a Three loss-of-function (LoF) alleles in zebrafish were generated by CRISPR/Cas9 injections targeting smchd1. b Endogenous spatial-temporal expression pattern of smchd1 in zebrafish embryos as determined by qPCR and WISH. n = 3 biological samples (qPCR), 3 independent experiments with at least 20 embryos each (WISH). Data are presented as mean values ± SD. c Maternal zygotic (MZ) smchd1^lof embryos lack smchd1 mRNA (qPCR and WISH) and protein (Western blot), suggestive of total nonsense-mediated decay. Scale bar = 200 μm. n = 2 biological samples (qPCR, Western blot), 3 independent experiments with at least 20 embryos each (WISH). d MZ smchd1^lof zebrafish are viable and fertile, and do not model arhinia at 3 months. Scale bar = 1 mm.

a K-means clustering of differentially expressed genes at 4-8-cell stage. b Gene ontology: biological process (GO:BP) enrichment of cluster 5 from K-means clustering at 4-8-cell stage. RNA-seq of wt and MZ lof1 embryos show changes in hox gene expression at both 4-8-cell stage (c) and sphere stage (d). e hox genes show persistent upregulation at 11-somites. n = 3 biological samples, p values were calculated by 2-tailed unpaired Student’s t test. f hoxb2a shows expanded expression boundary at 10-somites (top) and hoxc10a shows anteriorly expanded expression at 17-somites (bottom). krox20 marks rhombomeres 3 and 5. g–h Adult fish show defects in axial skeletal patterning, including loss of a rib (arrow) and loss of a supraneural vertebra (arrowhead). Scale bar = 200 µm. P values were calculated by the Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test. Bars without p values are not significant. All data are presented as mean values ± SD.

a, b Skeletal preparations of fish of different genotypes. Phenotypes include loss of a rib (filled arrow), loss of a supraneural vertebra (arrowhead) and loss of a caudal vertebra (open arrow) Scale bar = 1 mm. P values were calculated by the Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test. c qPCR shows that there is little to no zygotic smchd1 expression. p: paternal allele; m: maternal allele. n = 3 biological samples. d qPCR shows that hox derepression is already observed in unfertilized oocytes and that oocytes from ± females show intermediate level of hox mis-expression. n = 3 biological samples, p values were calculated by 2-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. All data are presented as mean values ± SD. e In situ hybridization of smchd1 shows that smchd1 is expressed in developing oocytes. Roman numerals denote stages of oocyte development. Scale bar = 200 µm. f Bisulfite sequencing in oocytes shows hypomethylation of hoxc10a locus when smchd1 is knocked out.

a A new Smchd1 loss-of-function allele was generated using CRISPR/Cas9. This produced a 6 nt deletion including the splice donor site of intron 3. This resulted in a number of aberrantly spliced transcripts. The protein variation listed is the smallest in-frame mutation detected, a 14 amino-acid deletion including the catalytic residue of the GHKL ATPase domain p.E147. b Western blot from heads of E10.5 embryos show a markedly reduced level of SMCHD1 protein in the Δ6nt/Δ6nt, which runs slightly faster than wildtype SMCHD1. n = at least 5 embryos per genotype. c Observed and expected numbers of pups born of each genotype. Axial skeletal patterning analysis of + /+ and Δ6nt/+ animals. Homeotic transformations observed in the animals are marked in red in the schematic (d). Transformations include C7 → T1, T13 → L1 and L6 → S1. C7 rib is labelled with a *, the missing T13 rib is marked with an arrow and the hypomorphic T13 rib is labelled with an arrowhead. Number of animals analysed with each phenotype are in (e). Animals showing any of the homeotic transformations are considered as transformed. Most Δ6nt/+ animals show all of the homeotic transformations marked in (d). Scale bar = 500 µm.

a Two loss of function alleles of lrif1 were generated in zebrafish by CRISPR/Cas9 injections. b Endogenous spatial-temporal expression pattern of lrif1 in zebrafish embryos as determined by qPCR and WISH. n = 3 biological samples (qPCR), 3 independent experiments with at least 20 embryos each (WISH). c Maternal zygotic (MZ) lrif1^lof embryos lack lrif1 mRNA suggestive of total nonsense-mediated decay. n = 3 biological samples. P values were calculated by 2-tailed unpaired Student’s t test. d, e Axial skeletal patterning defects in adult fish. Phenotypes include loss of a rib (filled arrow), loss of a supraneural vertebra (arrowhead) and loss of a caudal vertebra (open arrow). P values were calculated by the Kruskal-Wallis test followed by Dunn’s Multiple Comparison Test. f qPCR shows that hox derepression is detected in unfertilized oocytes from both +/− and −/− females. P values were calculated by 2-tailed unpaired Student’s t test. Inset shows expression of lrif1 in Stage I and II oocytes. Scale bars = 200 µm. n = 3 biological samples (qPCR), 5 ovaries of adult fish (WISH). g Model of Smchd1/Lrif1-driven transgenerational inheritance for skeletal patterning. Smchd1/ Lrif1 sets up an epigenetic state during oogenesis to ensure correct expression of hox genes during embryogenesis which in turn patterns the vertebrae. *p < 0.05, **p < 0.01, ***p < 0.001. All data are presented as mean values ± SD.

a RNA-seq performed on controls and FSHD2 patient fibroblasts reveal dysregulations of HOX expression when SMCHD1 was mutated in the germline. b Schematic of experimental design to determine whether HOX dysregulation is the result of germline or somatic SMCHD1 haploinsufficiency. SMCHD1 was CRISPR/ Cas9-inactivated ex vivo in control fibroblasts using two distinct guide RNAs (KO1 and KO2). Yellow box represents maternal contribution of SMCHD1 which diminishes rapidly post-fertilization. c Western blot in fibroblasts cell extract show successful knockout of SMCHD1, resulting in protein-null alleles. FSHD2 cells used here are + /V1826Gfs*19. d qPCR shows that, unlike in FSHD2 cells, somatic knockout of SMCHD1 in adult cells does not lead to the upregulation of HOX. P values were calculated by 2-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean values ± SD.