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. 2023 Jan 23;12:e76863. doi: 10.7554/eLife.76863

Figure 1. subdued and white walker (CG15270) are anoctamin/TMEM16 channels expressed in class III (CIII) neurons.

(A) Alleles, chromosomal deficiencies, and enhancer trap or T2A GAL4s used in this study, as well as approximate location for primers (F and R) used to validate mutants. (B) Class III expression of subdued and CG15270 from cell-type-specific microarray (GSE69353), expressed as mean log fold-change difference between isolated CIII and whole-larval samples. Positive values indicate enrichment/upregulation and negative values downregulation. (C) qRT-PCR validating CIII expression of subdued and CG15270 (n=4, each condition). (D) UAS-GFP driven under the control of a subdued enhancer trap, GAL4c240 or T2A-GAL4wwk, evidences that subdued and white walker are expressed in CIII neurons. (E) Species, cladogram, and number of sequences used in phylogenetic analysis. (F) Maximum likelihood phylogeny of animal anoctamins, with weak branches rearranged in a species-aware manner against cladogram in E. subdued is a member of the ANO1/2 clade of nephrozoan calcium-activated chloride channels and of the clade of metazoan anoctamins that includes mammalian ANO1/2/3/4/5/6/9. Mammalian ANO8 is homologous to CG15270. Additionally, this phylogeny evidences a separate clade of ANOs of unknown function we have deemed ANO11 (cyan).

Figure 1.

Figure 1—figure supplement 1. Overlapping expression of GAL4nompC and the newly described class III (CIII)::tdTomato fusion.

Figure 1—figure supplement 1.

Figure 1—figure supplement 2. GAL4c240 driven UAS-GFP expression.

Figure 1—figure supplement 2.

Consistent with previous reports, GAL4c240 also shows expression in class IV (CIV) neurons. CIII, class III.
Figure 1—figure supplement 3. T2A-GAL4wwk driven UAS-GFP expression.

Figure 1—figure supplement 3.

T2A-GAL4wwk does not singularly drive expression in class III (CIII) neurons but rather appears to broadly mark neural tissues. Examples: (A) T2A-GAL4wwk>GFP expression patterns in the dorsal sensory neuron cluster. "ES" indicates external sensory neurons. (B) T2A-GAL4wwk>GFP in the larval central nervous system; expression appears through the central nerve cord and in restricted regions of the brain lobes.
Figure 1—figure supplement 4. Maximum likelihood phylogeny of anoctamins corresponding to Figure 1.

Figure 1—figure supplement 4.

Figure 1—figure supplement 5. NOTUNG-generated phylogeny of anoctamins corresponding to Figure 1.

Figure 1—figure supplement 5.