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.