Fig 1: Loss of Akirin2 in the limb epithelium leads to soft-tissue syndactyly. (a) Interdigital tissue regression is impaired in Akirin2 Emx-KO forelimb (digits 2/3) and hindlimb (digits 2/3/4). (b) Alcian Blue/Alizarin Red staining at P0 and in one of the few surviving mutants at P10 shows that the Akirin2 Emx-KO limb does not have fused bone or cartilage elements, though broader Alizarin Red (bone) staining is observed in the phalanges (red arrows). (c) A very rare Akirin2 Emx-KO that survived until P30 contains clear syndactyly of the mature forelimb and hindlimb. (d–g) Quantification of interdigital tissue regression shows impaired tissue regression between digits 2–3 of the forelimb (d) and 2–4 of the hindlimb (f,g) in Akirin2 Emx-KO mice. The difference in forelimb interdigital regression between digits 3–4 approaches statistical significance (e; p = 0.054). n = 3 animals/genotype for E12 & E13; 4 animals/genotype for E14, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test. (h) Immunostaining of Akirin2 Emx-KO distal limb buds at E12.5 shows that Akirin2 expression is extensively lost in the interdigital space between digits 2–3 in both fore- and hindlimb. In the forelimb, most cells between digits 3–4 remain Akirin2-positive, while in the hindlimb more Akirin2-negative cells are observed (interdigital regions indicated between arrowheads). Scale bar: 1 mm in a-c; 50 µm in h.
Fig 2: Akirin2 loss in limb epithelium leads to decreased programmed cell death between digits that display syndactyly. (a) Cleaved caspase-3 staining is nearly absent between digits 2–3 in Akirin2 Emx-KO forelimb and reduced between the other digits. (b) Sytox Green cellular stain of the limb sections in (a). (c) High magnification views of the distal limb bud region between digits 2 and 3 demonstrates reduced cleaved caspase-3 staining in the Emx-KO interdigital tissue (red circle). (d) Quantification of the number of cleaved caspase-3 cells within the fixed region outlined in (c; Con = Control). Key: CC3, cleaved caspase-3; n = forelimb E13: Con 21 sections, 5 animals; KO 19 sections, 5 animals; hindlimb E14: Con 14 sections, 3 animals; KO 18 sections, 3 animals. **p < 0.01, ***p < 0.001, t-test. Scale bar: 200 µm.
Fig 3: Akirin2 expression in the developing limb bud epithelium. (a) RT-PCR using two different primer sets that generate amplicons across multiple exons. Akirin2 is expressed in the limb at E10.5 and E14. (b) In wild type E10.5 cryosections, strong Akirin2 immunoreactivity is seen in the somites (denoted by *) and in the forelimb bud (FL), including expression in the AER (arrows). (c) At E12.5, Akirin2 immunostaining is lost in most cells of the ectoderm overlying the interdigital tissue of the knockout forelimb compared with control; as expected, mesodermal expression is unaffected as Cre is limited to the ectoderm. (d) Expression of a tdTom Cre reporter allele in the developing limb at E11.5 in both control and mutants, counterstained with the nuclear marker Sytox Green, shows epithelium-restricted Cre activity. (e) High magnification image of the tdTom reporter showing widespread, but patchy, expression in the ventral ectoderm of E10.5 control forelimb (arrows indicate tdTom-negative cells). Key: A, anterior; Di, distal; D, dorsal; ec, ectoderm; FL, forelimb; me, mesoderm; P, posterior; Pr, proximal; V, ventral. Scale bar: 100 µm in b; 50 µm in c; 200 µm in d; 50 µm in e.
Fig 4: Akirin2 loss in limb epithelium leads to increased cell proliferation between digits that display syndactyly. (a–c) Immunohistochemistry of EdU with DAPI counterstain shows increased EdU-positive cell density at the distal end of the limb at E13 and E14 (a, low magnification, white arrowhead; and b,c, high magnification, red circles; digits are numbered). (d) Quantification of percent DAPI-positive cells that are also EdU-positive in control and Akirin2 Emx-KO in the E13 forelimb and E14 forelimb and hindlimb. Cell counts were made in the distal interdigital areas, shown outlined by the red circles. n = forelimb E13: Con 21 sections, 3 animals; KO 25 sections, 4 animals; forelimb E14: Con 17 sections, 3 animals; KO 18 sections from 3 animals; hindlimb E14: Con 18 sections, 3 animals; KO 18 sections, 3 animals. ****p < 0.0001, t-test. Scale bar (in a): 300 µm in a; 100 µm in b,c.
Fig 5: Fgf8 expression is aberrantly retained in Akirin2 knockout limb epithelium. Whole mount in situ hybridisation using Fgf8 riboprobes comparing control and Akirin2 Emx-KO limb at E10.5 (a), E11.5 (c–f), E12.5 (g–n) and E13 (o–v). Fgf8 signal is retained in the Akirin2 Emx-KO limb distal epithelia in the regions that display syndactyly from E12.5 onwards (g–v): digits 2–3 in forelimb and digits 2–4 in hindlimb (black arrowheads). Insets at E12.5 (k–n) and E13 (s–v) show higher magnification images of the distal limb. Panel (b) is a schematic showing the KO phenotype of perduring Fgf8 expression (blue). Key: A, anterior; D, dorsal; Di, distal; P, posterior; Pr, proximal; V, ventral. Scale bar: 500 µm in each row.
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