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doi: 10.1242/10.1242/dev.00245

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The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb

¹ Departamento de Anatomía y Biología Celular, Universidad de Cantabria, 39011 Santander, Spain
² Department of Anatomy, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706, USA
³ Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA

View larger version (68K): [in a new window]   Fig. 1. Skeletal pattern and chondrogenesis in the ozd mutant limbs. (A) A homozygous ozd embryo at day 10 of development showing limb-specific skeletal deficiencies. (B) Skeletal preparations of ozd wing and leg compared with stage-matched wild-type limbs. (C) Higher magnification image comparing morphology of the ozd leg digit (asterisk) with wild-type leg d1. (D) Alcian Green preparations comparing chondrogenic condensations in st. 25 and 27 wild-type and ozd limbs. f, femur; fi, fibula; h, humerus; r, radius; t, tibia; u, ulna.

View larger version (80K): [in a new window]   Fig. 2. Pattern of cell death and affected tissue layer in ozd limbs. (A-D) TUNEL analysis of sectioned, st. 24 wild-type and ozd limb buds reveals an abnormal pattern of cell death in ozd. Beneath each section is a picture of the limb bud prior to embedding, shown to better understand its shape. Arrows in B and D indicate the lack of cell death along the posterior border. (E) Normal skeletal pattern of a recombinant limb constructed with wild-type ectoderm and mesoderm. (F) Recombined ozd ectoderm and wild-type mesoderm result in a normal skeletal pattern. (G) Recombined ozd mesoderm and wild-type ectoderm produce wings with an ozd skeletal pattern. Axial orientations are indicated in A. A, anterior; Di, distal; P, posterior; Pr, proximal.

View larger version (68K): [in a new window]   Fig. 3. Shh expression is undetectable in ozd limb buds. (A) Normal pattern of Shh expression in a wild-type st. 18 embryo. (B) Comparable stage ozd embryo lacks detectable Shh expression in the limbs (arrows). In situ hybridization to sectioned limb buds confirms this result, showing posterior Shh expression in a wild-type st. 18 wing bud (C) while failing to detect Shh expression in ozd wing buds (D). Normal Shh expression is shown for wild-type st. 19 (E) and 22 (G) embryos, while comparable ozd (F) and (H) embryos exhibit normal domains of Shh expression except in the limb buds (arrow).

View larger version (79K): [in a new window]   Fig. 4. Expression of putative Shh target genes in ozd limb buds. Both Ptc1 (A,B) and Gli1 (C,D) expression is undetectable in ozd limb buds at early (A,C) or later stages (B,D). (E-F) Gli3 expression is normal in mutant limbs at st. 18/19 (E), but abnormally extends to the posterior border at later stages (F). (G,H) dHAND expression is normal in early stage ozd wing buds (G), but is posteriorly restricted at later stages. (I,J) ozd limb buds express Bmp2, but expression levels are reduced and the spatial domain posteriorly restricted relative to wild type. In every panel anterior is up.

View larger version (90K): [in a new window]   Fig. 5. Expression of Hoxd11, Hoxd12 and Hoxd13 in ozd limb buds. (A-D) Hoxd11 expression in wild-type and ozd limbs. (A,B) Up to st. 25, Hoxd11 shows a normal pattern of expression in the mutant wing, although its expression level is slightly reduced. (C,D) From st. 27, Hoxd11 expression in the ozd wing is confined to the posterior border. (A-D) In ozd leg buds, Hoxd11 expression is more affected, showing a reduced domain of expression by st. 21/22 (A), and becomes undetectable by st. 24/25 (B-D). (E-H) Hoxd12 expression in wild-type and ozd limbs. (E) The pattern of Hoxd12 expression is close to normal in st. 21/22 ozd wing buds, but expression levels are reduced. (F-H) From st. 24/25, Hoxd12 becomes confined to the posterior border of the mutant wing. (E-H) Hoxd12 expression in the ozd leg bud is only observed at early stages and at very reduced levels. (I-L) Hoxd13 expression in wild-type and ozd limbs. (I) Hoxd13 expression occurs at low levels and is posteriorly restricted in st. 21/22 ozd wing and leg buds, relative to wild type. (J-L) From st. 23/24 Hoxd13 expression becomes undetectable both in wing and leg buds. (K-L) Expression is re-initiated at st. 27 in the distal mesoderm of ozd legs. In all the panels anterior is up.

View larger version (114K): [in a new window]   Fig. 6. Expression of Fgf8, Fgf4 and Gre in ozd limb buds. (A,B) Fgf8 is expressed at high level throughout the ozd AER. The AP extent of the AER is reduced in the narrowed ozd limb. (C) At st. 27, Fgf8 appears down regulated in the anterior AER but expression persists at high levels in the posterior AER. (D,E) Fgf4 expression in the mutant AER is reduced except at the most posterior edge, where a spot of elevated expression became apparent by st. 23. Note that the specimens in D and E have been analyzed for both Fgf4 and Shh expression. (F) By st. 25, Fgf4 expression has declined in the mutant AER except for the posterior spot of high-level expression. (G-I) Gre expression in ozd and wild-type limbs. ozd limb buds express Gre but, contrary to normal, its spatial domain of expression expands to the posterior border. In all the panels anterior is up.

View larger version (48K): [in a new window]   Fig. 7. ozd mesoderm is Shh-responsive, but cannot express Shh. The ozd mutant wing phenotype (A) was completely rescued by a ZPA graft (B). The ZPA graft was of leg origin and contributed a leg digit marked here as 3*. (C) Application of an Shh-N-soaked bead to posterior st. 20 ozd wing buds restores zeugopod development. (D) Two sequential applied SHH beads rescues both zeugopod and autopod formation in ozd wings. (E) Reduction of Shh expression 24 hours after application of an RA bead to the posterior border of a wild-type wing bud. (F) ozd posterior mesoderm does not express Shh 24 hours after RA application. (G) Induction of Shh at the anterior border of a wild-type leg bud 24 hours after implantation of a RA bead. (H) RA application at the anterior border of ozd leg buds does not induce ectopic Shh expression. (I-L) RA application induces ectopic anterior dHAND expression in both normal (I) and mutant (J) wing buds. dHAND expression in the unmanipulated contralateral wild-type (K) and ozd (L) limb buds. The position of the RA bead is indicated by the red arrow.

View larger version (29K): [in a new window]   Fig. 8. SHH function in generating the amniote limb skeleton. ozd legs (top) develop with a single, identifiable, Shh-independent skeletal element at each PD level (red), forming a linear series of the anterior-most elements found in wild-type legs (bottom). Therefore, given a functional AER, the limb field contains all the information necessary to form a distally complete limb. In normal development, Shh signaling is differentially required at each PD level to elaborate the formation and patterning of additional limb skeletal elements (blue) along the AP axis. We propose that while the femur (f) is Shh independent, Shh acts in the zeugopod context of the prepatterned tibia (ti) to specify the fibula (fi). In the autopod, Shh acts in the context of the prepatterned d1 to progressively specify the posterior digits (d2, d3, and then d4). We note that Shh is also required to elaborate the posterior fibulare (fl) and basal commune (bc) elements of the tarsus, which respectively fuse to the distal tibia (ti) and proximal metatarsals (mt) by 8.5 days of development (data not shown). tl, tibiale.

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