Comparative functional and evolutionary analysis of essential germline stem cell genes across the genus Drosophila and two outgroup species

Abstract

In Drosophila melanogaster, bag of marbles (bam) encodes a protein essential for germline stem cell daughter (GSC) differentiation in early gametogenesis. Despite its essential role in D. melanogaster, direct functional evaluation of bam in other closely related Drosophila species reveal this essential function is not necessarily conserved. In D. teissieri, for example, bam is not essential for GSC daughter differentiation. Here, we generated bam null alleles using CRISPR-Cas9 in a species more distantly related to D. melanogaster, D. americana, to interrogate whether bam's essential GSC differentiation function is novel to the melanogaster species group or a function more basal to the Drosophila genus. To further characterize the extent of the functional flexibility of other GSC regulating genes, we generated a gene ortholog dataset for 366 GSC regulating genes essential in D. melanogaster across 15 additional Drosophila and two outgroup species. We find that bam's essential GSC function is conserved between D. melanogaster and D. americana and therefore originated prior to the formation of the melanogaster species group. Additionally, we find that ~8% of the 366 GSC genes essential in D. melanogaster are absent in at least one of the 17 species in our ortholog dataset. These results indicate that developmental systems drift (DSD), in which the specific genes regulating a function may change, but the final phenotype is retained, occurs in stem cell regulation and the production of gametes across Drosophila species.

Keywords: CRISPR; bam; comparative functional analysis; germline stem cells.

Figure 1.

Fertility and cytological analyses of bam function in adult D. americana. Fertility is presented as adult D. americana progeny per fly for each bam genotype and presented separately for females (a) and males (d). The raw data as progeny per fly is plotted on the upper axes with the mean difference for the three genotype comparisons against the shared control wildtype illustrated in the Cumming estimation plots on the lower axes. Mean differences are plotted as bootstrap resampling distributions. Each mean difference is depicted as a dot, and 95% confidence intervals are indicated by the vertical black bars. Immunostaining of ovaries (b and c) and testes (e and f) of wildtype (b and e) and null bam genotypes (c and f). Composite Z-projections for ovaries and testes show staining for the germline (vasa), fusome (1B1), and nuclei (DAPI) with separate single channels for each image illustrated in the side panels (i. vasa, ii. DAPI, iii. 1B1). Wildtype tissue phenotypes are indicated with arrows and mutant tissue phenotypes are indicated with arrowheads.

Figure 2.

Predicted absences of 366 GSC regulating genes essential in D. melanogaster (predicted by Yan et al. 2014) by species plus bgcn and Yb using different ortholog detection strategies. a. Percentage of GSC genes predicted absent by species for three ortholog identification strategies. Light grey bars represent ortholog predictions using Ensembl, dark grey represent predictions with RBBH alone, and black represent RBBH plus syntenic evaluation. The bar below the species indicates divergence time from D. melanogaster (MY). b. Presence and absence of GSC genes across 15 Drosophila and two outgroup species that are predicted absent in at least one species after RBBH and syntenic evaluation. Blue indicates gene presence and black indicates gene absence.

Figure 3.

Percentage of GSC genes absent by functional category. Categories are pulled from the gene-interaction network generated in Yan et al. (2014). The “No subcategory (105)” group includes genes represented in the interaction network map without clear categorical associations and the “No functional category (57)” includes GSC genes that do not appear in the interaction network map.

Figure 4.

Interaction network size and absences in absent GSC genes. Genes with at least one absence in the included species are listed with white bars representing the size of their interaction networks including genetic and physical interactors. The number of interaction network genes absent are represented with black bars.

Figure 5.

GSC gene and associated network absences across species. GSC genes with absences in at least one included species are highlighted in dark grey. Associated interaction network (I.N.) genes are indented, italicized, and highlighted in light grey. Gene presence is indicated by light blue and absence is indicated by black. First, GSC genes with no absences in their interaction networks are arranged by increasing interaction network size. Next, GSC genes with absences in their interaction networks are arranged in the same manner. GSC genes with no interaction networks (six genes) are excluded from this figure.