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Fig. 2 creb3l1 mutant zebrafish are normal size and exhibit normal overall skeletal morphology and bone density. (A) Exon 2 of the zebrafish creb3l1 gene was targeted to generate creb3l1ΔbZIP/ΔbZIP fish with a +7 bp insertion (in green) that causes a frameshift mutation at amino acid 65 (D to P change). This mutation generates a Creb3l1 fragment encoding only the N-terminal 65 amino acids of Creb3l1, followed by 10 additional amino acids not present in wild-type Creb3l1 (in gray). Yellow highlights wild-type codons while gray highlights mutant codons and amino acids. TGA stop codon is in red. cDNA, consensus coding sequence and protein sequences are shown. (B) creb3l1 mRNA levels were measured by RT-PCR in 7 dpf creb3l1+/+ and creb3l1ΔbZIP/ΔbZIP larvae. Expression of creb3l1 is significantly elevated in the creb3l1ΔbZIP/ΔbZIP larvae. n = 3–4, ***p < .001. Each data point represents a pool of ~30 larvae. (C) Larvae from heterozygous creb3l1ΔbZIP/+ incrosses were genotyped at 7 dpf and show the expected Mendelian ratios. n and p values are indicated. (D) The length of age-matched 6 mpf creb3l1+/+ and creb3l1ΔbZIP/ΔbZIP fish was measured from the snout to the posterior-most point of the tail. There was no significant difference in length between the wild-type and the creb3l1 mutant fish. n = 27–34. Each data point represents an individual fish. (E) Age-matched 3 mpf creb3l1+/+ and creb3l1ΔbZIP/ΔbZIP fish were analyzed by μCT and representative scans of male and female animals are shown. The creb3l1ΔbZIP/ΔbZIP fish exhibit overall normal skeletal morphology. Scale bar = 5 mm. (F) Bone density (mgHA/cm3) of the entire skeleton of male and female creb3l1+/+ and creb3l1ΔbZIP/ΔbZIP fish was determined from the μCT scans. n = 9–11. No significant difference was detected. Each data point represents an individual fish.