View Program - 63rd Annual Drosophila Research Conference

The study of histamine and its function in Drosophila melanogaster has primarily been focused on its role as a neurotransmitter, both in photoreceptors and neurons of the CNS, leading to the identification of histamine’s role in a number of functions such as vision, grooming behavior, thermal preference, and sleep¹. Histidine decarboxylase is the enzyme that synthesizes histamine, and is encoded by the Hdc gene. Mutations in the Hdc gene have been identified that result in the inability to synthesize histamine in the entire fly². We have set out to determine how tissue-specific expression of Hdc could be regulated by creating deletions in the 5’ region of the Hdc gene where 3 distinct transcription start sites (TSSs) have been reported for Hdc³. A 9.4 kb genomic Xba1 fragment was cloned into the P{CaSpeR-3} vector and used to generate germline transformants (P{CaSpeR3-Hdc^9.4+}) in a mutant Hdc^JK910 background. Histamine immunostaining of the P{CaSpeR3-Hdc^9.4+} transformant indicated that all CNS neurons contained histamine in both larvae and adults, suggesting that the Hdc gene was intact. A series of deletions, in ~300 bp steps, were generated in the 9.4 kb Xba1 Hdc genomic fragment from the 5’ end (~4.3 kb from the start of the HDC coding region) towards the coding sequence and transformed back into Hdc^JK910 mutant flies. Various P{CaSpeR3-Hdc^Δx} transformants were subjected to histamine immunodetection to determine which histaminergic cells in the CNS were no longer detected as each successive deletion step was taken in the transgene. Larval and adult stages were examined for alterations in the CNS and PNS pattern of histamine staining to correlate expression in specific cells with the removal of genomic regions containing predicted TSSs. Deletions that removed the Hdc-RD TSS had minimal effect in the tissues examined, while the removal of the TSS associated with the Hdc-RB, -RC isoforms appeared to cause loss of histamine staining in most of the CNS cells, while leaving PNS expression (photoreceptors) intact. Additionally, we have identified 3 consecutive deletions that disrupt expression in single pairs of neurons that span between the Hdc-RD and Hdc-RB, -RC isoform TSSs³. Elimination of these specific regions in the 5’ region of the Hdc gene do have incremental effects on Hdc expression, suggesting that some of the TSSs identified are tissue-specific. These transgenic deletion-bearing flies could be useful as tools for examining the contribution of some histaminergic CNS neurons to a variety of behaviors ascribed to histamine in the CNS as photoreceptor expression appears to be retained as the Hdc-RA TSS was not disrupted.
References:
1. Oh et al., 2013, PLoS ONE 8(7):e68269
2. Burg et al., 1993, EMBO J. 12(3):911-919.
3. Larkin, A. et al. (2021), Nucleic Acids Res. 49(D1) D899–D907
CL was supported by a McNair Scholars Program Grant through GVSU.

Use of transformants bearing deletions in the 5’ upstream region of the Hdc gene to identify regions required for CNS expression of Hdc