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First published online November 21, 2006
doi: 10.1242/10.1242/dev.02696

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LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction

¹ Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
² Howard Hughes Medical Institute, University of Colorado Health Sciences Center, Department of Pediatrics, Section of Developmental Biology, Aurora, CO 80045, USA.

View larger version (76K): [in this window] [in a new window]   Fig. 1. mte and mitt mouse embryos have limb defects due to mutations in the Lrp4 gene. (A) Schematic diagram of mouse Lrp proteins. Positions of mutations in the mitt and mte alleles of Lrp4 are indicated next to the proposed Lrp4 structure. The intracellular region of Lrp4 contains motifs predicted to mediate clustering to clathrincoated pits and a terminal SXV motif predicted to mediate interactions with PDZ domain-containing proteins. (B-E) E18.5 Limb skeletal preparations highlight the abnormalities in Lrp4mitt autopods (C,E) compared with wild type (B,D). (B,C) Forelimbs; (D,E) hind limbs. Digit numbers are reduced, phalanges and metacarpals/metatarsals are fused, and ossification is decreased in Lrp4mitmutant autopods (C,E). At earlier stages of limb development, Lrp4 mutants display AER expansion and a failure of AER refinement and maturation. (F-K) Longitudinal sections through E9.5 limb buds. Dorsal is to the top, distal to the right. Fgf8, a marker of AER cells, is normally restricted to the distal region of E9.5 forelimbs (F), but is expanded both dorsally (red arrowhead) and ventrally (black arrowhead) in Lrp4 mutants (G) and no morphologically distinct AER forms (see Fig. S1 in the supplementary material). In parallel, dorsal expression of Msx2 (H) is expanded in Lrp4 mutants (I, red arrowhead) and there is a loss of Wnt7a in the most distal region of mutant forelimbs (compare K to J).

View larger version (65K): [in this window] [in a new window]   Fig. 2. Lrp4 is expressed in multiple tissues and organs during mouse development. (A) Brightfield image of a radioactive in situ hybridization for Lrp4. A cross-section of an E9.5 forelimb bud shows expression of Lrp4 (dark grains) throughout the ectoderm. (B) Dorsal view of an E11.5 hind limb bud shows strong Lrp4 expression (blue) in the AER. (C,D) Whole-mount in situ hybridizations show expression of Lrp4 at the tips of the ureteric buds at E11.5 (C, black arrows) and E16.5 (D). (E) A lateral view of the snout of an E12.5 embryo shows Lrp4 is expressed in whisker primordia. (F) Transverse section through the dorsolateral back region of an E14.5 embryo, counter-stained with Fast Red (pink). Lrp4 is expressed in developing hair follicles (blue). Inset, high magnification view of a hair follicle. (G) Upper palate of an E12.5 embryo. Lrp4 is strongly expressed in the developing incisors and molars. (H) Whole-mount in situ hybridization shows expression of Lrp4 in mammary buds (white arrowheads) at E13.5. (I) Radioactive in situ hybridization (darkfield) for Lrp4 on a sagittal section of a wild-type E12.5 embryo. Lrp4 is expressed strongly in the proximal airways of the lungs (red arrows) and in the diaphragm (white arrow). (J) Transverse section through an E10.5 embryo counter-stained with Fast Red (pink) shows expression of Lrp4 in the dorsal neural tube and floorplate. di, diaphragm; he, heart; in, incisor; lb, limb bud; li, liver; lu, lung; mb, mammary bud; mn, motorneuron; mo, molar; wh, whisker vibrissae.

View larger version (103K): [in this window] [in a new window]   Fig. 3. Lrp4 is required for neuromuscular synaptogenesis. (A) Newborn wild-type pups display motile activity and have begun feeding shortly after birth (indicated by the presence of a milkspot). By contrast, Lrp4 mutant pups (B) are cyanotic, do not move, feed or breathe and retain their in utero posture. (C,D) Longitudinal histological sections through the left lung lobe of E18.5 wild-type (C) and mutant embryos (D). The future alveolar spaces (al) are smaller and the interstitial mesenchyme is thicker in Lrp4 mutant (D) embryos compared with wild type (C). E18.5 intercostal (E,F) and hind limb (G,H) muscles stained with -bungarotoxin (red) to mark acetylcholine receptors and co-visualized with HB9-GFP (E-F, green), which is expressed specifically in motoneurons, or synaptophysin (G-H, green) which marks synaptic vesicles. E18.5 wild-type (I-K) and mutant (L-N) diaphragm muscles stained with -bungarotoxin (red) and co-visualized with HB9-GFP (green). Lrp4 mutants show extensive branching of the phrenic nerve in diaphragms and lack AChR clustering in the synaptic endplate (N). In all types of muscle examined, Lrp4 mutants lack normal AChR clustering in the synaptic endplate.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Lrp4 is required for the initiation of AChR clustering. E13.5 (A-F) and E14.5 (G-L) wild-type (A-C,G-I) and mutant (D-F,J-L) diaphragms, with neuronal projections visualized with neurofilament (A-F, green) or HB9-GFP (G-L, green) and AChRs marked by -bungarotoxin (red). Before innervation, pre-clustering of AChRs does not occur in Lrp4 mutants (compare B to E). At E14.5, mutant phrenic nerves show longer primary branches that do not stop in the endplate region. AChR clusters are still undetectable in Lrp4 mutants at this stage of NMJ initiation (K). (M-P) Wild type (M,O) and Lrp4 mutant (N,P) cultured myotubes, either untreated (M,N) or following overnight treatment with recombinant Agrin (O,P). AChR clusters (visualized with -bungarotoxin in red) form in myotubes derived from wild type but not Lrp4 mutants.

View larger version (71K): [in this window] [in a new window]   Fig. 5 . Rapsn, utrophin and MuSK localization is disrupted in Lrp4 mutants. Immunofluorescence analysis of NMJs in control (A-C) and Lrp4 mutant (D-F) embryos. Sections of E18.5 hind limb muscles were stained with antibodies against Rapsn (A,D) and utrophin (Utrn; B,E). MuSK localization was visualized on sections of E18.5 intercostal muscles (C,F). Synaptic sites were identified by co-staining with antibodies against synaptic vesicle proteins, SV2 (A,D,C,F) or synaptophysin (B,E).

View larger version (107K): [in this window] [in a new window]   Fig. 6 . Synaptic nuclei are not transcriptionally specialized in Lrp4 mutant muscles. E14.5 (A-H) or E18.5 (I-P) diaphragms showing in situ hybridizations using probes specific to AChR (A,E,I,M) AChR (B,F,J,N), Musk (C,G,K,O) and Lrp4 (D,H,L,P). At E14.5, transcripts of these genes become restricted to the endplate band region in wild-type muscles (A-D) but are more broadly expressed in Lrp4 mutants (E-H). At E18.5,transcripts of these genes continue to be upregulated in the endplate band region in wild-type muscles (I-L) but are expressed at low, uniform levels in mutants (M-P).

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