Abu-Daya A , Nishimoto S , Fairclough L , Mohun TJ , Logan MP , Zimmerman LB .

1 R01 HD4 2276-01 NICHD NIH HHS , U117560477 Medical Research Council , U117560482 Medical Research Council , U117562103 Medical Research Council , MC_U117560482 Medical Research Council , MC_U117562103 Medical Research Council , R01 HD042276 NICHD NIH HHS , MC_U117560477 Medical Research Council , MRC_MC_U117562103 Medical Research Council , MRC_MC_U117560477 Medical Research Council , MRC_MC_U117560482 Medical Research Council

Xtr.npnt{insZimml} + npnt MO (Fig 4. B)

	Fig. 5. Xdm mutants fail to express tbx5 in the forelimb-forming region. (A–C) WISH analysis of stage 48 wild type embryos detects tbx5 in the nascent forelimb bud and heart (white arrow). (D–F) Xdm embryos lack tbx5 expression in equivalent flank region. (B, E) Magnified view of boxed regions in (A) and (D) showing pronephros (black arrow). (C) 10× view of a transverse section through wild type flank showing tbx5 expression in the nascent forelimb (gray arrow) below the pronephros (black arrow). (F) 10× view of a transverse section of xdm at equivalent level of the forelimb region shows pronephros (black arrow) but no tbx5 expression.
	Fig. 6. Npnt is widely expressed during development. (A) Npnt WISH shows strong expression in neural tissue and axial mesoderm in the late neurula (stage 19). (B) Tailbud (stage 30) and tadpole (C, stage 39) express in the head/gill arches, CNS, somites, and pronephros (arrow). (D–F) Transverse sections of npnt WISH at stages 46–49 show broad staining including pronephros (black arrow) but exclusion from the epidermis and the prospective forelimb-forming region (open arrow).
	Fig. 7. Integrin α8β1 receptor expression at limb forming stages. (A–F) WISH of wild type tadpoles at stages 44, 45, and 47 with probes for the α8 (itga8, A, C, E) and β1 (itgb1, B, D, F) receptor subunits. (G, H) Transverse sections of α8 and (I, J) β1 WISH at stages 45 and 47. β1 expression is virtually ubiquitous. α8 integrin shows more regionalized expression at stages 44 and 45 in the liver, head cartilage and other structures before becoming broadly expressed by stage 47.
	Fig. 8. Npnt is not required for pronephros or mesonephros formation. (A, B) Specification of pronephros occurs normally as shown by pax8 WISH in both wt (left panels) and xdm (right panels) at the tailbud stage (black arrowheads; white arrowheads show the otic vesicle). (C, D) At stage 40, xdm pax8 expression in pronephros (black arrowheads) is indistinguishable from wild type (white arrowheads mark hindbrain expression). (E, F) Dissected mesonephros from metamorphosing mutant tadpoles (F) is morphologically indistinguishable from wild type (E); the anterior is to the left.
	Fig. 1. A transgene insertion ablates all forelimb structures. (A) Metamorphosing froglets heterozygous for an Nkx2.5FP transgene show normal forelimb development while (B) homozygous siblings lack forelimbs. (C, E) Skeletal preparation of a wild type froglet showing limb skeletal elements including the scapula (sc), humerus (hu), ulna (ul), and digits (dit); clavicle is hidden beneath the scapula. (D, F) All forelimb skeletal structures, including the scapula and clavicle, are missing in xdm froglets, but the ribs are unaffected (r1, r2, and r3).
	Fig. 2. Forelimb buds do not form in xdm. (A, F, K, P) Dorsal views of stages 47, 48, 50 and 53 wt tadpoles showing sites of the forelimb (black arrowheads) and the hindlimb (white arrowheads). (D, I, N, S) Hindlimb development occurs normally in the mutant compared with the wt (B, G, L, Q). (C, E) Stage 47 wild type and mutant tadpoles immediately prior to forelimb bud initiation. (H) Forelimb buds (dashed outline) first appear at stage 48 in wt tadpoles but not in xdm homozygotes (J). (M) Forelimbs are more distinct in stage 50 wt larvae but are still absent in xdm (O). (R) By stage 53 the wt forelimb has formed a handplate, while mutant tadpoles still lack forelimb buds (T).
	Fig. 3. Xdm maps to a transgene insertion in nephronectin. (A) Schematic showing the wild type npnt genomic locus (above), and the structure of the Nkx2.5FP transgene insertion in the xdm allele (below). Genotyping primers which amplify the wild type npnt allele (NF + NR) and the junctions at each end of the insertion (NR + TR and TF + NR) are indicated. (B) Npnt sequence spanning the insertion can be amplified with npnt primers (NF + NR) from wild type (wt) but not transgenic (tgn) haploid embryos; chimaeric DNA at either end of the insertion can be amplified from transgenic but not wild type haploids using an npnt primer with a transgene primer (NF + TR and TF + NR). (C) Genotyping wild type and forelimbless siblings from an xdm cross with NF + NR and NF + TR shows that the xdm phenotype is tightly linked to the transgene insertion; sample of 14 shown (175 total genotyped). (D) Npnt mRNA is truncated by the transgene insertion. Top panel, 5 transgenic and 2 non-transgenic st. 40 tadpoles genotyped as in (C). Asterisk (left lane) marks heterozygous transgenic tadpole. RT-PCR (2nd and 3rd panels) shows that npnt message upstream of transgene insertion (exons 1) appears reduced in homozygous xdm tadpoles compared to heterozygous or wild type, while exons downstream of the insertion (exons 4) are not detected. See Fig. S1 for sequences across junctions of npnt intron and transgene. (E) The truncated protein encoded by the xdm allele lacks the 5 EGF-like domains, the RGD/linker, and the MAM domain of nephronectin.
	Fig. 4. Npnt knockdown phenocopies xdm. Npnt morpholino antisense oligonucleotides were injected into one blastomere of two-cell wild type embryos. (A, ventral view) Injection of two different non-overlapping morpholino sets resulted in unilateral forelimb ablation in a small but statistically significant number of metamorphosing tadpoles. (B) Morpholino knockdown ablating all forelimb skeletal elements, recapitulating the xdm phenotype. (C) Upper lanes, RT-PCR of control (c) and npnt splice-blocking (n) morpholino injected embryos indicates that npnt knockdown is virtually complete at stages 24 and 37, recovering at stage 42, and approaching wt levels at stage 45. Lower lanes, odc control.
	itga8 (integrin, alpha 8 ) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 44, lateral view, anterior left, dorsal up, head and trunk region only.
	itga8 (integrin, alpha 8 ) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 45, lateral view, anterior left, dorsal up, head and trunk region only.
	itga8 (integrin, alpha 8 ) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 47, lateral view, anterior left, dorsal up, head and trunk region only.
	itgb1 (integrin, beta 1) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 44, lateral view, anterior left, dorsal up, head and trunk region only.
	itgb1 (integrin, beta 1) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 45, lateral view, anterior left, dorsal up, head and trunk region only.
	npnt (nephronectin) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 19, dorsal view, anterior down.
	npnt (nephronectin) gene expression in Xenopus tropicalis embryo via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.

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