Characterization of transgenic alk8 reporter zebrafish

Objectives: Our goal was to generate and characterize stable heat shock inducible transgenic zebrafish lines expressing wild type (WT), constitutively active (CA), and dominant negative (DN) forms of Alk8, a novel type I TGFb family member receptor.� We will use these lines to investigate roles for Alk8 in bone and tooth development and regeneration.�

Methods: Heat shock inducible wild type, constitutively active, and dominant negative Alk8-mCherry constructs were generated using Gateway cloning methods.� Each construct was injected into Bmp-Response-Element-GFP (BRE-GFP) zebrafish (kindly provided by Dr. Thomas Schilling).� Microinjected fish were heat-shocked at 37�C for 1 hour at the 50% epiboly stage, and screened at 24 hpf for Alk8-mCherry fluorescence, and characteristic dominant negative (DN) dorsalized, and constitutively active (CA) ventralized phenotypes.� Once functionally confirmed, Alk8 construct injected BRE-GFP fish were raised to adulthood (3 months), and screened to identify germline transgenic founder fish. Embryos obtained from incrossed F0 founder fish were heat-shocked, and screened for strong fluorescence and dorsalized (DN-Alk8) and ventralized (CA-Alk8) phenotypes.�

Results: Out of eighteen hs-CA-alk8-mCherry injected F0 zebrafish analyzed to date, progeny from five (16.7%) showed both positive mCherry fluorescence and ventralized phenotype. In addition, progeny from seven out of twenty (35%) hs-DN-alk8-mCherry injected F0 founders showed both positive fluorescence and dorsalized phenotypes.� The F1 progeny from transgenic founder fish are currently being raised to adulthood for further characterizations of tooth and bone development and regeneration.

Conclusions: Expression of positive mCherry fluorescence and DN-Alk8 dorsalized and CA-Alk8 ventralized phenotype in the F1 generation was used to monitor germline transmission of each Alk8 construct. Both alk8-CA and alk8-DN RNA injected founder fish were successful in passing the transgene to F1 offspring.� Ongoing studies are characterizing the developmental requirements for Alk8 in bone and tooth development, homeostasis, and regeneration.�