montréal, 110 avenue des Pins Ouest, H2W 1R7, Montréal, Quebec, Canada.
⁹ Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

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Fig. 1. Combined activity of Sall1 and Sall3 contributes to the development of the autopod. Alcian Blue-stained E15.5 forelimbs of Sall1;Sall3 mutants are shown. Genotypes of Sall1;Sall3 are indicated on the top: (A) +/+;+/+, (B) +/-;+/-, (C) +/+;-/-, (D) +/-;-/-, (E) -/-;+/+, (F) -/-;+/- and (G) -/-;-/-. Middle panels show lateral views of entire forelimb skeletons, and the bottom panels show dorsal views of the autopod. In A, the stylopod, zeugopod and autopod are indicated as S, Z and A, and digits are indicated with 1-5. Metacarpal fusions in the Sall1^-^/-;Sall3^+/+ (E), Sall1^-^/-;Sall3^+/- (F) and Sall1^-^/-;Sall3^-^/- (G) mutants are indicated by arrows. Two small carpal elements left in the Sall1^-^/-;Sall3^-^/- (G) mutant are indicated by asterisks. Skeletal phenotypes become more severe from the left (Sall1^+/+;Sall3^+/+; A) to the right (Sall1^-^/-;Sall3^-^/-; G).

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Fig. 2. Expression of Sall1 and Sall3 is regulated by Shh-Gli3. Dorsal views of E10.5 forelimbs stained with Sall1 (A-C) and Sall3 (D-F) with the anterior towards the top. Wild-type (WT; A,D), Shh^-^/- (B,E) and Gli3^-^/- (C,F) limbs are shown. Normal expression of Sall1 and Sall3 is restricted to the distal-posterior mesenchyme (A,D; black arrows). Both Sall1 and Sall3 are downregulated in Shh^-^/- limbs (B,E; red arrows), and are ectopically expressed in the anterior mesenchyme in the Gli3^-^/- limbs (C,F; blue arrows).

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Fig. 3. Reduced Shh signaling in Sall1^-^/-;Sall3^-^/- mutant limbs. Dorsal views of E10.5 (A,B) and E11.5 (C-H) limb buds stained with Grem1 (A-D), Sox9 (E,F) and Fgf8 (G,H), with the anterior towards the top. Wild-type (WT; A,C,E,G) and Sall1^-^/-;Sall3^-^/- (B,D,F,H) limbs are shown. (A-D) Grem1 expression is downregulated in the mutant limb (B,D; arrows), compared with control limbs (A,C). (E,F) Morphological alteration was visible at E11.5 by Sox9 in situ hybridization. The control limb has primordia for digit1-digit5 (E). The mutant limb lacks digit1 primordia, exhibits fused digit2 and digit3 primordia, and has delayed separation of digit4 and digit5 primordia (F). (G,H) The Fgf8 expression domain is shorter along the anterior-posterior axis in the AER in the mutant (H), compared with the control limb (G). The anterior and posterior margins of Fgf8 expression domain are indicated by arrows.

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Fig. 4. Expression of Epha3 and Epha4 is upregulated in the Hoxa13 and Hoxd13 mutants. Dorsal views of E11.5 forelimbs stained with Epha3 (A-C) and Epha4 (D-F) with the anterior towards the top. Wild-type (WT; A,D), Hoxa13^-^/- (B,E) and Hoxd13^-^/- (C,F) limbs are shown. Normal Epha3 expression in the anterior edge (arrow) and prospective wrist region (arrowhead) (A) is upregulated and expanded posteriorly in the Hoxa13^-^/- (B, arrow) and Hoxd13^-^/- (C, arrow) limbs. Normal Epha4 expression in the distal-anterior mesenchyme (D, arrow) is upregulated and expanded distal-posteriorly in the Hoxa13^-^/- (E, arrow) and Hoxd13^-^/- (F, arrow) limbs.

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Fig. 5. Expression of Epha3 and Epha4 is downregulated in Sall1;Sall3 mutant limbs. Dorsal views of E11.5 forelimbs stained with Epha3 (A-C) and Epha4 (D-F) with the anterior towards the top. Wild-type (A,D), Sall1^-^/-;Sall3^+/- (B,E) and Sall1^-^/-;Sall3^-^/- (C,F) limbs are shown. (A) Normal expression of Epha3 is detected in the anterior edge (arrow) and prospective wrist region (arrowhead). (B) Epha3 expression is downregulated in the Sall1^-^/-;Sall3^+/- limb. (C) The anterior edge expression is more severely downregulated and the prospective wrist region expression is undetectable in the Sall1^-^/-;Sall3^-^/- limb. (D) Normal Epha4 expression in detected the distal anterior mesenchyme (arrow). (E,F) Epha4 expression is downregulated in the Sall1^-^/-;Sall3^+/- limb (E), and is more severely downregulated in the Sall1^-^/-;Sall3^-^/- limb (F).

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Fig. 6. Hox represses the expression of Sall1 and Sall3. Dorsal views of E11.5 hindlimbs stained with Sall1 (A-D) and Sall3 (E-H) with anterior towards the top. Wild-type (A,E), Hoxa13^-^/- (B,F), Hoxd13^-^/- (C,G) and Hoxa13^-^/-;Hoxd13^-^/- (D,H) limbs are shown. (A) Normal Sall1 expression starts to regress from the most distal mesenchyme (arrow). (B) In the Hoxa13^-^/- limb, the Sall1 expression domain became larger and the signal stronger. (C) In the Hoxd13^-^/- limb, the Sall1 signal is detected in the distal region (arrow) and is stronger than that in the wild type. (D) In the Hoxa13^-^/-;Hoxd13^-^/- limb, a large domain in the distal mesenchyme expresses significantly high levels of Sall1 (arrow). (E) Normal Sall3 expression also starts to regress from the most distal mesenchyme (arrow). (F) In the Hoxa13^-^/- limb, higher level of Sall3 expression is detected in the anterior mesenchyme (arrow). (G) In the Hoxd13^-^/- limb, higher level of Sall3 expression is detected in the distal-middle region (arrow). (H) In the Hoxa13^-^/-;Hoxd13^-^/- limb, strong expression of Sall3 is detected in the wide region of the distal mesenchyme.

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Fig. 7. Sall1 modulates Hox activity post-transcriptionally. (A) EMSA assay with the HA-Sall1 protein. Sall1 recognizes the Epha4 upstream element (arrow). The specificity was confirmed by supershift induced by anti-HA antibody (asterisk). The Sall1-probe complex became weaker by introducing mutations in the probe (M1, M2), and is abolished by introducing multiple mutations (M3). (B,C) EMSA assay with Flag-Hoxa13 protein (B) and Flag-Hoxd13 protein (C). Both Hoxa13 and Hoxd13 recognize the Epha4 upstream element (arrows). The specificity was confirmed by supershift induced by anti-Flag antibody (asterisks). The Hox13-probe complex was weaker with the M1 mutant probe, but was not severely affected with the M2 mutant probe. The binding was abolished with the M3 mutant probe containing multiple mutations. M1, mutant1; M2, mutant2; M3, mutant3 (see Materials and methods). (D) Sall1 and Hox13 compete for an Epha4 upstream element. Specific bands formed between ³²P-labeled wild-type probe and Sall1 (arrow), and between ³²P-labeled wild-type probe and Hox13 (broken arrow) were detected. By co-incubating with Sall1, the Hoxa13-DNA complex and the Hoxd13-DNA complex became weaker. (E) Luciferase-reporter assay showing Hox-activity modulation by Sall1. The reporter construct was co-transfected with 100 ng of Hoxa13, Hoxd13 and/or Sall1 expression constructs, together with 20 ng pRL-TK. Data are shown as mean±s.d. Significant differences between mock transfected (moc), and Hoxd13, Hoxd13 and Hoxd13+Sall1 are detected (P<0.001).

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