DEV02696 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Expression of patterning genes in all axes is disrupted in Lrp4-mutant limbs. (A-L) E10.5 forelimbs from wild-type (A-F) and Lrp4-mutant (G-L) embryos. Anterior is to the left. Distal (A,B,G,H) and dorsal (C-F,H-L) limb views showing the expression of limb patterning genes. Topgal expression (A,G), marking Wnt signaling (DasGupta, 1999 #130), and Fgf8 expression expanded dorsally and ventrally in Lrp4 mutants (G-I) compared with wild type (A-C). Msx2 expression also expands in Lrp4 mutants (J). The posterior domain of sonic hedgehog (Shh) expression is reduced in Lrp4 mutants (K) as is patched expression (L). (M-R) Longitudinal sections through E11.5 hindlimbs from wild-type (M-O) and Lrp4-mutant (P-R) embryos showing dorsal-ventral patterning defects. Lrp4-mutant limbs extend from the body at a different angle than in wild type. Dorsal is to the top. Wnt7a expression does not extend to the dorsal-ventral boundary in Lrp4-mutant limbs (P) but is restricted to proximal ectoderm (red arrow). Distal expression of Lmx1b, which is dependent upon Wnt7a, is reduced in Lrp4-mutant mesenchyme (Q). engrailed 1 (En1) is normally restricted to the ventral AER at this stage (O). The expansion of En1 expression in Lrp4 mutants (R) reflects the broadened AER (Fgf8-expressing cells).

• Supplemental Figure 2 -

  Fig. S2. Early lung formation appears normal in Lrp4-mutant animals but late-term Lrp4-mutant lungs are reduced in size. Dissociated lung lobes from E18.5 wild-type (A) and Lrp4-mutant (B) embryos. Lrp4-mutant lungs are reduced in size compared with wild-type. (C-P) Longitudinal histological sections through the left lung lobes of E18.5 wild-type (C,E,G,I,K,M,O) and Lrp4 mutants (D,F,H,J,L,N,P). Cell density is higher in Lrp4-mutant lungs, but there is no obvious change in cell division based on phospho-histone H3 expression in the epithelium of mutant lungs (D) compared to wild-type (C). Proliferating cell nuclear antigen (PCNA) also showed comparable patterns of expression in wild-type and mutant lungs at E18.5 (not shown). Cell death, as visualized by TUNEL staining, was not significantly different between wild-type (E) and mutant (F) lungs, showing that the difference in size of the lungs was not due to an increase in cell death. (G-P) Phalloidin staining (green) was used to denote cell outlines and protein expression is shown in red. There was no difference in the number of ciliated cells expressing acetylated α-tubulin in Lrp4 mutants (H). There was a mild reduction in expression of the 26 kD clara cell protein in the secretory clara cells in Lrp4 mutants (J) compared with wild-type (I). Surfactant protein C (SP-C), a marker of type II alveolar epithelial cells (AECs) was normal in mutant lungs (L). T1- α, a marker of type I AECs was expressed in Lrp4 (N) mutant lungs in a similar manner to wild type (M). To investigate whether the delayed lung sacculation also involved abnormal blood-vessel development, we examined platelet endothelial cell adhesion molecule (PECAM-1) expression in Lrp4-mutant lungs. PECAM-1 staining revealed extensive lung vascular development in Lrp4 mutants (P).

• Supplemental Figure 3 -

  Fig. S3. MuSK is expressed at similar levels in Lrp4-mutant embryos as compared with wild type. RNA derived from whole E12.5 wild-type and Lrp4^mitt mutant embryos from the same litter were analyzed for MuSK expression compared to an internal control (HPRT). Wild-type MuSK/HPRT ratios ranged from 2.36 (±0.34) to 2.47 (±0.06), whereas Lrp4^mitt ratios ranged from 1.78 (±0.07) to 2.06 (±0.17).