Functional Analysis of Forkhead Transcription Factor Fd59a in Spermatogenesis of Drosophila melanogaster

1. Introduction

Forkhead box (Fox) transcription factor, which contains a highly conserved DNA binding domain of ~100 amino acids consisting of three α helices, three β folds and two ring connections, plays critical roles in organ development, innate immunity, and other processes [1]. Based on phylogenetic analysis, Fox proteins are assigned to different subclasses and named “Fox, subclass N, member X” [2]. FoxD subfamily members are mainly involved in metabolism and early organ development [3]. In mammals, FoxD1 regulates human early embryonic development and is associated with various diseases. For example, FoxD1 can promote SLC2A1 (Solute carrier family 2 member 1) transcription and inhibit degradation of SLC2A1 to facilitate proliferation, invasion, and metastasis of pancreatic cancer cells [4,5,6]. In planarian, FoxD gene expression was induced by wound signaling, and it was involved in head regeneration [7]. In Drosophila melanogaster, Fd59a/FoxD may regulate development of nervous system and control egg-laying behavior of females [8]. However, the functions of insect FoxD subfamily members are still largely unknown.

Spermatogenesis is a process to produce mature sperm for reproduction. The process of spermatogenesis is conserved from insects to vertebrates; thus, insect testis is an ideal model for studying mechanisms of spermatogenesis [9]. In D. melanogaster, germ stem cells (GSCs) differentiate into goniablasts under the control of stem cell niche, then goniablasts develop into spermatids through mitosis and meiosis. After nuclear elongation and individualization processes, round spermatids finally become mature sperm [10].

Spermatogenesis is regulated by various signaling pathways, such as JAK-STAT (Janus kinase-signal transducer and activator of transcription), BMP (Bone morphogenetic protein), and EGF (Epidermal growth factor) pathways [11], and by many genes [12,13]. Recent studies showed that different genes are involved in spermatogenesis of insects. For example, knockdown expression of ribosomal protein S3 (RpS3) strongly disrupted spermatid elongation and individualization process in D. melanogaster [14]. Knockdown or mutation of cytochrome c1-like (cyt-c1L) gene in early germ cells resulted in male sterility of D. melanogaster [15]. Moreover, BmHen1, a gene in Bombyx mori encoding methyltransferase that modifies piRNAs, was found to regulate eupyrene sperm development [16]. These results indicate that the molecular mechanism of insect spermatogenesis is much more complicated than what we have already known about.

In our previous study, we showed that B. mori FoxA participated in the development of wing disc [17]. Microarray data showed that Fox genes were expressed in B. mori testis and BmFoxD was expressed at a high level [18]. In Drosophila, expression of Fd59a/FoxD was about 2-fold higher in the testis than in the ovary [19], suggesting that Fd59a may play a role in testis development or spermatogenesis. In this study, we showed that mutation and knockdown expression of Fd59a caused swelling in the apical region of testis and decreased male fertility. More importantly, loss of function in Fd59a disrupted the homeostasis of testis stem cell niche and induced apoptosis of sperm bundles, resulting in only a few mature sperm in the seminal vesicle. By analyzing RNA sequencing from the testis of Fd59a^2/2 mutants, we found that Fd59a may regulate expression of genes related to reproductive and metabolic processes. Our findings suggest that Fd59a plays a role in Drosophila spermatogenesis.

2. Materials and Methods

2.1. Fly Lines

Wild-type w¹¹¹⁸ line was maintained in the laboratory [20]. Nos-Gal4 (TB00040) and UAS-GFP dsRNA (BDSC9331) fly lines were obtained from Tsinghua Fly Center in Beijing, China. Fd59a¹/CyO (BDSC56819), Fd59a²/CyO (BDSC56820) and UAS-Fd59a RNAi (BDSC31937) flies were obtained from the Bloomington Drosophila Stock Center in Indiana, USA. Bam-Gal4 fly line was kindly provided by Professor Yufeng Wang at the School of Life Sciences, Central China Normal University, Wuhan, China.

To analyze functions of Fd59a, Fd59a¹/CyO males were crossed to Fd59a¹/CyO females to generate Fd59a^1/1 loss of function flies, while Fd59a²/CyO males were crossed to Fd59a²/CyO females to generate Fd59a^2/2 loss of function flies. To knockdown expression of Fd59a, Nos-Gal4 and Bam-Gal4 flies were crossed to UAS-Fd59a RNAi flies, respectively.

All flies were reared on a fresh cornmeal/yeast/brown sugar diet with p-hydroxybenzoic acid methylester as a mold inhibitor at 25°C with a photoperiod of approximately 12 L : 12 D (light : dark) [21].

2.2. Bioinformatics Analysis

Amino acid homology search was performed by BLAST at the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov). Sequence alignment was done by Cluster W. Phylogenetic tree was constructed by RAxML (Random Axelerated Maximum Likelikhood) with 1000 bootstrap replications [22]. Functional protein domains were analyzed by the SMART (https://smart.embl.de/). Potential Fox binding sites in the promoter sequences of select genes were predicted using the JASPAR program (https://jaspar.genereg.net/).

2.3. RNA Isolation and Quantitative RT-PCR

To analyze the expression pattern of Fd59a at different developmental stages, around 50 adult flies of w¹¹¹⁸ (male : female ratio is ~2 : 1) were collected and mated in cage. Then flies were transferred to a new cage 2 h later. Based on the developmental stages, embryos at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h after egg-laying, and the first, second, third instar larvae, early, mid and late pupae, and 1, 3 and 5-day-old adults were collected. All samples were collected into RNAex Pro reagent (Accurate Biology, Changsha, China) and kept at -80°C.

Total RNA was extracted as described previously [20]. The first-strand cDNA was synthesized from 1 μg of total RNA using HiFiScript gDNA removal cDNA Synthesis Kit (cwbiotech, Taizhou, China). Gene specific primers (Table 1) for the tested genes were designed based on sequences from the Flybase and synthesized by Tsingke Biotechnology (Beijing, China).

Quantitative reverse transcription – polymerase chain reaction (qRT-PCR) was performed using QuantStudio™ 6 Flex (Thermo Fisher Scientific, Waltham, USA) with ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) following the manufacturer’s instructions. The qPCR cycling program was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s. Relative expression level of genes was calibrated against the reference gene Rp49 by using 2^−ΔΔCt calculation method [23].

2.4. Male Fertility Test

For fertility test, 10 1-day-old virgin females and 15 3-day-old males were placed in bottles with egg collection plates as described previously [24]. Egg collection plates were changed every 24 h, and the number of embryos and larvae in the plates were counted.

2.5. Immunofluorescence Staining

Testes from 3-day-old adult flies were dissected in 10 mM phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) and fixed in 4% paraformaldehyde for 30 min at room temperature. Testis samples were washed in 3‰ PBT (10 mM PBS containing 0.1% Triton X-100) for 3 or more times (each for 15 min) and blocked with blocking buffer (3‰ PBT containing 5% normal goat serum) for 1 h at room temperature. Next, all samples were incubated with primary antibody prepared in dilution buffer (3‰ PBT containing 3% normal goat serum) at 4°C overnight, washed three times in 3‰ PBT, and then incubated with secondary antibody at room temperature for 3 h in darkness. Finally, samples were washed three times with 3‰ PBT, mounted using VECTASHIELD® antifade Mounting Medium containing 2 μg/ml DAPI (Vector Laboratories, Newark, USA), and observed under confocal microscopy.

Primary antibodies used were anti-Vasa (1:50, Developmental Studies Hybridoma Bank, Iowa, USA), anti-Fasciclin III (Fas III) (1:100, Developmental Studies Hybridoma Bank, 7G10, Iowa, USA), anti-αSpectrin (1:50, Developmental Studies Hybridoma Bank, 3A9, Iowa, USA). The secondary antibodies were goat anti-rabbit 568 and goat anti-mouse 488, which were Alexa Fluor-conjugated (1:500, Invitrogen, Carlsbad, USA). Fluorescent images were captured using an FV3000 confocal microscope (Olympus, Tokyo, Japan).

2.6. TUNEL Assay

To analyze whether loss of function in Fd59a can induce cell death of sperm bundle, testes from 3-day-old adults of Fd59a^2/2 and Fd59a RNAi flies were dissected. The collected testes were fixed in 4% paraformaldehyde and washed with 3‰ PBT as described above.

Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was performed using the one-step TUNEL apoptosis assay kit from Beyotime Biotechnology (Shanghai, China). Testis samples prepared from above were incubated with 50 μL TUNEL reaction mixture containing 5 μL of TdT enzyme and 45 μL of fluorescent labeling solution at 4°C overnight, and then washed three times in 3‰ PBT. DAPI staining was performed as described above.

2.7. RNA Sequencing

Testes from 3-day-old Fd59a^2/2 and w¹¹¹⁸ flies were dissected in DEPC-treated PBS (10 mM, pH7.4). Total RNA was extracted, and RNA sequencing was performed at Majorbio Bio-Pharm Technology (Shanghai, China) using an Illumina HiSeq 2000 (Illumina, San Diego, CA, USA).

2.8. Bioinformatics Analysis of RNA-seq Data

Transcript abundances in this study were determined using Fragments Per Kilobase per Million mapped reads (FPKM) values. Differentially expressed genes (DEGs) were identified based on a log₂ fold-change > 1.5 (or log2 fold-change < −1.5) and a p-adjust < 0.05 with three biological replicates. Gene Ontology (GO) enrichment analysis of DEGs was performed using the GOseq R package.

2.9. Statistical Analysis

The size of apical region from Fd59a^2/2 and w¹¹¹⁸ testes was quantitated by ImageJ. Three biological replicates and three technical replicates for each biological sample were performed. Statistical significance was analyzed by the student’s t-test (for comparison of two groups) or one-way analysis of variance followed by a least significant difference test (for multiple comparisons among groups). Data were analyzed using GraphPad Prism version 9.0 and presented as mean ± S.E.

3. Results

3.1. Expression Profile of Fd59a in Drosophila

To understand functions of Fd59a, we first determined the expression profile of Fd59a at different developmental stages of Drosophila by qRT-PCR. The results showed that expression of Fd59a mRNA peaked twice during development from embryo to adult, with the first peak around mid-embryonic stage and then a plateau with a relatively high level from late-embryonic stage to 1^st instar larval stage, and the second peak just at the metamorphosis period and maintaining at a relative high level till mid pupal stage (Figure 1A), indicating that Fd59a may be involved in Drosophila development.

It has been reported that loss of function in Fd59a affected egg-laying of Drosophila females [8]. Interestingly, microarray data from the Flybase showed that expression level of Fd59a was about 2-fold higher in the testis than in the ovary [19]. We performed qRT-PCR and confirmed that mRNA level of Fd59a was significantly higher in the testis than in the ovary of 3-fay-old w¹¹¹⁸ flies (Figure 1B), suggesting that Fd59a may also have a function in the testis of Drosophila.

3.2. Sequence and Phylogenetic Analyses of Fd59a

Fd59a gene is in the 2^nd chromosome. The full-length cDNA of Fd59a is 1371 bp long, encoding a protein of 456 amino acid residues, with a calculated molecular weight of 49.1 kDa and pI of 5.19.

Fd59a belongs to the FoxD subfamily. To reveal the evolutionary relationship of Fd59a, Fd59a/FoxD homologous sequences from some insect and vertebrate species were blasted and downloaded from NCBI (Table 2), sequence alignment was performed, and phylogenetic tree was constructed. The results showed that all the select Fd59a/FoxD homologous proteins contain a conserved forkhead box domain (Figure 2A). Meanwhile, Drosophila Fd59a was most close to FoxD3-A of Papilio Xuthus and FoxD3-like of Blattella germanica (Figure 2B) and they contain similar functional domains (Figure 2C). These results suggest that Drosophila Fd59a is evolutionarily close to insect FoxD members.

3.3. Loss of Function in Fd59a Affects Testis Development and Male Fertility

To determine the role of Fd59a in testis development in D. melanogaster, testes from Fd59a^1/1 and Fd59a^2/2 loss of function mutant flies as well as w¹¹¹⁸ flies were dissected. Fd59a¹ and Fd59a² were two mutant types of Fd59a, with Fd59a¹ as a hypomorphic mutation and Fd59a² as a null allele [8]. We found that the apical region of testes from the Fd59a^1/1 and Fd59a^2/2 mutant flies was swelled (Figure 3A,B), with only a few mature sperm in the seminal vesicle of Fd59a^2/2 mutant testis compared to full of mature sperm in the w¹¹¹⁸ flies (Figure 3A). As the phenotype of Fa59a^2/2 flies was more significant than that of Fa59a^1/1 flies, we carried out our following studies in the Fa59a^2/2 mutant flies. When Fd59a^2/2 males were crossed with w¹¹¹⁸ females, the hatching rate of F1 flies was significantly decreased compared to the control (Figure 3C). Together, these results suggest that Fd59a plays a critical role in the development of testis and/or spermatogenesis of D. melanogaster.

3.4. Loss of Function in Fd59a Affects Spermatogenesis

In the apical tip of testis, hub cells are surrounded with GSCs and CySCs to form the stem cell niche, and proliferation and differentiation of testis stem cells are controlled by the niche. Disruption of niche homeostasis can lead to swelling of testis and defect in spermatogenesis [25]. To clarify the role of Fd59a in spermatogenesis, Fas Ⅲ and Vasa antibodies were used to label the hub cells and germ cells, respectively, and αSpectrin antibody was used to label spectrosomes and fusomes for early germ cell development. The results showed that distribution of GSCs and CySCs was scattered in the Fd59a^2/2 testis compared to the control testis (Figure 4A, a1-a4, b1-b4, a6, b6), and a strong pattern of fusome and spectrosome formation was displayed in the control testis, while only a few fusomes and spectrosomes were observed in the Fd59a^2/2 mutant testis (Figure 4A, a3, a5, b3, b5). These results suggest that Fd59a may play a role in maintaining the homeostasis of testis stem cell niche.

Mammalian FoxD1 is related to apoptosis, as knockdown expression of FoxD1 facilitated apoptosis in HNSCC (head and neck squamous cell carcinoma) cells [26]. To determine whether loss of function in Fd59a can induce apoptosis in the testis, TUNEL assay was performed. TUNEL positive signals were detected in sperm bundles of the Fd59a^2/2 mutant flies but not in the w¹¹¹⁸ flies (Figure 5A, a1-a4, b1-b4), suggesting that loss of function in Fd59a induced apoptosis of spermatid.

To further confirm the role of Fd59a in testis, expression of Fd59a in GSCs was knocked down by Nos-Gal4 (Figure 4B), and similar phenotypes, such as swelling in the apical region of testis, fewer mature sperm in the seminal vesicle and apoptosis of sperm bundles, were observed in the Nos-Gal4>Fd59a RNAi flies (Figure 4A, c1-c6, d1-d6, and Figure 5A, c1-c4, d1-d4). Meanwhile, knockdown expression of Fd59a in the 4–16 stage of spermatogonia by Bam-Gal4 (Figure 5C) also induced apoptosis of sperm bundles (Figure 5A, e1-e4, f1-f4). Moreover, only a few mature sperms were observed in the seminal vesicles of the Nos-Gal4>Fd59a RNAi and Bam-Gal4>Fd59a RNAi flies, while the control flies were filled with mature sperm (Figure 5B). These combined results suggest that loss of function of Fd59a in the testis resulted in disruption of the stem cell niche during spermatogenesis and increase in apoptosis of sperm bundles, finally leading to fewer mature sperm in the seminal vesicle.

3.5. Fd59a Regulates Gene Expression in the Testis

To better understand how Fd59a plays a role in regulating testis development and spermatogenesis, we carried out RNA-sequencing (RNA-seq) with RNAs isolated from the testes of w¹¹¹⁸ (control) and Fd59a^2/2 males. Totally 1863 differential expressed genes (DEGs) with at least 1.5-fold change (p-adjust < 0.05) were identified by RNA-seq, with 854 genes upregulated and 1009 genes downregulated in the testis of Fd59a^2/2 flies (Figure 6A). This result indicated that Fd59a serves as a transcription factor in the testis.

Gene Ontology (GO) analysis showed that 120 differentially expressed genes (DEGs) are involved in the reproductive process and 475 DEGs participate in the metabolic process (Figure 6B). Among the reproduction related DEGs, many genes have been shown to play a role in gonad development and spermatogenesis, such as Fz2 and Zpg [27,28]. There are also 57 DEGs which are involved in cell death, including Rnrs and Ptp52F [29,30].

To confirm RNA-seq data, totally 20 DEGs associated with reproductive process and cell death were selected for qRT-PCR analysis (Table 3). The results showed that expression profiles of all the select DEGs in the testis detected by qRT-PCR were consistent with those of the RNA-seq data (Figure 6C). Then, 2000 bp promoter sequences upstream the transcriptional start sites of these select genes were downloaded, and the potential Fox binding sites were predicted using JASPAR database (the relative profile score threshold was set to 85%). Except for CG32817 promoter, several Fox binding sites were predicted in the promoter of each select gene, with more than 10 potential Fox binding sites in the promoters of Spd-2, Cal1, Blanks, Ptp52F, Lola and Debcl genes (Table 3). This result further supported that Fd59a is an upstream regulator of these genes. Taken together, our results suggest that Fd59a serves as a transcription factor to regulate expression of genes involved in reproduction, cell growth and death in the testis.

4. Discussion

Drosophila testis is an ideal system to study spermatogenesis. In this study, we found that FoxD transcription factor Fd59a contributes to spermatogenesis of Drosophila. So far, little is known about the functions of Drosophila Fd59a/FoxD and other insect FoxD members. It was reported that Fd59a was expressed in octopaminergic neurons and regulated egg-laying behavior of females [8]. Drosophila CHES-1-like/FoxN inhibited germline stem cell differentiation by upregulating Dpp expression and ectopic expression of CHES-1-like reduced male fertility significantly [31]. In B. mori, Fox family genes were expressed in the testis, with BmFoxL2-2 and BmFoxD at a higher level than other BmFox genes [18]. However, the function of BmFoxD in the testis is still unknown.

In this study, we showed that spermatogenesis was disrupted, and apoptosis of sperm bundles was induced in Fd59a mutant and RNAi flies. Spermatogenesis is a complex process regulated by multiple signaling pathways and many different genes. Over-activation of the JAK-STAT signaling pathway led to overgrowth of testis and disrupted structure of testis stem cell niche in Drosophila [25,32]. Over-activation of the JNK or loss of the Notch signaling caused cell death in the testis of Drosophila [33,34]. In mammals, deletion of Stat3 in Foxd1 cell lineage protected mice from kidney fibrosis [35]. Hypoxia-inducible factors (HIFs) regulated genes related to oxygen homeostasis, and lack of Hif-p4h-2 (HIF prolyl-4-hydroxylases) in FoxD1 lineage led to dysregulation of genes involved in the Notch signaling pathway [36]. These results suggest that there is a genetic interaction between mammalian FoxD subfamily members and the JAK-STAT and Notch pathways. However, mRNA levels of genes related to the above three signaling pathways did not change significantly in the Fd59a^2/2 testis (RNA-seq data), indicating that Fd59a is not involved in the JAK-STAT, JNK or Notch signaling pathway. Therefore, insect FoxD members may function differently from mammalian FoxD subfamily members.

Results from RNA-seq and qRT-PCR showed that many genes related to reproduction and cell death were differentially expressed in the testis of Fd59a^2/2 flies. Among the reproduction related DEGs, Fz2 and Zpg have been reported to regulate germ stem cell development in Drosophila testis [27,28], while Blanks functioned in sperm individualization [37]. Among cell death related DEGs, Ptp52F enhanced autophagy and apoptosis in Drosophila midgut [30]. In addition, serval potential Fox binding sites were predicted in the promoters of select DEGs. Thus, Fd59a acts as a transcription factor to regulate expression of genes involved in spermatogenesis and in maintaining survival of sperm cells.

It was reported that octopamine was essential for increasing GSCs in mating Drosophila females [38], and β-adrenergic-like octopamine receptor (OctβR) was strongly expressed in adult testis [39]. In Fd59a^2/2 adult testis, Octβ2R expression was down-regulated, thus, it is possible that Fd59a regulates spermatogenesis partly through regulating expression of Octβ2R, and Fd59a may be a key factor linking the nervous system to male reproduction system.