Late-onset skeletal defects observed in the koliber^nu7 (kol^nu7) mutant are the result of downregulation of msmo1 expression. (A) Compared to wild-type (wt) siblings (top), adult kol^nu7 mutants display a reduced body length and small head size. wt n=300, kol^nu7 n=300. (B) Whole-mount skeletal preparations reveal gross malformations and hyperossification throughout the adult kol^nu7 craniofacial and axial skeleton after Alcian Blue (cartilage) and Alizarin Red (ossified bone and mineralized tissues) staining. wt n=100, kol^nu7 n=100. (C-F) Early larval mutants do not display patterning defects or premature ossification. Whole-mount Alizarin Red staining of 4.7 mm (∼8 dpf) wt (C,E) and kol^nu7. (D,F). Ventral view (C,D) and lateral view (E,F). wt n=3, kol^nu7 n=4. (G) Positional cloning reveals that the kol^nu7 locus is located to the ∼457 kb critical region flanked by polymorphic markers with one or two recombinants out of 1844 meioses, corresponding to a genetic distance of 0.16 cM. (H) Screen of gene expression using quantitative RT-PCR from RNA extracted from hypural complex of ∼18 mm SL kol^nu7 and wt siblings. wt n=2, kol^nu7 =2. Only msmo1 level was significantly different out of 11 tested genes, located in the ∼1.2 Mb region encompassing the kol^nu7 locus. Initial screen results: grhprb not detected (ND); uba6 mean difference 0.64, s.d. 0.36; abpp2 −1.62, s.d. 1.00; mettl14 1.16, s.d. 0.68; prss12 1.20, s.d. 0.72; ndst3 1.51, s.d. 1.50; ugt8 −1.03, s.d. 0.07; spock3 ND; tll1 0.67, s.d. 0.27; cpe 0.90, s.d. 0.42; msmo1 −10.07, s.d. 4.32. Confirmation test of msmo1 expression (msmo1 −11.47, s.d. 8.49; P=0.0012), wt n=3, kol^nu7 =5. Three technical replicates were included for all assays. Gene expression was normalized to the reference gene eefla1. Fold change was calculated using Livak method (Livak and Schmittgen, 2001). P-value calculated using unpaired Student's t-test on dCt values. (I) The msmo1^nu81 mutant allele is not able to complement the kol^nu7 mutation. Adult kol^nu7/+:msmol^nu81/+ transheterozygotes phenocopy the kol^nu7 mutant. wt n=100, kol^nu7/+:msmol^nu81/+ n=100. (J) Whole-mount skeletal preparations reveal gross malformations throughout the kol^nu7/+:msmol^nu81/+ craniofacial and axial skeleton, similar to those observed in kol^nu7. wt n=10, kol^nu7/+:msmo1^nu81/+ n=10. (K) The msmo1^nu81 allele is the result of a 37 bp insertion, allowing for allele-specific expression analysis between msmo1^nu81 and kol^nu7. PAM, protospacer adjacent motif (underlined in red). (L) Strong downregulation of the kol^nu7-linked allele (asterisks) in kol^nu7/+:msmo1^nu81/+ compared to the wt allele in msmo1^nu81/+ suggests that the kol^nu7 mutation is cis-acting. kol^nu7/+:msmo1^nu81/+ n=3, msmo1^nu81/+ n=3. The top band is a heterodimer of wt and mutant strands.

The suppression of early lethality of the msmo1^nu81 mutation yields adult fish with kol^nu7-like phenotype. (A) Survival of larvae from msmo1^nu81/+ in-crosses, from 7 dpf to 70 dpf. Most msmo1^nu81 mutants die by 9 dpf. [7 dpf wild type (wt; black) n=19, heterozygotes (het; pale gray) n=49, knockout mutants (KO; dark gray) n=16, P=0.02794; 8 dpf wt n=21, het n=39, KO n=17, P=0.8065; 9 dpf wt n=12, het n=40, KO n=3, ***P=0.0008; 10 dpf wt n=37, het n=44, KO n=3, ****P<0.0001; 15 dpf wt n=26, het n=51, KO n=3, ****P<0.0001; 70 dpf wt n=18, het n=35, KO n=0, ***P=0.001.] (B) Overexpression of msmo1 driven by daily heat shock of the transgenic line Tg(hsp70l:msmo1:IRESnlsGFP)^nu99 rescued the lethality of msmo1^nu8 mutants. Transgenic screening based on cardiac GFP. (Control non-transgenic siblings 14 dpf wt n=14, het n=32, KO n=2, ***P=0.0035; transgenic siblings 14 dpf wt n=18, het n=44, KO n=28, P=0.3214.) cont, control; hs, heat shock. (C) Dietary cholesterol supplementation does not improve the survivability of msmo1^nu81 mutants. Clutches from msmo1^nu81/+ in-crosses were fed either a high-cholesterol diet (hcd) or a control standardized diet (cont) beginning at 5 dpf until collection at 10 dpf. (HCD wt n=26, het n=50, KO n=9, P=0.0089; control diet wt n=22, het n=66, KO n=5, P<0.0001.) All two-tailed P-values were calculated using chi-squared test. (D-F′) Whole-mount in situ hybridization during the first 5 days of development shows msmo1 expression predominately in the yolk syncytial layer (YSL) and liver. Expression is first detected during early somitogenesis in the YSL (D) and continues there at 3 dpf (E). At 4 dpf, strong expression is observed in the differentiated liver (F,F′). (G-I) Generation of msmo1^nu81/wild-type chimeras using endoderm replacement rescues early lethality of msmo1^nu81 mutants and reveals a strong kol^nu7-like phenotype. (G) Schematic of the procedure. Wild-type Tg(ubi:Zebrabow-M)^a131 donor embryos were injected with sox32 RNA to force an endodermal fate. At high stage (∼3 hpf), cells were transplanted from donor to host embryos collected from msmo1^nu81/+ in-crosses. (H) Surviving msmo1^nu81 mutants display a strong kol^nu7-like phenotype. msmo1^nu81 n=4. (I) The majority of organs of endodermal origin displayed a high enrichment in transplanted Tg(ubi:Zebrabow-M)^a131 cells (red). msmo1^nu81 n=4. (J-L) Liver-specific msmo1 expression in msmo1^nu81 mutants rescues early lethality and produces juvenile msmo1^nu81 mutants with strong kol^nu7-like phenotype. (J) Liver-specific regulatory element fabp10a was used to drive msmo1 expression in msmo1^nu81 mutants. (K) Adult Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 mutants phenocopy kol^nu7 mutant. Tg(fabp10:msmo1:pA)^nu100 n=50, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=50. (L) Whole-mount skeletal preparations reveal gross malformations throughout Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 craniofacial and axial skeleton, similar to those observed in kol^nu7. Tg(fabp10:msmo1:pA)^nu100 n=3, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=7.

Loss of Lss activity phenotype is epistatic to msmo1^nu81 mutation. (A) The lss^nu60 allele. PAM, protospacer adjacent motif (underlined in red). (B) Survival analysis of clutches of lss^nu60/+ in-crosses, from 9 dpf to 23 dpf, reveals that most lss^nu60 mutants die by 12 dpf. [9 dpf wild type (wt; black) n=21, heterozygotes (het; pale gray) n=30, knockout mutants (KO; dark gray) n=15, P=0.4412; 10 dpf wt n=15, het n=27, KO n=16, P=0.8563; 11 dpf wt n=20, het n=44, KO n=14, P=0.3320; 12 dpf wt n=24, het n=48, KO n=10, *P=0.0277; 13 dpf wt n=33, het n=51, KO n=10, **P=0.0026; 23 dpf wt n=8, het n=27, KO n=0, ***P=0.0009.] (C) Survival analysis of clutches of msmo1^nu81/+;lss^nu60/+ in-crosses reveals that loss of Lss activity increases survivability of msmo1^nu81 mutants at 10 dpf. [wt observed (O) n=11, expected (E) n=11; msmo1^nu81 O n=4, E n=11; msmo1^nu81;lss^nu60/+ O n=9, E n=22; msmo1^nu81;lss^nu60 O n=10, E n=11; lss^nu60 O n=12, E n=11; msmo1^nu81/+;lss^nu60 O n=30, E n=22; msmo1^nu81/+;lss^nu60/+ O n=40, E n=44; msmo1^nu81/+ O n=18, E n=22; lss^nu60/+ O n=18, E n=22; *P=0.0431.] Two-tailed P-values were calculated using chi-squared test. (D,E) Suppression of early lethality of lss^nu60 mutants by liver-specific expression of lss. (D) Adult Tg(fabp10:lss:pA)^nu101;lss^nu60 mutants phenocopy kol^nu7. Tg(fabp10:lss:pA)^nu101 n=12, Tg(fabp10:lss:pA)^nu101;lss^nu60 n=12. (E) Whole-mount skeletal preparations reveal gross malformations throughout Tg(fabp10:lss:pA)^nu101;lss^nu60 craniofacial and axial skeleton, similar to those observed in kol^nu7. Tg(fabp10:lss:pA)^nu101 n=3, Tg(fabp10:lss:pA)^nu101;lss^nu60 n=5.

Loss of Msmo1 and Lss activity is associated with abnormal endochondral bone formation. (A-T) The hyomandibular (A,E,I,M,Q), ceratohyal (B,F,J,N,R) and hypural complex (C,D,G,H,K,L,O,P,S,T) were compared between wild type (A-D), kol^nu7 (E-H), kol^nu7/+:msmol^nu81/+ (I-L), Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (M-P) and Tg(fabp10:lss:pA)^nu101;lss^nu60 (Q-T). (E-T) The endochondral bones of the mutants are compressed and irregularly shaped with ectopic ossification and fusions throughout the growth plates, marked with arrows. Images represent disarticulated whole-mount adult skeletal preparations at ∼4 months of age. Scale bars: 100μm. Tg*, Tg(fabp10:msmo1:pA)^nu100; Tg**, Tg(fabp10:lss:pA)^nu101. wt n=4, kol^nu7 n=4, kol^nu7/+:msmol^nu81/+ n=4, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=4, Tg(fabp10:lss:pA)^nu101;lss^nu60 n=4.

Loss of Msmo1 and Lss activities disrupts growth plate patterning. (A-P) Expression domains of col2a1a (A,D,G,J), col10a1a (B,E,H,K,M-P) and msmo1 (C,F,I,L) were analyzed within growth plates of wild type (A-C), kol^nu7 (D-F), Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (G-I) and Tg(fabp10:lss:pA)^nu101;lss^nu60 (J-L) using RNAscope in situ hybridization. (A) Strong col2a1a expression is observed in round and stacked chondrocytes in wild type, corresponding to presumed resting and proliferating chondrocytes. (B) Expression of col10a1a in wild type is observed complementary to col2a1a expression in presumed hypertrophic chondrocytes. The expression of col2a1a is expanded in kol^nu7 (D) and Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (G) growth plates, with a near-complete loss of col10a1a expression in kol^nu7 (E) and Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (H). Expression of col2a1a (J) and col10a1a (K) in Tg(fabp10:lss:pA)^nu101;lss^nu60 appears less affected when compared to kol^nu7(D,E) and Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (G,H). Expression of msmo1 is detected primarily in pre-hypertrophic chondrocytes of wild type (C); msmo1 expression is undetectable in kol^nu7 growth plates (F), but is upregulated in transgenically rescued msmo1^nu81 (I) and lss^nu60 (L) mutants. (M-P) Expression of col10a1a in osteoblasts is unchanged between wild type and mutants. Images represent paraffin sections of the pterotic at 2 months of age (∼15 mm SL). Boxed areas in A-L are shown at higher magnification in A′-L′. Scale bars: 100 μm. Tg*, Tg(fabp10:msmo1:pA)^nu100; Tg**, Tg(fabp10:lss:pA)^nu101. wt n=6, kol^nu7 n=6, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=3, Tg(fabp10:lss:pA)^nu101;lss^nu60 n=3.

Ihh signaling within endochondral growth plates is present despite loss of Msmo1 and Lss activity. (A-L) Expression domains of ihha (A,D,G,J), ihhb (B,E,H,K) and ptch1 (C,F,I,L) were analyzed within growth plates of wild type (A-C), kol^nu7 (D-F), Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (G-I) and Tg(fabp10:lss:pA)^nu101;lss^nu60 (J-L) using RNAscope in situ hybridization. (A-C) In wild type, expression of ihha and ihhb is observed in the pre-hypertrophic chondrocyte zone, with ptch1 expression seen to either side of these domains. (D-L) Expression of ihha, ihhb and ptch1 is present within mutant growth plates and similar to domains observed in wild type. Images represent paraffin sections of the pterotic at 2 months of age (∼15 mm SL). Scale bars: 100 μm. Tg*, Tg(fabp10:msmo1:pA)^nu100; Tg**, Tg(fabp10:lss:pA)^nu101. wt n=3, kol^nu7 n=3, kol^nu7/+:msmol^nu81/+ n=3, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=3, Tg(fabp10:lss:pA)^nu101;lss^nu60 n=3.

New zebrafish models for CDP. (A-D) Abnormal ossification, marked with arrows, within endochondral growth plates of kol^nu7 (B), kol^nu7/+:msmol^nu81/+ (C) and Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 (D) mutant phenotypes resembles defects observed in patients with chondrodysplasia punctata. Images represent lateral views of palatoquadrate growth plates of craniofacial bones from disarticulated whole-mount adult skeletal preparations at ∼4 months of age. Scale bars: 100 μm. Tg*, Tg(fabp10:msmo1:pA)^nu100. wt n=4, kol^nu7 n=6, kol^nu7/+:msmol^nu81/+ n=4, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=4.

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