Characterization of AK2 Gene Function in Zebrafish Hematopoiesis

The Adenylate Kinase (AK) gene family consists of 7 different members (AK1-7) that contribute to energy metabolism of the cells by converting ATP (or GTP) and free AMP to ADP (or GDP) and free ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 is uniquely located in the mitochondrial intermembrane space and has been implicated in Caspase 10-mediated apoptosis, although the published data remain controversial. More recently, it was demonstrated that mutations of the AK2 gene cause reticular dysgenesis, an autosomal recessive form of severe combined immunodeficiency (SCID). Reticular dysgenesis is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation resulting in overwhelming infections and high lethality in affected patients. Moreover, patients commonly present with bilateral sensorineural deafness. The mechanisms underlying the biological consequences of AK2-defieincy remain unclear and the generation and characterization study of model systems is expected to provide useful insigths. Ak2 gene-targeted mice have not been reported in the literature, but lines carrying homozygous inactivating retroviral insertions have been shown to be embryonically lethal (our unpublished observations).

Because of the known advantages of zebrafish as model system for developmental studies and the similarities of hematopoiesis in zebrafish and higher vertebrates, we set out to investigate the function of the zebrafish ak2 gene in development, with particular emphasis on hematopoiesis.

Similar to humans, we found that two different alternatively spliced isoforms of the ak2 gene (Isoform A and Isoform B) are expressed in zebrafish. By Real-Time PCR and In situ Hybridization (ISH) we analyzed the expression of both ak2 isoforms during embryo development. Preliminary data indicate that Isoform A is more abundantly represented than Isoform B during embryo development. ISH analysis showed that the two isoforms have different spatial expression patterns. These data suggest different functionalities for ak2 isoforms during embryo development.

To explore such hypothesis, we injected two different morpholinos (MOs) targeting the ak2 isoforms. Downregulation of both ak2 isoforms phenocopied the human disease and resulted in a strong reduction of developing lymphocytes. Moreover we observed a hypochromic phenotype that also suggested impairment of the erythroid lineage. ISH experiments are underway to better define the affected hematopoietic lineages. Interestingly, ak2 MOs-injected embryos showed also developmental defects beyond the hematopoietic system, such as abnormal jaw development. Future studies will focus on the characterization of the specific function of the alternatively spliced ak2 isoforms.

We show that the transcription features of the AK2 gene are conserved in zebrafish. The observed differential expression patterns of the zebrafish ak2 isoforms may provide new insights into the function of AK2 in the development of the hematopoietic system, as well as other organs and offers prospects for the understanding of the molecular mechanisms involved in reticular dysgenesis.

No relevant conflicts of interest to declare.