CRISPR-Cas9 induced lrp5 zebrafish knockout line. (A) Schematic representation of the ZF lrp5 gene structure with Sanger sequence chromatogram; (B) demonstrating the del8bp (Ala53fs) mutation, that results in a frameshift and a premature stops codon; (C) Schematic representation of the ZF LRP5 wild-type and mutated protein demonstrates the lack of functional domains in LRP5 of lrp5^-/- (LY -low-density lipoprotein receptor; EGF – epidermal growth factor; LDLa- Low-density lipoprotein receptor domain class (A, D) Western-blot results demonstrated the absence of LRP5 protein in lrp5^-/-.(E) Each column in the graph represents the proportion of genotype (%) per developmental stage. Only 3.7% of lrp5^-/- in survive and reach adolescence using instead of expected 25%.

lrp5^-/- adult fish display low bone mineral density. (A) Whole-mount alizarin red staining at 1.5 mpf display the distribution of mineralized matrix through the head bones. In the skull of lrp5^-/- distribution of mineralized matrix throughout whole head bones differs from lrp5^+/+ Scale bar=1mm. (B) coronal view of the cranial vault. The alizarin red signal is distributed ubiquitously in lrp5^+/+ lower jaw (C) and opercle (D), meanwhile in lrp5^-/- fish the lower jaw demonstrated discontinues alizarin red signal. Scale bar=500µm. (E) Representative photos of lrp5^+/+ and lrp5^-/- siblings at 4mpf; lrp5^-/- present with deformed head and body curvatures. Scale bar=1cm. (F) 3D reconstruction of X-ray images of lrp5^+/+ and lrp5^-/- skeleton at 3 and 6mpf. Scale bar= 1cm (G, H) Total BMD is decreased in adult lrp5^-/- fish. (I, J) the skull BMD of lrp5^-/- mutant fish is significantly lower both at 3 and 6mpf. Results in G-J are expressed as mean SD (3mpf n =13-15 fish, 6mpf n =12-13 fish, t-test).

Craniofacial deformities of adult lrp5 mutant. (A) Adult lrp5^-/- display major deformities of neuro- and viscera- craniums. (B) Schematic representation of morphometrical parameters measured na- nasofacial angle, pd- parasphenoid distance between its edges. (C, D) Nasofacial angle at 3mpf and 6mpf is significantly bigger in lrp5^-/- compared to lrp5^+/+, meanwhile parasphenoid distance is significantly shorter in lrp5^-/- adult fish at 3 and 6mpf (E, F). (G) Parasphenoid bones of lrp5^-/- are severely deformed and display a similar fracture pattern, outlined in yellow. (H) 40% of lrp5^-/- fish displayed malformed parasphenoid at 3mpf and 38.4% at 6mpf within lrp5^-/- group. At 3mpf and 6mpf lrp5^+/+ display no indication of parasphenoid deformities.(Scale bar 500µm, 3mpf n =13-15 fish, 6mpf n =12-13 fish, t-test).

RNA-seq results. (A) The overall distribution of differentially expressed genes in the cranial bones between the lrp5^+/+ and lrp5^−/− groups. Red and green represent up-regulated and down-regulated transcripts, respectively, in the clustering analysis. The color intensity is directly proportional to the change. (B) The volcano plot shows DEGs in the lrp5^−/− cranial bones. The red part indicates up-regulated genes, and the green part indicates down-regulated genes. (C) Box plot of pathway analysis for all DEGs.

RT-qPCR validation of RNA-Seq data. Genes relevant to osteoclasts activity (A-C) and osteoclasts differentiation (D-F). (n=6-7, Mann-Whitney test, dots represent each individual value distributed around mean (line): in black lrp5^+/+ control, dark red lrp5^-/-). Mann-Whitney test p-values: *p ≤ 0.05, **p≤ 0.01.

lrp5^-/- scales display a larger demineralized area and TRAP positive area. (A) von Kossa staining of lrp5^-/- scales revealed significantly more demineralized area (t-test, n of scales per group=120-130) than of control siblings scales. (B) TRAP staining of lrp5^-/- scales indicates significantly larger TRAP area compared to lrp5^+/+ (t-test, n of scales=95-101). Scale bar=100µm. (C) Branching of lepidotrichia in lrp5^+/+ fins occurs in distal segments (red arrows), meanwhile lrp5^-/- fish display bifurcation in proximal segments of lepidotrichia (C, red arrows). The number of normally branched fin rays is significantly lower in lrp5^-/- compared to lrp5^+/+ siblings. (D) Schematic representation of excessive branching in lrp5^-/-. Results in A, B and C are expressed as mean SD.