The Ntan1 gene is expressed in perineural glia and neurons of adult Drosophila

Abstract

The Drosophila Ntan1 gene encodes an N-terminal asparagine amidohydrolase that we show is highly conserved throughout evolution. Protein isoforms share more than 72% of similarity with their human counterparts. At the cellular level, this gene regulates the type of glial cell growth in Drosophila larvae by its different expression levels. The Drosophila Ntan1 gene has 4 transcripts that encode 2 protein isoforms. Here we describe that although this gene is expressed at all developmental stages and adult organs tested (eye, antennae and brain) there are some transcript-dependent specificities. Therefore, both quantitative and qualitative cues could account for gene function. However, widespread developmental stage and organ-dependent expression could be masking cell-specific constraints that can be explored in Drosophila by using Gal4 drivers. We report a new genetic driver within this gene, Mz317-Gal4, that recapitulates the Ntan1 gene expression pattern in adults. It shows specific expression for perineural glia in the olfactory organs but mixed expression with some neurons in the adult brain. Memory and social behavior disturbances in mice and cancer and schizophrenia in humans have been linked to the Ntan1 gene. Therefore, these new tools in Drosophila may contribute to our understanding of the cellular basis of these alterations.

Figure 1

Gene expression analysis of the Ntan1 gene in Drosophila melanogaster. (A) Schematic representation of the insertion sites of the Gal4 elements of Mz317-Gal4; Mz97-Gal4,UAS-Stinger and Ntan1^NP1111-Gal4 fly lines in the Ntan1 gene. The P{GawB}Mz317 insertion and the P{GawB}Mz97 are located ∼ 150 bp and ∼ 100 bp upstream of the second transcriptional start site within the first intron, respectively. The P{GawB}Ntan1^NP1111 insertion is located ∼ 1300 bp upstream of the second transcriptional start site within the first intron. 5′ UTR and 3′ UTR regions, exons and introns of each transcript and the location of the different primers used in RT-PCR are shown. (B) Gene expression of the Ntan1 gene by RT-PCR in different developmental stages and adult organs of wildtype flies (Canton-S). (C) Gene expression of Ntan1 transcripts by RT-PCR in different developmental stages and adult organs of wildtype flies (Canton-S). M: Molecular marker, G: Genomic DNA, E: Embryonic cDNA, L: Larval  cDNA, P: Pupal cDNA, A: Third antennal segment cDNA, B: Brain cDNA, EY: Eye cDNA, RT−: RT-PCR without retrotranscriptase, C−: Negative control.

Figure 2

Comparative analysis of the aminoacid sequences of the NTAN1 isoforms of various insect and non-insect species. (A) Neighbor Joining tree of insects and other mammalian species NTAN1 orthologs. Dm (Drosophila melanogaster), Cc (Ceratitis capitata), Bd (Bactrocera dorsalis) Ag (Anopheles gambiae), Aa (Aedes aegypti), Cq (Culex quinquefasciatus), Bm (Bombyx mori), Am (Apis mellifera), Nv (Nasonia vitripennis), Mm (Mus musculus) and Hm (Homo sapiens). The tree was built with MEGA11. Bootstrap support (1000 replicates) is indicated. The scale bar units show aminoacid substitutions per site. (B) Comparison of sequence similarity and identity between the DmNTAN1-1 isoform and the other isoforms of the different species studied with the LALIGN/PLALIGN software.

Figure 3

Analysis of GFP expression in the Mz317-Gal4 line. (A) Immunohistochemistry on antennal cryosections (14 µm) with anti-GFP antibody (green) on the following genotypes: Mz317-Gal4;UAS-mCD8::GFP, Mz317-Gal4/repo-Gal80;UAS-mCD8::GFP, Mz317-Gal4/elav-Gal80;UAS-mCD8::GFP and UAS-mCD8::GFP. Scale bar represents 40 µm. (B) Immunohistochemistry on brain dissections with anti-GFP (green) and anti-nc82 (magenta) antibodies on Mz317-Gal4;UAS-mCD8::GFP, Mz317-Gal4/repo-Gal80;UAS-mCD8::GFP, Mz317-Gal4/elav-Gal80;UAS-mCD8::GFP and UAS-mCD8::GFP. Scale bar represents 100 µm.

Figure 4

Analysis of GFP expression in different Ntan1-related Gal4 lines in Drosophila melanogaster. (A) Immunohistochemistry on antennal cryosections (14 µm) with anti-GFP antibody (green) on the following genotypes: Mz317-Gal;UAS-mCD8::GFP, Mz97-Gal4,UAS-Stinger;UAS-mCD8::GFP and Ntan1^NP1111-Gal4;UAS-mCD8::GFP. Scale bar represents 40 µm. (B) Immunohistochemistry on brain dissections with anti-GFP (green) and anti-nc82 (magenta) antibodies on Mz317-Gal;UAS-mCD8::GFP, Mz97-Gal4,UAS-Stinger;UAS-mCD8::GFP and Ntan1^NP1111-Gal4;UAS-mCD8::GFP. Scale bar represents 100 µm.

Figure 5

Gene expression analysis of Ntan1 in Mz97-Gal4 and Mz317-Gal4 fly lines. Expression of the Ntan1 gene (A) and Ntan1 transcripts (B) by RT-PCR in Mz97-Gal4 and Mz317-Gal4 fly lines. M: Molecular marker, G: Genomic DNA of wildtype genotype, Mz317: Mz317-Gal4 cDNA, Mz97: Mz97-Gal4,UAS-Stinger cDNA and C−: Negative control.