Duplicated zebrafish co-orthologs of parathyroid hormone-related peptide (PTHrP, Pthlh) play different roles in craniofacial skeletogenesis

In mammals, Parathyroid Hormone-related Peptide (PTHrP, alias parathyroid hormone like hormone, Pthlh) acts as a paracrine hormone that regulates the patterning of cartilage, bone, teeth, pancreas and thymus. Beyond mammals, however, little is known about the molecular genetic mechanisms by which Pthlh regulates early development. To evaluate conserved pathways of craniofacial skeletogenesis, we isolated two Pthlh co-orthologs from the zebrafish (Danio rerio) and investigated their structural, phylogenetic and syntenic relationships, and expression and function. Results showed that pthlh duplicates originated in the teleost genome duplication (TGD). Zebrafish pthlha and pthlhb were maternally expressed and showed overlapping and distinct zygotic expression patterns during skeletal development that mirrored mammalian expression domains. To explore the regulation of duplicated pthlh genes, we studied their expression patterns in mutants and found that sox9a and sox9b are both upstream of pthlha in arch and fin bud cartilages, but only sox9b is upstream of pthlha in the pancreas. Morpholino antisense knockdown showed that pthlha regulates both sox9a and sox9b in the pharyngeal arches but not in the brain or otic vesicles and that pthlhb does not regulate either sox9 gene, which is likely related to its highly degraded nuclear localization signal. Knockdown of pthlha but not pthlhb caused runx2b over-expression in craniofacial cartilages and premature bone mineralization. We conclude that in normal cartilage development, Sox9 up-regulates Pthlh, which down-regulates Runx2, and that the duplicated nature of all three of these genes in zebrafish creates a network of regulation by different co-orthologs in different tissues.