Sex Determination gene expression in Drosophila larval gonads at single cell resolution

A. Model of initial somatic sex determination signals. The karyotype in female (XX) versus male (X, also has a Y, where Y is not a part of sex determination signal but involved in fertility). In response to XX karyotype, Tra protein is produced and binds to dsx mRNA along with Tra2 protein leading to the production of DsxF isoform. In response to single X in the absence of Tra protein, leads to the production of DsxM isoform.

B. Cartoons of third instar larval ovary and testis.

Ovary representing sheath cells, terminal filaments, cap cells, germline stem cells, germline cells with intermingled cells and swarm cells. Testis representing hub, germline stem cells, spermatogonia with somatic cyst cells, primary spermatocytes, terminal epithelial cells, and pigment cells.

C., D., and E. are the gene expression (Average Transcript per Million (TPM) from scRNASeq) of tra, tra-2 and dsx, in different cell types of larval ovary and testis.

F. and G. Immunostained gonads where dsx-Gal4 is driving uas-GFP expression in ovary and testis. White arrows representing Dsx expressing cells. The TF region in the ovary is the Terminal Filament region represented by the dotted line).

Gal4-dsx > uas-GFP: Green (fatbody, sheath cells, intermingled cells, terminal epithelium), VAS: Magenta (germ cells). Scale bars 40 μm

Sex transformed Drosophila larval gonads

A. Visualizing the gene expression (reads) of dsx locus on the Genome browser from the RNA-seq data of larval gonads from wild type ovary (XX), testis sex transformed into ovary (XY; traF), wild type testis (XY) and ovary sex transformed into testis (XX; tra-). The dsxF mRNA is represented by Red and dsxM mRNA is represented by blue genomic tracks to follow female and male specific splicing patterns.

XX; tra- (Df(tra)/tra1) is a progeny from w1118; Df(3L)st-j7, Ki/TM6 females and w1118/Y; tra[1]/TM6 males.

XY; traF (U2af-traF/+; tra5) is a progeny from X/X; tra[F.U2af50]/+; tra[5] females and FM7, act-gfp/Y males.

B, C, D and E are Immuno stained XX, XY; traF, XY and XX; tra- gonads to observe the morphology VAS: green (Germ cells), TJ: Magenta (cyst cells), DAPI: gray (Nuclei). Scale bar 40 μm

F, G, H and I are the Immuno stained XX, XY; traF, XY and XX; tra- gonads to observe Niche regions VAS: Green (Germ cells), Engrailed: Red (terminal filaments), Ncad: Red (hub), Scale bars 20 μm

White arrows in F and G are pointing to similar looking niche regions in XX and XY; traF.

White arrow in H is pointing no-gap between the hub to the nearest germ cells. White arrow in I is pointing the gap between the hub to the nearest germ cells.

J. Volume (V=4/3!abc) of XX, XY; traF, XY and XX; tra- larval gonads.

K. Total number of germ cells in the larval gonads of the genotype represented.

L. The distance between the somatic cells in the niche region to the nearest germ cell.

Two-sample Wilcoxon Rank Sum (Mann-Whitney) test is utilized to find the statistical significance in J - L. In L, triangles indicate numbers less than 1 were raised to 1.

Transcriptomics analysis of wild type and sex-transformed gonads.

A. Heatmap representing significantly differentially expressed genes (p-value<0.05) in XX, XY; traF, XY and XX; tra- larval gonads by two-way ANOVA analysis of bulk RNA-seq data

B. The summary of ANOVA analysis for XX, XY; traF, XY and XX; tra- gonads representing the number of genes belonging to the Karyotype, TRA only or their Interaction categories.

C. Representing encoded Dsx isoforms in different genotypes. XX encodes DSXF and dsxD/dsx1 and XX; tra- encode DSXM. The summary of ANOVA analysis for XX, XX; dsxD/dsx1 and XX; tra- gonads represent the number of genes in dsx only, tra only or ‘dsx by tra’ categories.

XX; dsxD (dsx[1]/dsx(D)) is a progeny from w1118; dsx[1] p[p]/TM6 females and w1118/Y; dsx(D),Sb1,e1.1(3)e1/TM6 males.

D. Splicing analysis comparing XX; dsxD/dsx1 and XX; tra- representing all significantly differential splicing events of TRA (Percent spliced in PSI with log10 Adj.P-value)

E. The gene expression (Average Transcript per Million (TPM)) of nos, nsl-1, wwox Npc1a in different cell types of larval ovary and testis generated utilizing the scRNASeq.

Germ line cell division in the sex-transformed gonads.

A - D., The gene expression (Transcript per Million (TPM) or Average TPM) of dup and bam in bulk RNAseq (from XX, XY; traF, XY and XX; tra- gonads) and in different cell types of wild type larval ovary and testis by scRNASeq

G. - N., Immunostained XX, XY; traF, XY and XX; tra- gonads to identify dot, bar spectrosomes, and branched fusomes in Germ cells.

VAS: green (Germ cells), H3ser10P: Magenta (marks entry into metaphase, mitotically active germ cells,), DAPI: blue (Nuclei), HTS; gray (spectrosome)

Scale bar 20 μm in G and H, 40 μm in I and J, 5 μm in K - N.

O. The ratio of the number of H3ser10P positive cells to the number of total germ cells in a gonad.

P. The ratio of the number of Dot, bar spectrosomes, and branched fusomes to the total number of germ cells in a gonad.

Two-sample Wilcoxon Rank Sum (Mann-Whitney) test is utilized to find the statistical significance in O - P

Sexual identity of germ cells in the sex-transformed gonads.

A. The sxl expression (Transcript per Million (TPM) or Average TPM) in bulk RNAseq and in different cell types of larval ovary and testis by scRNASeq

B. Splicing pattern of sxl (Percent spliced) in XX, XY; traF, XY and XX; tra- larval gonads analyzed from Illumina (short reads) and PacBio (long read) RNA-sequencing data

C. - F., The gene expression (TPM) of Hp6, CG42857, RpL22-like and blanks in bulk RNAseq and in different cell types of larval ovary and testis by scRNASeq

Expression of genes responding to the presence of tra and/or karyotype.

A. – L., The gene expression (TPM) of Ovo, CG1894, bNACtes1 and dec-1 in bulk RNAseq and in different cell types of larval ovary and testis by scRNASeq

M. – T., Immunostained larval ovary and testis to identify different genes.

VAS: blue (Germ cells), TJ: Red (cyst cells), N-cad: red (hub), Spectrin; red(fusome), H2A-V5 tagged Ovo, CG1894, BNACtes1 and dec-1: green Scale bar 20 μm

Primary Spermatocytes in sex transformed gonads.

A.-F. The gene expression (TPM) of early spermatocyte-specifically expressed genes can, fzo and kl-3 in bulk RNAseq and in different cell types of larval ovary and testis by scRNASeq

G. - J. Germ line nuclear structure in XX, XY; traF, XY and XX; tra- larval gonads.

DAPI: grey (DNA), kl-3: green (kl-3 transcript in the nucleus). White circle representing the periphery of the nucleus

K. - N. Centrioles

ASL: grey (Asterless)

O. - R. Immuno stained XXY, XY; traF, XY and XXY;tra- gonads

DAPI: grey (DNA), kl-3: green (kl-3 transcript in the nucleus)

The model explaining the interaction between soma and germ line.

A. Nature of the sex-reversed gonad phenotype

B. Multiple pathways regulate gene expression during gonad development and controlling sex determination and differentiation of the germline in gonads