Loss of CRISPLD2 in Zebrafish Causes Craniofacial Anomalies

Craniofacial development is a tightly regulated process that involves the complex orchestration of genetic and environmental factors.  The tissues that form the craniofacies are derived from cells that migrate from the neural tube and direct craniofacial patterning.  Perturbation of neural crest cell (NCC) migration can cause abnormal craniofacial development, such as nonsyndromic cleft lip with or without cleft palate (NSCLP).  NSCLP is a common, complex birth defect that is caused by genetic and environmental factors.  We have recently described an association between the CRISPLD2 (cysteine rich secretory protein LCCL domain containing 2) gene and NSCLP and showed that this gene is expressed in developing murine craniofacies.  The function of this gene during craniofacial development is unknown. Objectives: To describe the expression pattern and loss of function of crispld2 in zebrafish.  Methods: In situ hybridization studies were performed to identify gene expression of crispld2 and NCC marker dlx2 (distal-less homeobox 2) in zebrafish.  Three morpholinos were designed to truncate crispld2 protein for partial and complete gene knockdown.  Results: zcrispld2 is ubiquitously expressed in developing zebrafish during the somite stages and localizes to the anterior region by post fertilization day five.  Morpholino knockdown of zcrispld2 resulted in (1) lower survival rates, (2) craniofacial abnormalities, and (3) altered NCC patterning.  Conclusion: We show that zcrispld2 plays a significant role in zebrafish craniofacial development and that loss of this gene alters jaw and palate formation, supporting the role of CRISPLD2 in NSCLP.