Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.

BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood. RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration. CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

INTRODUCTION: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. METHODS: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. RESULTS: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. CONCLUSION: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.