An exploratory analysis of the effects of human FYN and its Drosophila ortholog in Drosophila tissues

Abstract

Many cancers are initiated by the activation of oncogenes, which cause cancer phenotypes through a variety of disruptive mechanisms. FYN is a human proto-oncogene implicated in many cancers, especially those of the central nervous system and ovary. FYN encodes a non-receptor tyrosine kinase from the Src tyrosine kinase family, and has been shown to promote proliferation and metastasis. Our previous publication demonstrated that introduction of human FYN in the Drosophila eye resulted in a deleterious effect on eye structure, suggesting potential conserved functions across species. This study utilized the Gal4-UAS system to express a wildtype version of human FYN and a constitutively active form of its Drosophila ortholog, Src64B, in the eye and ovary of Drosophila melanogaster, to further explore their conserved functions. Our findings suggested that overexpression of FYN and Src64B in Drosophila eye tissue exhibited conserved developmental defects and a rough eye phenotype. FYN overexpression in the Drosophila ovary demonstrated accumulation of anterior egg chamber squamous cells without associated proliferative activity. These accumulated squamous cells exhibited altered expressions of apical, basolateral, and Par-complex polarity factors. Constitutive expression of Src64B showed similar phenotypes. These results indicate that the conserved oncogenic potential of FYN may be linked to changes in cell polarity signaling, which is considered a hallmark of cancer.

Introduction

Drosophila melanogaster is a well-established model organism often used in genetic studies, as its genome is 60% homologous to the human genome overall, and approximately 75% homologous regarding disease-causing genes^1,2. Drosophila is particularly useful in genetic research due to its ease of manipulation and a wide variety of available resources. The variety of tissue types coupled with short generation time allows a significant amount of data to be gathered with lower cost and time investment².

The Drosophila eye is an organ constructed from hundreds of ommatidia, which are individual symmetrical units arranged in a hexagonal pattern. Uniformly distributed eye bristles are interspersed between the ommatidia. This tissue has been used for over a century to study a variety of developmental mechanisms, as well as disease-causing genes³. Approximately two-thirds of Drosophila genes contribute to eye development, making this tissue particularly sensitive to perturbations in gene expression. The resultant phenotypic changes can include different eye colors, textures, and sizes, and are generally readily observable via light or scanning electron microscope⁴.

Drosophila ovarian tissue is another well-studied and effective model system. The Drosophila ovary is composed of approximately 16 ovarioles, each of which is composed of 6–7 egg chambers developing in succession. Each egg chamber is composed of one developing oocyte and fifteen nurse cells, and the entire egg chamber is enclosed within a unicellular layer of somatic follicle cells. Throughout its fourteen stages of development, the egg chamber increases in size and the follicle cells migrate⁵. During stages 8–10, an anterior group of follicle cells named border cells (BCs) detach from the anterior end of the egg chamber and migrate through the germline nurse cells to the medial point. Meanwhile, another group of anterior follicle cells adjacent to BCs, referred to as squamous cells or stretched cells (SCs), also complete migration across the exterior of the anterior half of the egg chamber.

More interestingly, the Drosophila egg chamber model system contains all three major types of epithelial cells at various stages of development. During early stages of development, all main body follicle cells have a cuboidal shape. Following differentiation at stage 5 of development, posterior follicle cells transition to a columnar shape to line the developing oocyte. Anterior follicle cells differentiate into BCs and SCs, and SCs further flatten from cuboidal shape to squamous shape. These transitions in shape are the result of a complex interplay of developmental factors which have been the subject of our recent research⁶.

One of our recent research projects studied the effects of human genes on the Drosophila eye. We documented a disturbance in the Drosophila eye phenotype when FYN, a known human proto-oncogene⁷, was selectively expressed in the tissue. The selective expression of wildtype FYN resulted in a rough eye texture and disrupted and lost eye bristles⁴. These deleterious effects suggest potential conserved functions across species. Here, we further validate and explore the role of FYN in the Drosophila eye and ovary. Moreover, this study will demonstrate conservation of function in the Drosophila eye and ovary between FYN and its Drosophila ortholog, Src64B.

Results

Human FYN and its fly homology

Human FYN was identified as a gene of interest in the Drosophila eye⁴ by our recent research suggesting potential conserved functions across species. To inform further investigation of homologous gene functions, a Harvard Medical School DIOPT Ortholog Finder query^8,9 was performed to identify a potential Drosophila ortholog of FYN. Fly Src64B was identified as a particular gene of interest. Results of the query are summarized in Table 1.

Table 1.

Bioinformatic analysis of potential FYN orthologs in Drosophila. A Harvard DIOPT query was generated to identify potential orthologous genes to FYN in Drosophila. Src64B was selected as the correct ortholog, as it returned the best score.

Gene	DIOPT Score	DIOPT weighted score	Homology rank	Best score?	Best score reversed?
Src64B	8	7.95	High	Yes	Yes
Src42A	7	6.74	Moderate	No	No
Csk	4	3.73	Moderate	No	No
Btk	3	2.83	Low	No	No
FER	3	2.83	Low	No	No
Abl	3	2.83	Low	No	No
Shark	3	2.73	Low	No	No
drk	2	1.9	Low	No	No
hop	2	1.8	Low	No	No

Of the nine potential orthologs identified by the DIOPT query, Src64B was most closely related to FYN, with a score of 8. Src64B also returned the Best Score and Best Score Reversed, which indicated that the two genes were both most closely related to each other in their respective genomes. Src42A, a paralog of Src64B, demonstrated a lesser degree of homology with FYN with a score of 7. Src64B was chosen for further analysis as the correct ortholog of FYN.

FYN and Its Drosophila ortholog display conserved eye phenotypes

The eyes of GMR-Gal4 control flies are well-organized, with hundreds of ommatidia arranged in a hexagonal pattern, with uniformly distributed eye bristles interspersed (Fig. 1A, A’). Using the GMR-Gal4 driver, we induced expression of wildtype FYN and a constitutively active (CA) form of Src64B in the Drosophila eye. Consistent with our previous publication⁴, induction of FYN in the Drosophila eye resulted in a rough eye phenotype (Fig. 1B). Further SEM results identified loss and shortening of bristles, as well as disruptions to bristle patterning. Results also demonstrated disorganization of ommatidia patterning, with infrequent structural degeneration of ommatidia (Fig. 1B’). Constitutive expression of Src64B in the Drosophila eye also resulted in a rough eye phenotype (Fig. 1C), and demonstrated more pronounced disorganization of ommatidia, as well as increased incidence of patterning defects such as shortening and loss of bristles (Fig. 1C’). These findings confirmed conserved phenotypes between FYN and constitutively active Src64B in the eye.

Fig. 1.

Expression of FYN and its Drosophila ortholog in the Drosophila eye. Phenotypic defects were observed following overexpression of FYN and constitutive expression of Src64B in the Drosophila eye. (A-C) Eyes imaged with light microscopy at 5x magnification. Scale bar = 0.1 mm. (A’-C’) Eyes imaged with SEM at 1000x magnification. Scale bar = 50 μm.

Role of FYN and Src64B in the Drosophila egg chamber

While it has been previously well-established as a driver of human cancers in a variety of organs including the breast, prostate, and central nervous system^7,10, FYN has recently also been implicated in studies of human ovarian cancer^11,12. As such, the Drosophila egg chamber system provides a particularly interesting model system to further analyze FYN and potential homologous functions. In this study, FYN and its Drosophila ortholog, Src64B, were expressed in the Drosophila anterior follicle SCs using an A90-Gal4 driver.

As shown in the A90-Gal4 control (Fig. 2A, A’), SCs completed cell migration at stage 10, and are evenly distributed and well-separated at the anterior half of egg chamber. However, expression of wildtype FYN in the SCs caused accumulation of SCs at the anterior end of the egg chamber (Fig. 2B, B’). A similar, more pronounced phenotype was observed when Src64B was constitutively expressed in the anterior SCs (Fig. 2C, C’). The consistent phenotypes again suggest a conserved underlying mechanism between FYN and its Drosophila ortholog Src64B.

Fig. 2.

Expression of wildtype FYN and CA Src64B in the anterior egg chamber. Expression of FYN and constitutive Src64B CA in the anterior squamous cells resulted in accumulation of anterior squamous cells and border cell migration deficits. (A-C) RFP in white marks the expression pattern of the A90 driver, and therefore the localized expression of the gene of interest. (A’-C’) DAPI staining in white demonstrates the locations of cell nuclei. The area of normal distribution of anterior SCs is indicated by red dotted lines. The accumulative phenotype of SCs is indicated by white arrows. Scale bar = 20 μm.

Accumulation of SCs is not associated with inappropriate proliferation

The anterior accumulation of SCs was first theorized to be caused by two possible scenarios: either extensive and inappropriate proliferation of follicle cells at the anterior, or delayed SC migration. Phosphohistone H3 (PH3) is expressed in cells in late G2 and mitotic phases, and it is commonly used to detect cells actively undergoing mitosis¹³. In normal Drosophila egg chamber development, follicle cells cease to undergo mitosis during stage 5⁵. Thus, any mitotic activity after stage 5 would be considered abnormal. A90-Gal4 drivers were applied to drive expression of our genes of interest in the Drosophila anterior follicle SCs, and were labeled with RFP (Fig. 3A, B, C). Control SCs demonstrated no PH3 expression (Fig. 3A’, 100%, n = 42), which suggests no cells were actively undergoing mitosis. PH3 activity was similarly absent in stage 10 egg chambers with FYN (100%, n = 59) and Src64B (100%, n = 38) induction (Fig. 3B’,C’). Consistent with prior observations (Fig. 2A’, B’, C’), control SCs demonstrated no accumulative phenotype (Fig. 3A’’), but accumulation of SCs was observed when inducing wildtype FYN and constitutive Src64B, respectively (Fig. 3B’’, C’’). Therefore, we posited that the accumulation of SCs is the result of other factors.

Fig. 3.

Expression of mitotic marker PH3 in anterior egg chamber. Accumulated SCs did not demonstrate inappropriate mitotic activity. (A-C) RFP expression in white marks the expression pattern of the A90 driver, and therefore the expression locations of the gene of interest. (A’-C’) The expression of mitotic marker PH3 is shown in white. (A”-C”) DAPI staining in white demonstrates the locations of cell nuclei. Scale bar = 20 μm. (A’-C’,A’’-C’’) The area of normal distribution of anterior SCs is indicated by red dotted lines. The accumulative phenotype of SCs is indicated by white arrows.

Accumulated SCs retain their SC cell fate

The accumulated SCs were found to have changes to their cell morphology, as they did not flatten and spread as they would during normal development. We posited that these changes to morphology may have been linked to changes in cell fate, and therefore investigated whether the accumulated cells retained the appropriate SC fate. A90-Gal4 drivers were applied to drive expression of our genes of interest in the Drosophila anterior follicle SCs, and were labeled with RFP (Fig. 4A, B, C). Cell fate marker Eyes Absent (Eya)¹⁴ is normally expressed in anterior SCs, as shown in Fig. 4A’ (100%, n = 55). Our results indicate appropriate Eya staining was observed in anterior SCs overexpressing FYN (100%, n = 130) and constitutively expressing Src64B (100%, n = 32) (Fig. 4B’,C’), indicating no change in cell fate. Consistent with prior observations (Fig. 2A’, B’, C’), control SCs demonstrated no accumulative phenotype (Fig. 4A’’), but accumulation of SCs was observed when inducing wildtype FYN and constitutive Src64B, respectively (Fig. 4B’’, C’’).

Fig. 4.

Expression of squamous cell fate marker in anterior egg chamber. accumulated SCs demonstrated normal expression of SC cell fate marker Eya. (A-C) RFP expression in white marks the expression pattern of the A90 driver, and therefore the expression locations of the gene of interest. (A’-C’) The expression of SC cell fate marker Eya is shown in white. (A”-C”) DAPI staining in white demonstrates the locations of cell nuclei. Scale bar = 20 μm.

Accumulation of SCs may not be caused by improper ecdysone, JAK/STAT and Notch signaling

We next examined the expression patterns of downstream targets of a variety of essential signaling pathways in Drosophila egg chamber development: Notch, Ecdysone, and JAK/STAT. These signaling pathways have been previously well-characterized for their important roles in normal egg chamber development and SC morphological changes^6,15, and therefore may have influenced the observed accumulative phenotype. A90-Gal4 drivers were applied to drive expression of our genes of interest in the Drosophila anterior follicle SCs, and were labeled with RFP (Fig. 5A, B, C, D, E, F). The protein Hindsight (Hnt) is a downstream target of Notch signaling¹⁵, and is normally expressed in the SCs¹⁶ (Fig. 5A’, 100%, n = 36). Broad (Br) was identified by our lab as an important protein in Drosophila egg chamber development, and its expression is controlled by a complex interplay of multiple signaling pathways. It functions as a nuclear hormone receptor of the insect steroid hormone ecdysone, and is expressed in the egg chamber follicle cells beginning in stage 4^17–19. Upstream ecdysone and JAK/STAT signaling suppresses Br expression in development stages 8–10 (Fig. 5D’, 100%, n = 65) to allow for appropriate shape change of anterior SCs from cuboidal to squamous⁵. Br expression can also serve as a secondary evaluation of Notch signaling, as it is a downstream target of Notch in early oogenesis¹⁷.

Fig. 5.

Expression of downstream targets of Notch, Ecdysone, and JAK/STAT signaling in anterior egg chamber. No overt changes were identified to Br or Hnt signaling in the accumulated SCs. (A-C, D-F) RFP expression in white marks the expression pattern of the A90 driver, and therefore the expression locations of the gene of interest. (A’-C’) The expression of the protein Hnt is shown in white. (D’-F’) The expression of the protein Br is shown in white. (A”-C”,D’’-F’’) DAPI staining in white demonstrates the locations of cell nuclei. Scale bar = 20 μm.

No overt changes to the expression of Hnt were observed in the accumulated SCs (Fig. 5B’, 100%, n = 54; Figure C’, 100%, n = 33), nor any significant changes to Br expression (Fig. 5E’, 100%, n = 77; Figure F’, 100%, n = 49). Therefore, major disruptions to Notch, ecdysone, and JAK/STAT signaling pathways were not evident from these markers. Consistent with prior observations (Fig. 2A’, B’, C’), the accumulative phenotype of SCs was identified in cells expressing wildtype FYN and constitutive Src64B (Fig. 5B’’, C’’, E’’, F’’), but not in the controls (Fig. 5A’’, D’’).

Polarity markers are disrupted in accumulated SCs

The accumulated SCs displayed obvious deviations from normal SC morphology. As the determination of cell shape and cellular polarity are often intertwined^20,21, we tested the expression of various cell polarity markers in the accumulated SCs to determine whether changes in cell polarity signaling were responsible for the observed morphological changes. Bazooka (Baz) belongs to the Par-complex and functions as an apical polarity marker²². Discs Large (Dlg) is a member of the Scribble complex and is a basolateral polarity marker²³. Finally, Armadillo (Arm) plays an important role in establishing cell polarity through its essential function in adherens junction assembly^24,25. It is expressed in the apicolateral region of the cell²⁶. The results of this analysis are summarized in Fig. 6. A90-Gal4 drivers were applied to drive expression of our genes of interest in the Drosophila anterior follicle SCs, and were labeled with RFP (Fig. 6A, B, C, D, E, F, G, H, I).

Fig. 6.

Expression of cell polarity markers in the anterior egg chamber. The accumulated SCs demonstrated increased expression of various cell polarity markers, with changes also observed to the spatial expression of each marker. (A-C, D-F, G-I) RFP expression in white marks the expression pattern of the A90 driver, and therefore the expression locations of the gene of interest. (A’-C’) The expression of Par-complex apical polarity marker Baz is shown in white. (D’-F’) The expression of basolateral polarity marker Dlg is shown in white. (G’-I’) The expression of apicolateral polarity marker Arm is shown in white. (A”-C”,D’’-F’’,G’’-I’’) DAPI staining in white demonstrates the locations of cell nuclei. Scale bar = 20 μm.

Normal expression of the Par-complex, including Baz, is limited to the apical surface of cells. In the control SCs, due to their flattened shape, Baz-GFP is weakly expressed in apical surfaces (Fig. 6A’, 100%, n = 126). In this study, overexpression of FYN and constitutive expression of Src64B led to multiple accumulated layers of SCs, which also demonstrated an increased and disrupted expression pattern of Baz-GFP. Additionally, its expression was no longer limited to the apical surface, but extended to all surfaces among the SCs (Fig. 6B’, 100%, n = 167; Figure C’, 91%, n = 136).

Expression of Dlg is normally limited to basolateral surfaces of the cell (Fig. 6D’, 100%, n = 120). However, overexpression of FYN and constitutive expression of Src64B induced Dlg expression indiscriminately over the surface of the multiple accumulated layers of SCs. Additionally, the accumulated SCs demonstrated higher levels of Dlg expression compared to controls (Fig. 6E’, 100%, n = 50; Figure F’, 100%, n = 18).

Armadillo (Arm) participates in adherens junction formation and is apicolaterally expressed in normal follicle cells (Fig. 6G’, 100%, n = 108). Overexpression of FYN and constitutive expression of Src64B led to elevated Arm expression in the multiple layered SCs, and Arm was no longer restricted to the apicolateral surface of the cells (Fig. 6H’, 100%, n = 46; Figure I’, 100%, n = 92). Consistent with prior observations (Fig. 2A’, B’, C’), the accumulative phenotype of SCs was induced by expression of wildtype FYN and constitutive Src64B (Fig. 6B’’, C’’, E’’, F’’, H’’, I’’), but not in the controls (Fig. 6A’’, D’’, G’’).

Discussion

FYN and other Src family tyrosine kinases

Src family tyrosine kinases (SFKs) have been a subject of oncology research for several decades and have been linked to the development and proliferation of many human cancers. They are named after the Rous sarcoma virus, first discovered in the early 20th century²⁷. Normally, tyrosine kinases phosphorylate tyrosine residues on select proteins in response to signals from a number of different upstream molecules, and the proteins they phosphorylate are involved in a variety of cellular mechanisms such as cell growth, adhesion, and motility^28–30. Tyrosine kinases are composed of several conserved domains, including a ligand-binding and ATP-binding domain⁷. Autophosphorylation of their own kinase domain allows enzymatic activity²⁸, while phosphorylation of the C-terminal tail by a C-terminal Src kinase (Csk) results in downregulation^7,31. Oncogenic activity of Src family tyrosine kinases has been linked to mutations of the C-terminal tail that render the protein incapable of negative regulation, resulting in constitutive enzymatic action³².

FYN is a human non-receptor protein tyrosine kinase which acts in the cytoplasm of cells, and is overexpressed in many human cancers, including those of the brain, central nervous system, and ovaries. It is linked to several hallmarks of cancer including proliferation and migration, apoptosis, invasion, loss of adhesion, and deregulation of cell cycle checkpoints⁷. RNAi knockdown of FYN has also been linked to increased susceptibility to chemotherapeutic agents in breast cancer³³, and several Src family kinase inhibitors are currently in clinical trials as anti-cancer agents. FYN activity is regulated by a wide array of molecules including other known oncoproteins such as Ras, and other tyrosine kinases such as epidermal growth factor receptor (EGFR). Downstream targets of FYN include calmodulin, histone proteins⁷, and MAPK signaling³³.

Src64B is a Src family kinase (SFK) present in Drosophila, orthologous to FYN and other mammalian SFKs. It is regulated by tumor suppressor dCsk (Drosophila C-terminal Src kinase), and has been previously shown in literature to promote overproliferation and cell apoptosis at varying levels of expression, and to cooperate with other known oncogenes such as Ras^31,34. The disorganized eye phenotype documented in this study, as well as changes to cell polarity expression in the egg chamber SCs, serve to further develop understanding of the function of FYN and its ortholog Src64B, and establish the Drosophila egg chamber as a model system to study them.

Expression of FYN and Src64B results in a conserved rough eye phenotype

As previously reported in our screen project⁴, expression of human FYN in the Drosophila eye resulted in a rough eye phenotype, comprised of disorganization of ommatidia and loss and shortening of eye bristles. Constitutive expression of ortholog Src64B in the Drosophila eye resulted in a similar, more pronounced rough eye phenotype. Our results are consistent with previous research conducted by Vidal et al. (2007)³⁴, who demonstrated a rough eye phenotype following the overexpression of Src64B in the Drosophila eye. These results imply that the effects of these orthologous genes on the eye phenotype likely result from a conserved underlying mechanism across tissues and species, which is further explored subsequently in the egg chamber model.

Overexpression of FYN and constitutive expression of Src64B are linked to changes in cell polarity signaling

Overexpression of FYN and constitutive expression of Src64B in the anterior SCs of the Drosophila egg chamber resulted in consistent accumulation of multiple layers of SCs. Appropriate cell polarity signaling is essential for cell morphological changes, and allows the cells to attach, communicate, and participate in cell migration. As such, changes in cell polarity signaling can have a significant effect on a tissue’s structure²¹. The overexpression of multiple polarity factors has been found in this study to be colocalized with the overexpression of FYN and constitutive expression of Src64B.

The accumulated and multi-layered SCs demonstrated disruptions to the expression of apical marker Bazooka (Baz), the Drosophila ortholog to Par-3. The Par complex in Drosophila is comprised of Baz, Par-6, and atypical protein kinase C (aPKC)³⁵, and serves as a scaffold in the establishment and maintenance of cell polarization in a variety of contexts³⁶. Baz has specifically been shown to play a significant role in asymmetrical division of the developing Drosophila oocyte^36,37, as well as appropriate localization of cellular development factors in Drosophila neuroblasts³⁸. In normal Drosophila epithelial tissue, Baz is localized to the apical surfaces of the cell, and influences the proper positioning of apical cell connections such as adherens junctions²² and tight junctions³⁵. The disruption of Baz signaling in the accumulated SCs demonstrated in Fig. 6 provides evidence that overexpression of FYN and constitutive expression of Src64B is associated with changes to cell polarity signaling.

Discs large (Dlg) is one of a set of proteins comprising the Scribble module, which assists in the establishment of basolateral polarity through appropriate localization of various cell junctions, including adherens and basal junctions³⁹. Dysfunction of the Scribble module has previously been linked to tissue overgrowth and polarity defects⁴⁰. In this study, Dlg demonstrated moderate overexpression in the accumulated anterior SCs compared to the control. Additionally, in the affected cells, expression of Dlg was no longer localized to the basal and lateral surfaces of the cell, but expressed indiscriminately.

A similar and more pronounced phenotype was documented in the anterior SCs’ expression of Arm, the Drosophila ortholog of β-catenin. Arm plays an essential role in the assembly of apicolateral cell adherens junctions, and is therefore normally apicolaterally localized^24,25. Absence of Arm has been previously linked to breakdowns in cell polarity and cell-to-cell adhesion^25,26, while overexpression of Arm in the Drosophila eye has been associated with a rough eye phenotype and loss of bristles⁴¹. The similarity in eye phenotype documented by Greaves et al. and in this study lends further support to the theory that overexpression of FYN and its ortholog is linked to overexpression of polarity factors such as Arm.

In addition to its role in adherens junction formation, Arm also functions as an effector of the Wingless signaling pathway, which is homologous to the well-established proto-oncogenic Wnt pathway in humans⁴². In its capacity within the Wingless pathway, Arm influences cell fate decisions through downstream factors dTcf/Pangolin⁴³, and this complex has been linked to tumor suppression in Drosophila⁴⁴. Given the Wingless pathway’s established oncogenic potential, further exploration of Wingless participants could provide additional insight into the underlying mechanisms of FYN and its homologs.

These disruptions to the cell polarity factors tested in this study, each with potential deleterious effects to important intercellular junctions, provide a possible rationale for the subsequent observed effects on cellular and tissue morphology.

Effects of FYN and Src64B on downstream signaling are still largely unknown

Our findings suggest that the observed accumulation of anterior SCs in the egg chamber is not associated with inappropriate proliferation, alterations to the cell fate of SCs, or major disruptions to Notch, ecdysone, and JAK/STAT signaling in the ovary, but is associated with changes to cell polarity. Both FYN and Src64B are members of the Src family of non-receptor tyrosine kinases, which are known to act primarily as upstream regulators in various signaling cascades^28–30. It is unlikely that FYN or Src64B directly regulate cell polarity. Rather, they are more likely to influence polarity indirectly by phosphorylating downstream effectors, which in turn control cytoskeletal organization and cellular architecture. Further studies will be necessary to identify and characterize the downstream signaling pathways and target proteins affected by overexpression of FYN and Src64B, in order to fully understand how these kinases contribute to tissue development, cellular behavior, and cell migration. Future research could also explore how differing levels of expression of FYN and its related genes contribute to the observed phenotypes.

Conserved human and fly gene functions in Drosophila melanogaster

Drosophila melanogaster has remained a powerful model organism in human genetics and disease research for many reasons. One primary advantage is the degree of conservation between our genomes, particularly with regards to disease-causing genes. The results of this study further support conservation of function between human FYN and Drosophila Src64B. Our findings also provide further insight into the mechanism of action of FYN and its ortholog, namely its association with changes to cell polarity signaling. The knowledge gained from this fly study may in turn contribute to the understanding of FYN and its function in human disease.

Materials and methods

Harvard medical school DIOPT ortholog finder query

Harvard Medical School DIOPT Ortholog Finder (www.flyrnai.org/diopt) is a resource which enables the user to identify potential animal orthologs to human genes of interest. The DIOPT ortholog finder integrates a variety of published algorithms to output a single DIOPT score for a given gene relationship, with a higher score indicating a higher predicted degree of homology. Once a gene of interest is entered, the DIOPT ortholog finder produces a list of potentially homologous genes organized by DIOPT score, from which researchers can select multiple targets for further investigation^8,9. In this study, a query was generated in DIOPT version 10.0 (last accessed 06 December 2025) with “Homo sapiens” as the input species and “Drosophila melanogaster” as the output species, with gene name “FYN” entered. The resultant table demonstrated that Src64B was the most likely ortholog of FYN, as it returned “Yes” for both Best Score and Best Score Reversed.

Targeting temporal and regional gene expression with The Gal4-UAS system

The Gal4-UAS system was used to selectively express FYN and Src64B in specific Drosophila tissues⁴⁵. Gal4, a transcriptional activator, binds an Upstream Activating Sequence (UAS) linked to a gene of interest, and activates transcription of the downstream gene. In this study, the GMR-Gal4 fly line was used to express target genes selectively in the Drosophila eye tissue, while the A90-Gal4 fly line was used to express target genes in the anterior follicle cells of the Drosophila egg chamber. A90 drives expression selectively in the anterior follicle cells (both SCs and BCs) beginning in stages 6–7, and therefore A90-Gal4 restricts expression of the UAS-linked gene of interest to only the anterior follicle cells of egg chambers in middle and late oogenesis^46,47.

Gal80ts was used in addition to Gal4-UAS to control temporal expression of genes of interest. Gal80 proteins inhibit the activation domain of Gal4, preventing it from binding to UAS and driving transcription of the downstream gene. At temperatures of 29 °C and above, Gal80ts is destabilized and can no longer effectively block the action of Gal4⁴⁸. This is especially valuable as it allows carrier offspring gene expression to be activated within a defined time period. In our study, A90 Gal4-UAS, Gal80ts F1 offspring were raised at 21 ​°C through eclosion, and held at 21​°C until prepared for dissection. Within 5 days of eclosion, offspring were shifted to 30 ​°C for 48 ​hours to allow expression of target genes, then immediately dissected.

Drosophila stocks

The Gal80ts/CyO; A90-Gal4, UAS-mRFP/TM6B stock used in this study was a gift from Dr. Wu-Min Deng’s laboratory at Tulane University. The following stocks were purchased from the Bloomington Drosophila Stock Center at the University of Indiana: GMR-Gal4 (BDSC #1104), UAS-FYN (BDSC #64380), UAS-Src64B CA/CyO (BDSC #82162), and Baz-GFP (BDSC #51572). Fly stocks were cultivated with standard Bloomington fly food at room temperature (21 °C).

Eye phenotype assessment

The GMR-Gal4 line was crossed with UAS-FYN and UAS-Src64B CA/CyO respectively. Offspring were imaged with Leica Ivesta 3 Light Microscope under CO₂ anesthesia, and subsequently imaged with a Tescan Vega 3 Scanning Electron Microscope. Images were processed using Fiji and PowerPoint.

Ovarian phenotype assessment

The Gal80ts/CyO; A90-Gal4, UAS-mRFP/TM6B line was crossed with UAS-FYN and UAS-Src64B CA/CyO lines, respectively. F1 offspring were raised at 21 ​°C through eclosion, and held at 21​°C until prepared for dissection. Within 5 days of eclosion, offspring were shifted to 30 ​°C for 48 ​hours to allow expression of target genes, then immediately dissected. Fresh yeast paste was supplemented during the heat shock period to encourage egg production. Fly dissection, ovary sample preparation, and image acquisition were conducted as previously described in Jia et al. (2015)⁴⁹.

The following primary antibodies were used: rabbit anti-PH3 (06–570) 1:1000 (EMD Millipore Corp.), mouse anti-Eya (eya10H6-s) 1:10, mouse anti-Sn (sn 7 C-s) 1:30, mouse anti-Br-core (25E9.D7-s) 1:30, mouse anti-Hnt (1G9-s) 1:15, mouse anti-Arm (N2 7A1) 1:40, and mouse anti-Dlg (4F3 anti-discs large) 1:50. Unless otherwise specified, antibodies were purchased from the Developmental Studies Hybridoma Bank. Appropriate Alexa Fluor secondary antibodies (1:400; Invitrogen) were used according to the primary antibody host species. Finally, DAPI (1:1000; Invitrogen) staining was applied for nuclear localization. Samples were imaged with a Zeiss LSM 900 Confocal Microscope, and processed using Fiji and PowerPoint.

Author contributions

D.J. designed the experiments; M.M., M.D., A.N., K.Y-S., and D.J. were involved with data collection; D.J., M.M., M.D., and A.N. participated in data analysis; M.M. and D.J. drafted the manuscript; M.M. created the figures; all authors contributed to the paper revision. All authors approved the manuscript for submission.

M.M. and M.D. were supported by the Birla Carbon Scholars Program. M.M., M.D. and A.N. were supported by the National Institute of Health grant (R15GM148962). D. J. is currently supported by Start-Up Funding from Kennesaw State University and the National Institute of Health grant (R15GM148962). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health and other funding agencies.

Data availability

The authors declare that the main data of this study are presented within the article.

Declarations

Competing interests

The authors declare no competing interests.