InteractiveFly: GeneBrief

Cadherin 96Ca: Biological Overview | References

BIOLOGICAL OVERVIEW

Juvenile hormone (JH) is important to maintain insect larval status; however, its cell membrane receptor has not been identified. Using the lepidopteran insect Helicoverpa armigera (cotton bollworm), a serious agricultural pest, as a model, this study determined that receptor tyrosine kinases (RTKs) cadherin 96ca (CAD96CA), a protein with one n-terminal extracellular cadherin domain, and fibroblast growth factor receptor homologue (FGFR1) function as JH cell membrane receptors by their roles in JH-regulated gene expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of JH intracellular receptor MET1 and cofactor Taiman, and high affinity to JH III (see Juvenile Hormone III). Gene knockout of Cad96ca and Fgfr1 by CRISPR/Cas9 in embryo and knockdown in various insect cells, and overexpression of CAD96CA and FGFR1 in mammalian HEK-293T cells all supported CAD96CA and FGFR1 transmitting JH signal as JH cell membrane receptors (Li, 2025).

Juvenile hormone (JH) plays a vital role in insect development and maintaining insect larval status. JH is an acyclic sesquiterpenoid known to enter cells freely via diffusion because of its lipid-soluble character. JH binds its intracellular receptor methoprene-tolerant protein (MET), a basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) family protein. MET forms a transcription complex with the transcription factor Taiman (TAI, also known as FISC, p160/SRC, and is a steroid receptor coactivator) to initiate gene transcription. An important gene in the JH pathway is Kruppel homologue 1 (Kr-h1), which encodes the zinc-finger transcription factor Kr-h1. Kr-h1 acts downstream of MET and is induced rapidly by JH to regulate larval growth and development. Other genes, for example, the early trypsin gene of Aedes aegypti (AaEt), JH-inducible 21 kDa protein (Jhp21), JH esterase (Jhe), vitellogenin (Vg), Drosophila JH-inducible gene 1 (Jhi-1), and JH-inducible gene 26 (Jhi-26) are regulated by JH (Li, 2025).

However, some studies suggest that cell membrane receptors also play essential roles in JH signaling. For example, in A. aegypti, receptor tyrosine kinases (RTKs) are involved in JH-induced rapid increases in inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation of calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate MET and TAI, resulting in Kr-h1 gene transcription in response to JH. JH III, also via RTKs, leads to rapid calcium release and influx in Helicoverpa armigera epidermal cells. JH induces MET phosphorylation to increase MET interacting with TAI, which enhances Kr-h1 transcription in H. armigera. In Drosophila melanogaster, JH through RTK and PKC protein kinase C (PKC) induces phosphorylation of Ultraspiracle (USP). The phenomenon that RTK transmits JH signal has long been predicted; however, the RTKs critical for JH signaling have yet to be identified from numerous RTKs in vivo (Li, 2025).

RTKs constitute a class of cell surface transmembrane proteins that play important roles in mediating extracellular to intracellular signaling. Humans carry approximately 60 RTKs, the Drosophila genome encodes 21 RTK genes, Bombyx mori has 20 RTKs, and the German cockroach genome identifies 16 RTKs. H. armigera has 20 RTK candidates with gene codes in the H. armigera genome. The cotton bollworm is a well-known and worldwide distributed agricultural pest in Lepidoptera, which threatens cotton and many other vegetable crops by rapidly producing resistance to various chemical insecticides and Bt-transgenic cotton. Using H. armigera as a model, this study focuses on identifying the RTKs functioning as the JH receptors and demonstrating the mechanism. The RTKs were screened sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH-regulated gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH-induced rapid increase of intracellular calcium levels, JH-induced phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH-regulated gene expression, maintaining larval status, JH-induced rapid increase of intracellular calcium levels, JH-induced phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously. These data not only improve knowledge of JH signaling and open the door to studying insect development but also present new targets to explore the new growth regulators to control the pest (Li, 2025).

JH regulates insect development through intracellular and membrane signaling; however, the cell membrane receptors and the mechanism are unclear. In this study, CAD96CA and FGFR1 were screened out from the total 20 RTKs in the H. armigera genome and identified as JH III cell membrane receptors, which transmit JH signal for gene expression to prevent pupation and have a high affinity to JH III (Li, 2025).

JH induces a set of gene expression, such as Kr-h1, Vg, Jhi-1, and Jhi-26 , a rapid intracellular calcium increase, phosphorylation of MET and TAI, and prevents pupation. Several RTKs are involved in JH III-induced gene expression and calcium increase; however, only Cad96ca, Nrk, Fgfr1, and Wsck are involved in the JH III-induced pupation delay, in which, only CAD96CA, NRK, and FGFR1 are involved in the JH-induced phosphorylation of MET1 and TAI, and only CAD96CA and FGFR1 can bind JH III. Therefore, CAD96CA and FGFR1 are finally determined as JH III receptors (Li, 2025).

CAD96CA (also known as Stitcher, Ret-like receptor tyrosine kinase) activates upon epidermal wounding in Drosophila and promotes growth and suppresses autophagy in the Drosophila epithelial imaginal wing discs. There is a CAD96CA in the genome of the H. armigera, which is without function study. This study reports that CAD96CA prevents pupation by transmitting JH signal as a JH cell membrane receptor. CAD96CA of other insects has a universal function of transmitting JH signal to trigger Ca2+ mobilization, as demonstrated by the study in Sf9 cell lines of S. frugiperda and S2 cell lines of D. melanogaster (Li, 2025).

FGFRs control cell migration and differentiation in the developing embryo of D. melanogaster. The ligand of FGFR is FGF in D. melanogaster. FGF binds FGFR and triggers cell proliferation, differentiation, migration, and survival. Three FGF ligands and two FGF receptors (FGFRs) are identified in Drosophila. The Drosophila FGF-FGFR interaction is specific. Different ligands have different functions. The activation of FGFRs by specific ligands can affect specific biological processes. The FGFR in the membrane of Sf9 cells can bind to Vip3Aa toxin. One FGF and one FGFR are in the H. armigera genome, which have yet to be studied functionally. The study found that FGFR prevents insect pupation by transmitting JH signal as a JH cell membrane receptor. Exploring the molecular mechanism and output by which multiple ligands transmit signals through the same receptor is exciting and challenging in future work (Li, 2025).

CAD96CA and FGFR1 have similar functions in JH signaling, including transmitting JH signal for Kr-h1 expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of transcription factors MET1 and TAI, and high affinity to JH III. CAD96CA and FGFR1 are essential in the JH signal pathway, and the loss-of-function of each is sufficient to trigger strong effects on pupation, suggesting they can transmit JH signal individually. The difference is that CAD96CA expression has no tissue specificity, and the Fgfr1 gene is highly expressed in the midgut. A possibility is that CAD96CA and FGFR1 play roles by forming homodimer or heterodimer with each other or with other RTKs in tissues, which needs to be addressed in future studies. CAD96CA and FGFR1 transmit JH III signals in three different insect cell lines, suggesting their conserved roles in other insects (Li, 2025).

Homozygous Cad96ca null Drosophila die at late pupal stagee, suggesting that CAD96CA is critical to insect pupation. This study further revealed that Cad96ca mosaic mutation by CRISPR/Cas9 caused precocious pupation in H. armigera, suggesting that CAD96CA plays roles to prevent pupation. Similarly, null mutant of Fgfr1 or Fgfr2 in mouse is embryonic lethal. Htl (Fgfr) homozygous mutant in D. melanogaster die during late embryogenesis, too, suggesting FGFR1 is important to embryogenesis. However, in H. armigera, Fgfr1 mosaic mutation mainly caused precocious pupation, suggesting FGFR1 is necessary to prevent pupation. The double mutation of Cad96ca and Fgfr1 caused earlier pupation and death compared to the single mutation of Cad96ca or Fgfr1. These data suggested that both CAD96CA and FGFR1 can transmit JH signal to prevent pupation independently and cooperatively. MET1 is the intracellular receptor of JH. Knockout of Met1 in the B. mori leads to precocious metamorphosis and death in the penultimate instar. In the Spodoptera exigua, knockout of Met1 also results in precocious metamorphosis and death in the penultimate and final instars. In D. melanogaster, both Met and Gce null mutants die during the larval-pupal transition. In the H. armigera, most of the Met1 mutants died during the transformation from the final instar larva to the pupa, too. These data suggest that JH via MET1 prevents pupation. In the triple mutants of Met1, Cad96ca, and Fgfr1, most larvae died at the 5th and 6th instar larval stages, which is much more serious than the Met1 mutation, or Cad96ca and Fgfr1 double mutation, because of the mutation both intracellular receptor MET1 and two membrane receptors CAD96CA and FGFR1 of JH. These data suggest that JH exerts a complete regulatory role through cell membrane receptors and intracellular receptor, because the cell membrane receptors regulate the phosphorylation of the intracellular receptor MET1 and the interacting transcription factor TAI. The results from different insects suggest that JH via MET1, CAD96CA, and FGFR1 play roles in preventing metamorphosis. In B. mori, after the knockout of JH acid methyltransferase (Jhamt) using TALENs, the larvae died during L1 or L2 larval stages. The knockdown of Jhamt in Drosophila by RNAi does not exhibit a visible effect on development. Knockdown of Jhamt by RNAi in the Tribolium larval stage results in precocious larval-pupal metamorphosis. Homozygous Jhamt -/- larvae in mosquitoes hatch normally and live to the final-instar larvae (L4), and die prior to pupation. The results from different insects suggest that the insect development in the early larval instars is less dependent on JH. The phenotypes from H. armigera CAD96CA and FGFR1 editing are consistent with the general knowledge that the primary role of JH is to maintain larval status and antagonize 20E-induced metamorphosis. The insulin/insulin-like growth factor signaling (IIS) plays a major role in promoting cell proliferation and determining the larval growth rate, and 20E promotes metamorphosis. These hormones cross talk to regulate insect growth and metamorphic development (Li, 2025).

The phenotypes of gene mutation in H. armigera are somehow different from those obtained by homozygous mutation in other animals, due to the mosaic mutation by CRISPR/Cas9. In addition, RNAi of Cad96ca and Fgfr1 was observed precocious pupation as was the case in CRISPR/Cas9, suggesting the RNAi can be used for the study of gene function in insects, especially when the gene editing is embryonic lethal (Li, 2025).

The knockdown of Cad96ca, Nrk, Fgfr1, and Wsck showed phenotypes resistant to JH III induction and the decrease of Kr-h1 and increase of Br-z7 expression, but knockdown of Vegfr and Drl only decreased Kr-h1, without increase of Br-z7. Br-z7 is involved in 20E-induced metamorphosis in H. armigera, whereas, Kr-h1 is a JH early response gene that mediates JH action and represses Br expression. The high expression of Br-z7 is possible due to the down-regulation of Kr-h1 in Cad96ca, Nrk, Fgfr1 and Wsck knockdown larvae. The different expression profiles of Br-z7 in Vegfr and Drl knockdown larvae suggest other roles of Vegfr and Drl in JH signaling, which need further study (Li, 2025).

This study found six RTKs that respond to JH induction by participating in JH-induced gene expression and intracellular calcium increase; however, they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH-induced phosphorylation of MET1 and TAI and binding to JH III. The RTKs transmitting JH signal were screened primarily by examining some of JH-induced gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential (Li, 2025).

In addition, GPCRs also play a role in JH signaling. JH triggers GPCR, RTK, PLC, IP3R, and PKC to phosphorylate Na+/K+-ATPase-subunit, consequently activating Na+/K+-ATPase for the induction of patency in L. migratoria vitellogenin follicular epithelium; JH activates a signaling cascade including GPCR, PLC, extracellular Ca2+, and PKC, which induces vitellogenin receptor (VgR) phosphorylation and promotes vitellogenin (Vg) endocytosis in Locusta migratoria. JH activates a signaling cascade including GPCR, Cdc42, Par6, and aPKC, leading to an enlarged opening of patency for Vg transport. In Tribolium castaneum, the dopamine D2-like receptor-mediated JH signaling promotes the accumulation of vitellogenin and increases the level of cAMP in oocytes. In H. armigera, GPCRs are involved in JH III-induced broad isoform 7 (Br-Z7) phosphorylation. In summary, these published results indicate that RTKs and GPCRs contribute to JH signaling on the cell membrane; however, the GPCR functions as JH receptor needs to be addressed in the future study. The RNAi of RTKs does not affect JH-induced Jhi-1 expression, which implies that other receptors exist, presenting a target for future study of the new JH III receptor (Li, 2025).

RTKs are high-affinity cell surface receptors for many cytokines, polypeptide growth factors, and peptide hormones. Up to now, there is no report that RTK binds lipid hormone. This study determined that CAD96CA and FGFR1 have a high affinity to JH III by MST and ITC methods after they were isolated from the cell membrane (Li, 2025).

The [3H]JH III detection method is used to determine Drosophila MET in vitro translation product binding JH III, and Tribolium MET binding JH III. However, the commercial production of [3H]JH III has ceased, whereas the microscale thermophoresis (MST) method is a widely used method to detect protein binding of small molecules. Therefore, MST was used in this study as the alternative method to measure the binding strengths of RTKs with JH III. Using the MST method, this study determined that the saturable specific binding of Helicoverpa MET1 to JH III is Kd of 6.38 nM, which is comparable to that report for Drosophila MET and Tribolium MET determined using [3H]JH III, confirming MST method can be used to detect protein binding JH III. The CAD96CA exhibited saturable specific binding to JH III with a Kd of 11.96 nM, and FGFR1 showed a Kd of 23.61 nM, which is higher than that of MET1 for JH III, suggesting lower binding affinity of RTKs than the intracellular receptor MET1 for JH III. A similar phenomenon is reported in another study, the binding affinities of steroid membrane receptors are orders of magnitude lower than those of nuclear receptors. NRK did not bind JH III. One possible explanation is that NRK has a low affinity to JH III and thus transmits JH signal without binding JH, or NRK requires association with other proteins to play roles. This study provides new evidence for the binding of lipid hormones by RTK and a new method to study the binding of ligands to receptors (Li, 2025).

This study also verified the affinity of CAD96CA and FGFR1 with JH III through the ITC method, determining their respective Kd values as 79.6 and 88.5 nanomolar. ITC is a versatile analytical method for the character of molecular interactions. ITC is applied in the membrane protein family, containing GPCRs, ion channels, and transporters. The ITC method requires relatively high ligand and receptor concentrations for better saturation curves. However, when a protein solution of 1000 nM, protein aggregation occurred, thus a protein solution was used with a concentration of 700 nM. The Kd value detected by ITC is slightly higher than the result of the MST method; the results are sufficient to confirm the high affinity of CAD96CA and FGFR1 binding to JH III (Li, 2025).

Although JH I and JH II are natural hormones for lepidopteran larvae, H. armigera and B. mori also respond to JH III. In B. mori Bm-aff3 cells, the effective concentrations (EC50) of JHs (JH I, JH II, JH III, JHA, or methyl farnesoate) to induce Kr-h1 transcription are 1.6x¹⁰10, 1.2x¹⁰10, 2.6x¹⁰10, 6.0x¹⁰8, and 1.1x¹⁰7 M, respectively. In cultures of wing imaginal discs from B. mori, 1-2 μM JH III promotes cuticle protein 4 gene expression (Deng et al., 2011). The effective concentration of JH III to induce rapid calcium increase in HaEpi cells is ≥1 &mi;M and 500 ng of 6th instar larva. JH III is a commercially available reagent; therefore, JH III was used to carry out the experiments in this study, and the results hypothesize the possibility of CAD96CA and FGFR1 binding other JHs in addition to JH III, which should be addressed in future study (Li, 2025).

MET is determined as JH’s intracellular receptor by its characters binding to JH and regulating Kr-h1 expression. In this study, cell membrane receptors CAD96CA and FGFR1 are also able to bind JH III and transmit JH III signal to regulate a set of JH III-induced gene expression including Kr-h1. Obviously, both intracellular receptor MET and cell membrane receptor CAD96CA and FGFR1 are involved in JH III signaling as receptors. JH III transmits signal by cell membrane receptor and intracellular receptor at different signaling stages, with cell membrane receptor CAD96CA and FGFR1 inducing rapid Ca2+ signaling, which regulates the phosphorylation of MET and TAI to enhance the function of MET for gene transcription, and the intracellular receptor MET regulates gene transcription by diffusion into cells based on its lipid characteristics (Li, 2025).

The study in human cell line HEK293 shows that overexpression of B. mori JH intracellular receptor MET2 and its cofactor SRC together in HEK293 cells may induce JH-dependent luciferase reporter expression through a plasmid that contains the JH specific kJHRE (JH response element containing the E-box core sequence for JH binding), suggesting JH can diffuse into cells to initiate a gene expression when the insect MET2 and SRC and kJHRE exist. This study showed that overexpressing CAD96CA or FGFR1 in HEK-293T cells elicits Ca2+ elevation under JH III induction, suggesting CAD96CA or FGFR1 transmit a rapid signal of JH III in HEK-293T cells, which might trigger further cellular responses of HEK-293T to JH III. These data suggest that both cell membrane receptors CAD96CA and FGFR1 and intracellular receptor MET of JH can respond to JH. These proteins might be used as switches to induce a gene expression or regulate cell fate in heterogeneous cells by JH induction when the side effects are determined (Li, 2025).

CAD96CA and FGFR1 were involved in JH III signaling, including maintaining larval status, JH III-induced rapid intracellular calcium increase, gene expression, and phosphorylation of MET and TAI. CAD96CA and FGFR1 had high affinity to JH III and were possible cell membrane receptors of JH III and other JHs. CAD96CA and FGFR1 had a general role in transmitting the JH III signal for gene expression in various insect cells, suggesting their conserved roles in other insects. JH III transmits the signal by either cell membrane receptor or intracellular receptor at different stages in the signaling, with JH III transmitting the signal by cell membrane receptor CAD96CA and FGFR1 to induce rapid Ca2+ signaling, which regulates the phosphorylation of MET and TAI to enhance the function of MET for gene transcription, and intracellular receptor MET regulates gene transcription by diffusion into cells based on its lipid characteristics. CAD96CA and FGFR1 can transmit JH signal to prevent pupation independently and cooperatively. This study presents a platform to identify the agonist or inhibitor of JH cell membrane receptors to develop an environmental-friendly insect growth regulator (Li, 2025).

Retinal calcium waves coordinate uniform tissue patterning of the Drosophila eye

Optimal neural processing relies on precise tissue patterning across diverse cell types. This study shows that spontaneous calcium waves arise among non-neuronal support cells in the developing Drosophila eye to drive retinal morphogenesis. Waves are initiated by Cad96Ca receptor tyrosine kinase signaling, triggering PLCγ-mediated calcium release from the endoplasmic reticulum. A cell-type-specific 'Innexin-code' coordinates wave propagation through a defined gap junction network among non-neuronal retinal cells, excluding photoreceptors. Wave intensity scales with ommatidial size, triggering stronger Myosin II-driven apical contractions at interommatidial boundaries in larger ommatidia. This size-dependent mechanism compensates for early boundary irregularities, ensuring uniform ommatidial packing critical for precise optical architecture. These findings reveal how synchronized calcium signaling among non-neuronal cells orchestrates tissue patterning in the developing nervous system (Choi, 2025).

Precise tissue patterning is crucial for the optimal functioning of biological systems. Sensory systems exemplify this principle with their arrangement of functional modules that is essential for processing sensory input. To establish the functional organization of sensory modules, the overall architecture of the organ, mainly composed of non-neuronal cells including glial and epithelial cells, must be precisely patterned and coordinated with sensory neurons. However, despite its importance, patterning of non-neuronal components in sensory tissues is less understood than patterning of neuronal circuits (Choi, 2025).

Synchronized calcium activity in neuronal cells during development is crucial for the patterning of neuronal circuits. In the mammalian visual system, retinal waves are essential for the proper wiring of visual pathways, not just within the retina but also in higher brain areas. While this calcium activity is also important for the development of surrounding non-neuronal cells, such as Müller glia or retinal pigment epithelium, its significance in instructing and coordinating architectural organization of the nervous system is still not well understood. Beyond the nervous system, synchronized calcium activity is known to be critical for the morphogenesis of various other tissues. For instance, calcium activity regulates the formation of skeletal muscle by coordinating the differentiation and fusion of myoblasts. Therefore, synchronized calcium activity might also have a significant developmental role in the structural patterning of non-neuronal cells in the nervous system (Choi, 2025).

In contrast to synaptic transmission-mediated synchronized calcium activity between neurons, intercellular calcium activity in non-neuronal cells is primarily mediated by gap junction channels. These channels form multicellular networks across diverse cell types, allowing them to synchronize their activities- a process essential for neural circuit development. Beyond their role in nervous system development, gap junction networks are crucial for various biological processes, including tissue regeneration, cancer metastasis, and aging. However, technical difficulties have hindered a comprehensive understanding of their roles. These challenges include the complexity of live-cell imaging needed to track network activity and the difficulty of specifically manipulating gap junction channels, given their subunit diversity and vital role in cellular physiology (Choi, 2025).

The Drosophila retina offers an excellent system for investigating the role of intercellular calcium activity among non-neuronal cells that could be important for its structural patterning. Its simplicity, combined with its stereotypic organization and genetic accessibility, allow for precise observation and manipulation of cellular processes through live imaging of intact specimens. It has recently been used to identify the role of calcium waves during late retinal development. The retina is composed of around 800 unit-eyes (ommatidia); each ommatidium houses eight photoreceptor neurons (PRs). These units are surrounded by specialized non-neuronal retinal support cells that exhibit features of glial and epithelial cells. They include four cone cells (CC), two primary pigment cells (1PCs), and a cluster of twelve inter-ommatidial cells (IOCs) that are shared among six adjacent ommatidia. These IOCs include six secondary pigment cells (2PCs), three tertiary pigment cells (3PCs), and three bristle cells (BCs) (Choi, 2025).

Both neuronal and non-neuronal cells in the retina are regionally patterned. For example, the dorsal rim area has specialized photoreceptor neurons for polarized light detection while the dorsal third of the eye contains R7 color photoreceptors that co-express two UV-sensitive opsins. The ventral retina features structurally larger ommatidia compared to the dorsal side. This regional patterning is evolutionarily conserved across fly species and is linked to optimized visual behavior of ground features. The optic lobe circuitry also exhibits regional polarity, with distinct synaptic densities and neuronal subtypes between the dorsal and ventral sides. These findings suggest that the fly visual system has robust regional patterning of both neuronal and non-neuronal populations that is crucial for the processing of visual information (Choi, 2025).

This study optimized a versatile imaging platform to monitor calcium dynamics throughout the pupal stages of the intact Drosophila retina. Robust early retinal waves of synchronized calcium activity in retinal support cells just prior to synaptogenesis of photoreceptors to their optic lobe targets. The molecular mechanisms and developmental significance of these waves were investigated. Calcium release from the endoplasmic reticulum via IP3 receptor is initiated by the receptor tyrosine kinase Cad96Ca and phospholipase Cγ. The calcium signal propagates to adjacent cells through gap junctions, with unique Innexin subunit combinations involved in distinct cell types. Retinal waves drive calcium-dependent apical contraction of inter-ommatidial boundaries through IOC morphogenesis via the Myosin II pathway. Wave activity is more frequent in the ventral region of the eye, where larger ommatidia generate stronger calcium signals that promote more robust contractions. This mechanism compensates for initial boundary irregularities, ensuring uniform inter-ommatidial boundaries across the retina, which is critical for establishing consistent lens architecture. Therefore, this study reveals the molecular and cellular mechanisms by which synchronized calcium activity in retinal support cells coordinate the overall architecture of the eye (Choi, 2025).

This study describes the intricate dynamics of calcium activity due to retinal waves during early Drosophila retinal development and showed how it shapes retinal architecture by controlling the maturation of interommatidial cells. This provides insight into how directed synchronized calcium activity orchestrates structural patterning in tissues (Choi, 2025).

While synchronized calcium activity is crucial for neuronal connectivity, its role in nervous system tissue organization remains unclear. Non-neuronal support cells such as radial glial cells in the rodent cortex, show strong synchronized calcium activity in early development. However, previous studies primarily focused on their roles in cellular proliferation and differentiation, often treating them as a permissive phenomenon for neuronal developmen (Choi, 2025).

In insects, ommatidial size plays a crucial role in optimizing visual function. Larger ommatidia have larger lenses that enhance light capture and improve visual sensitivity, whereas smaller ommatidia increase resolution by capturing finer spatial details. This regional variation in ommatidial size is an evolutionarily conserved feature across fly species with ventral ommatidia detecting closer and larger ground features that are darker than sky landscapes. Despite this regional heterogeneity in ommatidial size, this study found that inter-ommatidial boundaries remain uniformly spaced across the Drosophila retina, which is essential for maintaining a smooth and regularly patterned optical surface, ensuring proper lens alignment in the adult eye. Retinal waves mediate compensatory interommatidial cell (IOC) maturation, ultimately normalizing inter-ommatidial spacing across the retina. Therefore, early synchronized calcium activity in non-neuronal support cells actively shapes tissue architecture (Choi, 2025).

In vertebrates, non-neuronal support cells like radial glia or retinal pigment epithelium (RPE) cells exhibit gap-junction-mediated intercellular calcium waves prior to synaptogenesis. The embryonic chick RPE cells resemble the fly's IOCs, supporting PRs and maintaining retinal structure. While RPE function is well-documented with respect to eye diseases, neurodegeneration, and aging in humans, the dynamics and role of RPE calcium waves remain underexplored (Choi, 2025).

As synapses form, synchronized neuronal activity, driven by neurotransmission, emerges throughout the nervous system and is crucial for circuit formation. In flies, gap-junction-mediated calcium waves in retinal support cells occur before synaptogenesis. This is followed later in development by neurotransmission-dependent synchronized activity in PRs and higher-order neurons. This activity, known as patterned stimulus-independent neuronal activity (PSINA), is essential for synaptic connectivity. These parallels in sequential occurrences of synchronized activity from non-neuronal cells to neurons suggest that Drosophila could be a valuable model for studying vertebrate calcium dynamics during development (Choi, 2025).

The early retinal waves that were observed occur at a very precise stage, suggesting that they are a developmental process that must be synchronized with other developmental events. This activity is initiated via Cad96Ca-PLCγ-IP3R-mediated calcium release from the ER, suggesting that an extrinsic signal activating the receptor tyrosine kinase Cad96Ca orchestrates the timing of retinal waves coordinated with other developmental events. Although the ligand for Cad96Ca remains unidentified, recent studies suggest that insect juvenile hormone can activate Cad96Ca in cotton bollworm. Juvenile hormone exhibits multiple peaks, including one at the mid-pupal stage, which coincides with the onset of retinal waves. This raises the possibility that juvenile hormone, or a related signal, could control Cad96Ca pathway (Choi, 2025).

The developing fly retina undergoes two distinct calcium wave dynamics: early and late retinal waves. The function of late waves (occurring from ~73hAPF) has been explored based on the phenotype of IP3R mutants that show defective basal floor meshwork with stress fibers failing to contract. These defective contractions persist into adulthood, resulting in aberrant retinal morphology and an impaired extracellular matrix. In this study, PLCγ or inx mutants led to nearly identical basal floor meshwork defects at 72h APF, i.e. before the onset of late wave. Since IP3R is necessary for both early and late waves, the defects seen in IP3R mutants cannot be attributed solely to late waves. Although further studies are necessary, the current findings suggest that early waves, along with late retinal waves, contribute to basal floor meshwork development (Choi, 2025).

These findings indicate that retinal waves leading to calcium signaling are crucial in organizing retinal architecture by activating the non-muscle myosin II pathway in IOC. This represents a critical mechanism through which spatial signaling shapes cell morphology. As ectopic calcium signals via TrpA channels restored IOC contraction, this suggests that calcium has an instructive role in regulating cellular morphogenesis. It should be noted that retinal waves generate periodic signals whereas TrpA-induced activity is more sustained: Future studies using optogenetics and high-resolution calcium imaging will be essential to determine the role of calcium signals in tissue organization and morphogenesis (Choi, 2025).

This study has several limitations affecting the interpretation of retinal wave patterning. First, the lack of cell-type-specific manipulation limits comprehensive mapping of wave propagation pathways and gap-junction networks. Second, the underlying mechanisms generating regionally biased and size-matched wave activity remain unexplored. Third, the roles of photoreceptors or wrapping glia, which contact the retina but do not exhibit calcium activity during waves, require further investigation. Finally, downstream signaling beyond Myosin II remains insufficiently characterized. Addressing these limitations through advanced genetic tools and strategies would provide a more comprehensive understanding of retinal wave functions and their broader relevance beyond Drosophila (Choi, 2025).

Effect of receptor tyrosine kinase family member CAD96CA on hormone signalling and ontogeny of silkworm

Moulting and metamorphosis are fundamental physiological processes in the growth and development of holometabolous insects, primarily regulated by ecdysteroids and juvenile hormone (JH), which are synthesized and secreted by the prothoracic gland and corpora allata, respectively. However, the signalling regulatory network involved in the synthesis of these hormones and their effects is complex and interactive and involves many unidentified functional genes. This study conducted a basic bioinformatics analysis of the CAD96CA gene and obtained CAD96CA mutants at the individual level in domestic silkworms using the CRISPR-Cas9 tecshnology. The growth, development and silk protein synthesis phenotypes of the mutants were analyzed and the synthesis and signalling effects of 20E and JH were detected. The results revealed that knocking out the CAD96CA gene resulted in inhibited larval growth, reduced silk protein production, hindered larval-pupal transition and led to larval mortality. The synthesis of 20E and its signalling pathways, as well as the signalling pathways of JH, were all affected to varying degrees following CAD96CA knockout. This study elucidates the role of CAD96CA in the growth and development of silkworms and provides a reference for studying metamorphosis mechanisms (Chen, 2025).

Two-tiered control of epithelial growth and autophagy by the insulin receptor and the ret-like receptor, stitcher

Body size in Drosophila larvae, like in other animals, is controlled by nutrition. Nutrient restriction leads to catabolic responses in the majority of tissues, but the Drosophila mitotic imaginal discs continue growing. The nature of these differential control mechanisms that spare distinct tissues from starvation are poorly understood. This study revealed that the Ret-like receptor tyrosine kinase (RTK), Stitcher (Stit), is required for cell growth and proliferation through the PI3K-I/TORC1 pathway in the Drosophila wing disc. Both Stit and insulin receptor (InR) signaling activate PI3K-I and drive cellular proliferation and tissue growth. However, whereas optimal growth requires signaling from both InR and Stit, catabolic changes manifested by autophagy only occur when both signaling pathways are compromised. The combined activities of Stit and InR in ectodermal epithelial tissues provide an RTK-mediated, two-tiered reaction threshold to varying nutritional conditions that promote epithelial organ growth even at low levels of InR signaling (O'Farrell, 2013).

Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila

Metazoans have evolved efficient mechanisms for epidermal repair and survival upon injury. Several cellular responses and key signaling molecules involved in wound healing have been identified in Drosophila but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair is poorly understood. The Ret-like, receptor tyrosine kinase Stitcher (Stit; Cadherin 96Ca in FlyBase) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce extracellular barrier restoration. This study describes the immediate down-stream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src-family tyrosine kinases (see Src oncogene at 42A) bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src-family kinases control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier repair genes. This analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode of Src-kinases and their central functions during epidermal wound healing in vivo (Tsarouhas, 2014).

This analysis highlights the crucial role of a single phospho-tyrosine (pY) in the control of several aspects of wound healing by the Stit RTK. Four effectors bind to pY762 to activate multiple parallel intracellular pathways, each controlling distinct aspects of wound healing. Similarly, the SH2 domains of both Grb2 and Src42A bind to the same pY of ErbB (member of the epidermal growth factor receptor family), suggesting that this method of activating parallel pathways by docking several effectors to the same site is common to several RTKs (Tsarouhas, 2014).

Drk is an adaptor protein with one SH2 and two SH3 domains. In flies, it was first identified and characterized in the context of signaling from the Sevenless receptor tyrosine kinase, where it links the phosphorylated receptor at the plasma membrane (by its SH2 domain) to the Ras guanine-exchange-factor Sos (by its SH3 domain). Ras activation then leads to the activation of a phosphorylation cascade of MAPK kinase (Raf) and Rolled, the Drosophila homolog of ERK and MAPK. Based on the current analysis and on previously published work, it is proposed that the predominant role of Drk in wound healing is to mediate ERK activation and the transcriptional induction of wound-response genes. In the context of wounding, Stit mutants show reduced dpERK activation, and overexpression of Stit induces ectopic ERK accumulation. Overexpression of Stit induces dpERK accumulation but embryos expressing StitY762F, which does not bind to Drk (or to the other effectors), showed strongly diminished dpERK accumulation. Because the overexpression of the constitutively active version of Src42A can induce the transcriptional activation of wound-response genes without inducing detectable accumulation of dpERK, it is suggested that Drk is the major linker between Stit and ERK. ERK phosphorylates Grh in vitro, and this phosphorylation is important for the Grh-dependent activation of wound reporters. This suggests that the binding of Drk to phosphorylated Stit induces ERK-mediated Grh phosphorylation and Ddc activation. Drk might activate an additional pathway downstream of Stit. The SH3 domains of Drk could recruit other downstream effectors, such as Disabled or Cbl, to fulfill the functions of Drk upon Stit activation (Tsarouhas, 2014).

In addition to the anticipated roles of dpERK in gene activation following wounding, this study revealed a key function of SFKs in the repair of epidermal injuries. The activation of Src family kinases (SFK) has been investigated in several contexts. A standard model involves the de-phosphorylation of Y527 of chicken c-src, which unlocks the autoinhibitory interaction and allows trans-auto-phosphorylation. More recently, cysteine oxidation by reactive oxygen species has emerged as a mechanism for Src activation in epithelial cells and leukocytes during wound healing in zebrafish. The current results reveal an additional RTK-dependent mechanism for the immediate SFK activation at the wound site. Phosphorylation of Y762 of Stit might provide a high-affinity binding site for the SH2 domain of Src kinases, which would therefore compete with the autoinhibitory interaction of the SH2 domain with Y511 in Src42A. This would lead to full activation either by autophosphorylation or by trans-phosphorylation by Stit. This is the mechanism by which Src is recruited to and activated on the docking site of the PDGF receptor in porcine aortic endothelial cells (Tsarouhas, 2014).

Following wounding or Stit activation, SFKs control both the local induction of wound-response genes by the conserved transcription factor Grh, and the assembly of a distinctive actin cable around the wound edge. Interestingly, Src42A is the predominant player in transcriptional activation. During late embryonic development, Src42A restricts Ddc expression in epidermal cells. However, following injury, it is required for the local induction of Ddc. Additionally, Src42ACA (a constitutively active (CA) form of the Src42A protein in Drosophila) can ectopically activate wound-response genes without causing any detectable accumulation of dpERK. This suggests a novel dpERK-independent mechanism of Grh activation during wound healing. These results contrast with the observations that injury of src42A mutant embryos results in widespread activation of wound-response genes along the entire epidermis. There are several explanations for this discrepancy. First, a previous study recorded the GFP intensity of Ddc.47, a minimal wound enhancer reporter presumably lacking the cis-elements that drive the developmental expression of Ddc in the epidermis and other tissues. Second, the widespread activation of Ddc was recorded several hours after the interval of the assays. Thus, this analysis reveals the immediate role of Src42A in the activation of wound-response genes at the injury site, whereas the results of a previous study might suggest a later, direct or indirect, role of Src42A in restricting the spread of the response. The different mechanisms of Src activation by phosphorylation or cysteine oxidation might provide a clue as to how Src42A can act both as a repressor of epidermal Ddc expression during development and as an activator of Ddc and other genes upon injury. Basal low levels of Src42A phosphorylation in the epidermis might favor Ddc repression, whereas increased levels of p-Src by Stit or other RTKs following injury might lead to the Grh-dependent activation of wound-response genes (Tsarouhas, 2014).

This study found that re-epithelialization after injury is controlled by all three non-RTKs, because double mutants show more pronounced defects in wound closure than single src42AE1 mutants. The assembly of the actin ring requires the coordination of the cytoskeleton across the membranes of the epithelial cells surrounding the wound edge. Because Src42A has been implicated in the control of E-cadherin trafficking, it is tempting to speculate that its role in re-epithelialization is to control the adhesion of the leading cells, thereby controlling wound constriction. By contrast, Btk29A and Src64B control the growth of the actin-rich ovarian ring canals and microfilament contraction during cellularization, suggesting that they might preferentially control actin-filament assembly and contraction at the wound edge. Besides Stit and its effectors, the Rho GTPase and profilin and Karst (a Drosophila β-heavy spectrin homolog) have also been shown to participate in the formation of a continuous actin cable, suggesting that they might represent downstream targets of Src and Btk29A during re-epithelialization. This analysis reveals distinct roles for RTK effectors in wound-healing responses, and provides a molecular framework towards understanding and manipulating RTK signaling during wound healing (Tsarouhas, 2014).

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelfial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. This study identified stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses (Wang, 2009).

Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. The function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells has been analyzed. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length and results in large bundles of microvilli. Altered microvilli morphology correlates with defects in the assembly of the vitelline membrane, an extracellular layer secreted by follicle cells that is part of the eggshell. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. Cad99C is thus a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process (D'Alterio, 2005; Schlichting, 2006). a name="Schlichting">

Cadherin Cad99C is regulated by Hedgehog signaling in Drosophila

The subdivision of the Drosophila wing imaginal disc into anterior and posterior compartments requires a transcriptional response to Hedgehog signaling. However, the genes regulated by Hedgehog signal transduction that mediate the segregation of anterior and posterior cells have not been identified. The previously predicted gene Cad99C has been molecularly characterized and shown to be regulated by Hedgehog signaling. Cad99C encodes a transmembrane protein with a molecular weight of approximately 184 kDa that contains 11 cadherin repeats in its extracellular domain and a conserved type I PDZ-binding site at its C-terminus. The levels of cad99C RNA and protein are low throughout the wing imaginal disc. However, in the pouch region, these levels are elevated in a strip of anterior cells along the A/P boundary where the Hedgehog signal is transduced. Ectopic expression of Hedgehog, or the Hedgehog-regulated transcription factor Cubitus interruptus, induces high-level expression of Cad99C. Conversely, blocking Hedgehog signal transduction by either inactivating Smoothened or Cubitus interruptus reduces high-level Cad99C expression. Finally, by analyzing mutant clones of cells, it was shown that Cad99C is not essential for cell segregation at the A/P boundary. It is concluded that cad99C is a novel Hedgehog-regulated gene encoding a member of the cadherin superfamily in Drosophila (Schlichting, 2005).

Cad99C is expressed at low levels throughout the wing, haltere, and leg imaginal discs, whereas elevated levels of Cad99C expression are confined to a strip of cells along the A/P boundary of the wing imaginal disc pouch that is known to respond to the Hh signal. Even though anterior cells along the A/P boundary of haltere and leg imaginal discs as well as cells outside the pouch region of wing imaginal discs also respond to the Hh signal, no elevated level of Cad99C was observed in these cells, indicating that cad99C is a region-specific Hh target gene. The Cad99C protein profile resembles cad99C RNA levels, indicating that the elevated expression of Cad99C is mainly due to transcriptional and not translational or posttranslational regulation. High-level Cad99C expression was reduced to the low level present in cells far away from the A/P boundary in clones of cells lacking Hh signal transduction due to mutations in either smo or ci. Conversely, ectopic expression of either Hh or Ci was sufficient to increase Cad99C expression in the wing imaginal disc pouch, indicating that high-level cad99C expression is controlled by Ci-mediated Hh signaling (Schlichting, 2005).

Different Hh-regulated genes respond differently to Ci[act] and Ci[rep]. For example, the expression of dpp is regulated both by Ci[act] and Ci[rep], whereas hh and ptc only respond to one form of Ci, Ci[rep] or Ci[act], respectively. Like ptc, cad99c appears to respond exclusively to Ci[act]. This is inferred from five observations: (1) ectopic expression in posterior cells of Ci, which under the influence of Hh is converted to Ci[act], induces high-level cad99C expression; (2) misexpression of a constitutively active form of Ci, Ci^PKA4, also induces high levels of cad99C expression; (3) ci null mutant clones in the anterior compartment close to the A/P boundary, where Ci[act] is the predominant form of Ci, fail to upregulate cad99C expression; (4) expression of a constitutive repressor form of Ci, Ci^Cell, does not reduce the low-level expression of cad99C; (5) ci null mutant clones in the anterior compartment away from the A/P boundary, where Ci[rep] is the prevailing form of Ci, show no increase in the expression of cad99C. Taken together, it is concluded that cad99C expression is regulated by Ci[act] and not Ci[rep] (Schlichting, 2005).

The segregation of cells at compartment boundaries is thought to depend on the differential adhesiveness (affinity) of cells on both sides of the compartment boundaries. Based on thermodynamic considerations, it has been proposed that cells will maximize the total strength of their adhesive interactions with neighboring cells by replacing weak cell–cell interactions with stronger ones. Cells with strong adhesive interactions will thus associate preferentially with one another and will segregate from less avidly adhering cells. As predicted by this model, cells expressing different levels of the same adhesion molecule segregate from one another. However, few adhesion molecules have been identified that can promote the differential adhesiveness of cells at compartment boundaries (Schlichting, 2005).

The maintenance of the A/P boundary in the developing Drosophila wing requires Ci-mediated Hh signal transduction in anterior cells. This suggests that Hh signaling may regulate the transcription of one or more genes that in turn affect the adhesiveness of anterior cells. Members of the cadherin superfamily are known to mediate adhesion between cells and several cadherins have been shown to be involved in cell segregation. Even though most cadherins implicated so far in cell segregation contain cytoplasmic β-catenin binding sites, which are absent in Cad99C, several cadherins lacking β-catenin binding sites have also been shown to mediate cell segregation. The discovery of a gene that is both regulated by Hh signaling and encodes for a cadherin, therefore, provides an attractive candidate for mediating the segregation of anterior and posterior cells. However, cad99C expression is not elevated in cells along the A/P boundary of haltere and leg imaginal discs or outside the pouch region of wing imaginal discs, indicating that if the elevated expression of Cad99C were important for cell segregation, this could not be a general mechanism for segregating anterior and posterior cells. However, since wing imaginal disc pouch cells differ in their expression profile from wing imaginal disc cells outside of the pouch, it is not inconceivable that different molecules could operate to segregate cells at the A/P boundary in different regions of the wing imaginal disc or in different imaginal discs (Schlichting, 2005).

A mutant allele of cad99C, termed cad99C^57A, was generated in order to test whether Cad99C is required to segregate anterior and posterior cells. cad99C^57A appears to be a null allele of cad99C based on four criteria: (1) sequencing of the genomic DNA revealed that the predicted promoter region, the transcriptional start site, and the coding sequence for the first 101 amino acid residues were deleted; (2) an RNA probe recognizing the 3′ region of the cad99C transcript, outside of the deletion present in cad99C^57A, does not show detectable staining in wing imaginal discs from homozygous cad99C^57A mutant larvae, indicating that the cad99C transcript levels are highly reduced; (3) an antibody directed to the C-terminus of Cad99C does not recognize a protein of the predicted size for Cad99C in extracts from wing imaginal discs of homozygous cad99C^57A mutant larvae; (4) Cad99C immunoreactivity is highly reduced in homozygous cad99C^57A mutant clones within wing imaginal discs (Schlichting, 2005).

The identification of cad99C as an Hh-regulated gene provides a starting point to investigate a cell biological mechanism used by Hh signaling to control the development of the Drosophila wing. It also provides a further step towards the functional characterization of all remaining members of the cadherin superfamily present in Drosophila that have so far only been predicted based on the genomic sequence (Schlichting, 2005).

As a marker for follicle cell microvilli, Cad99C revealed that the length of these apical protrusions increases from stage 7 to late stage 10a, when they reach their maximum extent of 2-3 µm. At stage 10a, longer microvilli are found in the center and shorter ones in the periphery of the apical follicle cell surface. After stage 10, microvilli regress, but short microvilli remain until the end of oogenesis. Concurrent with the development of the microvillus brush border, follicle cells secrete vitelline membrane (VM) material into the extracellular space between microvilli, producing so-called vitelline bodies, which correspond in height to the microvilli and subsequently fuse into a continuous VM layer above the microvilli. The extracellular space seen between individual microvilli at the light-microscopic level suggests a separation of >400 nm. With a predicted length of 50 nm for the Cad99C extracellular region, a Cad99C trans-dimer would not be able to bridge such a gap. It therefore appears unlikely that Cad99C promotes homophilic adhesion between adjacent microvilli along their entire length, although spotlike sites of adhesion cannot be excluded (D'Alterio. 2005).

To begin to understand how Cad99C might contribute to the formation of a proper vitelline membrane, a determination was made of which cells of the ovary Cad99C RNA and protein are present. RNA in situ hybridization using a Cad99C-specific antisense RNA probe was performed on wild-type ovaries. Little hybridization signal was detected in the germarium and stage-2 and stage-3 egg chambers. During stages 4-8, a hybridization signal is present in the follicle cells located at the anterior and posterior poles of the egg chamber. During stages 9-14, a hybridization signal was detected in follicle cells surrounding the oocyte. No signal above background was detected in nurse cells or the stretched follicle cells surrounding the nurse cells, and only background signal was detected from a control sense Cad99C RNA probe (Schlichting, 2006).

To determine the distribution of Cad99C protein in wild-type ovaries, an anti-Cad99C antiserum was used. Little immunoreactivity was detected in the germarium and stage-2 and stage-3 egg chambers. During stages 4-8, the anti-Cad99C antiserum stained the border between the follicle cells and the oocyte at the anterior and posterior poles of the egg chamber. During stages 9-14, the anti-Cad99C antiserum immunoreacted with structures at the entire border between the oocyte and the follicle cells. Follicle cells covering the nurse cells, or the nurse cells themselves, were not stained. Little immunoreactivity was detected in homozygous mutant Cad99C^57A/57A egg chambers. The detection of Cad99C protein at the border between the oocyte and the follicle cells, along with the detection of Cad99C RNA only in follicle cells, indicates that Cad99C protein is most probably present only in the follicle cells but not in the oocyte. Thus, both Cad99C RNA and protein are present at the proper time and place for Cad99C to play a role in deposition of the vitelline membrane (Schlichting, 2006).

Cad99C protein localizes to the border between the oocyte and surrounding follicle cells. To determine more precisely the subcellular localization of Cad99C, stage-10 egg chambers were stained with a marker for the zonula adherens, DE-cadherin, and Cad99C. Cad99C immunoreactivity was found apical to DE-cadherin, indicating that, unlike DE-cadherin, Cad99C does not localize to the zonula adherens, but rather to the apical plasma membrane. To test whether Cad99C protein localizes to the apical plasma membrane of follicle cells, the follicle-cell-specific Gal4 line CY2 was used in conjunction with UAS-mCD8-GFP to express CD8-GFP, a transmembrane protein routinely used to mark plasma membranes, in follicle cells. CD8-GFP labeled the basolateral plasma membrane as well as the apical plasma membrane. On the apical surface of follicle cells, CD8-GFP labeled protrusions that presumably represent single microvilli, or bundles containing a few microvilli. Cad99C colocalizes with the apical protrusions labeled with CD8-GFP, consistent with the notion that Cad99C localizes to microvilli. To test whether Cad99C localizes to microvilli at the ultrastructural level, immunogold electron microscopy using an anti-Cad99C antiserum was performed on control and Cad99C^57A/57A egg chambers. Very little immunoreactivity was detected on the apical plasma membrane of Cad99C^57A/57A mutant egg chambers. By contrast, immunoreactivity for Cad99C was observed in control Cad99C^57A/+ stage-9 to stage-14 egg chambers on microvilli of follicle cells. At stage 10, 97% of the immunogold particles on the apical plasma membrane were present on microvilli, whereas 3% were detected on the apical plasma membrane outside microvilli, indicating that Cad99C is highly enriched on the microvilli of follicle cells. Cad99C was not detected on the oocyte microvilli. Thus, Cad99C specifically localizes to microvilli of follicle cells (Schlichting, 2006).

Microvilli are fingerlike protrusions on the apical surface of epithelial cells, where they can form a dense brush border. Microvilli are also used by several sensory cells as a basic module to form specialized structures that engage in the transduction of light and mechanosensory stimuli. Stereocilia of vertebrate inner ear hair cells are a prominent example. The core of a microvillus consists of a bundle of cross-linked parallel actin filaments, which have their barbed (+) ends inserted at the microvillus tip and their pointed (–) ends anchored in a terminal web of actin filaments. The actin filament bundle undergoes constant turnover through treadmilling. Growth of microfilaments at barbed ends is thought to push the membrane envelope forward, lengthening the protrusion (D'Alterio. 2005).

Among the molecules that serve a critical function in the formation and regulation of microvillus growth are several F-actin cross-linking proteins, including villin, epsin, fimbrin, and fascin. Epsin can induce microvillus elongation in vitro probably by affecting the actin treadmilling process, and epsin mutant deaf jerker mice have shortened hair cell stereocilia. Additional actin-binding factors that control microvillus size colocalize with the tip complex, which is thought to nucleate the microfilament bundle and to regulate actin polymerization at barbed ends. They comprise myosin XVa and its binding partner whirlin, which promote concentration-dependent elongation of hair cell stereocilia. EPS-8, another actin-binding protein located at a microvillus tip, regulates microvillus length through its barbed-end capping function in the intestine of Caenorhabditis elegans. In the early Drosophila melanogaster embryo, absence of the Abelson kinase causes abnormally long microvilli. This correlates with an ectopic accumulation of F-actin and its growth promoting factor Enabled in the apical cell cortex (D'Alterio, 2005 and references therein).

Observations like these suggested that microfilaments and their binding factors may be sufficient for microvillus formation and elongation and that the plasma membrane may serve only as an anchor for the core bundle. That coupling to the plasma membrane is important is suggested by the finding that ezrin, an ERM (ezrin-radixin-Moesin) protein that likely forms a link between actin filaments and the plasma membrane, induces microvilli in cell culture. Deficiency of ezrin, which appears to organize the terminal web of microvilli, causes shortened, irregular intestinal microvilli in mice. Similarly, the terminal web-associated ERM protein Moesin in Drosophila is required for normal organization of microvilli in rhabdomeres, and an excessive formation of irregular microvilli results from expressing constitutively active Moesin. However, there has been no evidence so far that would implicate specific integral membrane proteins as regulators of microvillus growth (D'Alterio, 2005 and references therein).

Some proteins that have received considerable attention in recent years as important organizers of hair cell stereocilia are protocadherin 15 (PCDH15), cadherin 23 (CDH23), myosin VIIa, harmonin, and SANS. Mutations in these genes are responsible for Usher syndrome type 1 (USH1), a genetic disorder that combines congenital deafness, vestibular dysfunction, and retinitis pigmentosa in humans. The phenotype of mice mutant for any USH1 gene is characterized by splayed and disorganized stereocilia and consequently it was proposed that the two USH1 cadherins may contribute to the links that visibly connect neighboring stereocilia. For CDH23, this model is supported by the observations that it colocalizes with lateral and tip links, is needed for tip link integrity, and can mediate homophilic adhesion. The molecular function of PCDH15, however, has not been elucidated. Cad99C, the fly orthologue of PCDH15, is a component of microvilli; loss- and gain-of-function analyses show that Cad99C promotes microvillus elongation in a concentration-dependent manner. The data also suggest that Cad99C acts through a mechanism that does not involve adhesion between microvilli (D'Alterio, 2005).

Loss of Cad99C results in shorter microvilli and overexpression in longer microvilli than in wild type, indicating that the concentration of Cad99C is positively correlated with the length of microvilli in follicle cells. Interestingly, modifications in the Cad99C mRNA expression level during oogenesis appear to be good indicators for changes in microvilli. During mid-oogenesis, prominent expression of Cad99C is seen in follicle cells that show forming and growing apical microvilli, and Cad99C expression culminates when microvilli reach their maximum extension. The following drop of mRNA levels in most follicle cells coincides with a regression of microvillus size, whereas centripetal cells express Cad99C strongly, consistent with the delayed formation of a microvillus brush border by these follicle cells. It is therefore proposed that transcriptionally regulated changes in the concentration of Cad99C are critically involved in the dynamic remodeling of follicle cell microvilli (D'Alterio, 2005).

The loss of Cad99C, which results in microvilli defects, also leads to defects in eggshell formation, suggesting that normal microvilli have an important function in eggshell development. Interestingly, the dynamic regulation of Cad99C expression correlates well temporally and spatially with described phases of eggshell secretion and with morphogenetic movements of follicle cells; this raises the question of how these processes are interrelated. Follicle cells undergo multiple morphogenetic movements to reach positions from which they secrete eggshell material, either while the movement is being completed or immediately afterwards. Therefore, the striking correlation between the Cad99C expression profile and morphogenetic movements likely reflects the close association between those movements and eggshell secretion (D'Alterio, 2005).

Cad99C specifically localizes to the plasma membrane of microvilli and is distributed throughout their entire length. PCDH15 shows a similar subcellular distribution in stereocilia on the surface of hair cells in the cochlea (Ahmed, 2003). In PCDH15 mutant mice (Ames waltzer) and zebrafish (orbiter), stereocilia are splayed and their arrangement is severely disturbed, causing deafness (Alagramam, 2001a; Raphael, 2001; Seiler, 2005). The function of PCDH15 in stereocilia, however, has remained unclear. This study indicates that its Drosophila orthologue Cad99C is a potent regulator of microvillus size. Interestingly, PCDH15 is found at a higher concentration in the longer stereocilia of a staircase-like bundle of hair cell stereocilia (Ahmed, 2003), and an irregular shortening of stereocilia in Ames waltzer mice has been described (Alagramam, 2001a; Raphael, 2001). This raises the possibility that PCDH15 regulates the length of stereocilia similar to Cad99C. In addition to being required for size regulation, Cad99C is also important for the normal shape and arrangement of microvilli. It will be interesting to determine in future studies whether the effect on size and on shape and arrangement of microvilli/stereocilia reflects two distinct functions of Cad99C/PCDH15 or whether they are two consequences of the same molecular function. Together, it is proposed that Cad99C/PCDH15-type cadherins have an evolutionarily conserved role in microvillus biogenesis (D'Alterio, 2005).

During the course of evolution, Cad99C/PCDH15 cadherins have adapted to act in apparently very different apical actin-based protrusions, such as the follicle cell microvilli of fly ovaries and the complex stereocilia of the vertebrate cochlea. Moreover, loss of Cad99C causes subtle defects in eye rhabdomeres and mechanosensory bristles, indicating that Cad99C is also required for other actin-based protrusions. Similar to PCDH15, which is more widely expressed in epithelial tissues (Murcia, 2001; Ahmed, 2003), Cad99C is found on the apical surface of several ectodermal epithelia during D. melanogaster development, including the imaginal discs (Schlichting, 2005). In wing imaginal discs as in the follicular epithelium, overexpression of Cad99C induces the formation of very large apical protrusions. However, there are epithelial tissues that possess a microvillus brush border but do not express Cad99C at detectable levels, including the midgut. Cad99C is therefore not a general component of microvilli and may serve a biological function that is specifically required in a subset of microvilli. Alternatively, another member of the cadherin superfamily or other membrane protein might take the place of Cad99C in the microvilli of tissues that lack this cadherin (D'Alterio, 2005).

It is speculated that, as a cadherin, Cad99C may mediate homophilic adhesion between microvilli. However, follicle cell microvilli in wild type and after overexpression of Cad99C are clearly separated from each other, implying that Cad99C mediates neither homophilic nor heterophilic interactions between microvilli while promoting their outgrowth. This conclusion is corroborated by the behavior of cell clones that either lack or overexpress Cad99C. In imaginal discs where Cad99C is concentrated at the apical interface between cells, cell clones with reduced or increased levels of Cad99C expression have wiggly boundaries, indicating that they do not sort out from wild-type cells (Schlichting, 2005). Furthermore, Cad99C located ectopically in the lateral membrane of follicle cells when overexpressed is not enriched at the border between Cad99C overexpressing cells compared with borders between wild-type and overexpressing cells as would be expected from a homophilic adhesion molecule. Similarly, in imaginal discs, the distribution of Cad99C along cell boundaries is uniform and independent of the concentration of Cad99C in neighboring cells. Hence, these findings argue against a function of Cad99C in adhesion between adjacent plasma membranes (D'Alterio, 2005).

A parallel bundle of actin filaments and associated factors that control their cross-linking and turnover are instrumental for the formation and stability of microvilli. To what extent the plasma membrane of microvilli contributes to microvillus morphogenesis either by linking to the actin core or independent of it remains largely unexplored. PCDH15 appears to influence the actin cytoskeleton of stereocilia as the amount of F-actin in stereocilia of PCDH15 mutant hair cells was reduced (Raphael, 2001). This effect is possibly mediated through its proposed interactions with the PDZ domain protein harmonin (Adato, 2005; Reiners, 2005). Among the USH1-associated proteins, harmonin has a central function, as it can also bind to CDH23 (Siemens, 2002), myosin VIIa, and SANS, and is able to interact with actin filaments promoting their bundling in cell culture, thereby potentially linking cadherins on the cell surface to the actin cytoskeleton (D'Alterio, 2005).

Unexpectedly, it was found that a truncated form of Cad99C that lacks most of the cytoplasmic tail, including the putative PDZ domain-binding sites, causes the same excessive lengthening of microvilli as the full-length protein, even in a Cad99C mutant background. This shows that the PDZ domain-binding sites do not have an essential positive regulatory function in microvillus outgrowth. It cannot be rule out that the remaining 31 juxtamembrane cytoplasmic amino acids interact with a cytoplasmic factor, but this short sequence contains no known motifs and is not conserved. Therefore, it seems likely that the membrane-bound extracellular domain of Cad99C is sufficient to promote microvillus extension independent of endogenous Cad99C. How does the extracellular domain of Cad99C control microvillus size? The data are consistent with two attractive models for Cad99C activity. The cadherin domains of Cad99C could influence the microvillus actin core by binding to an extracellular ligand, which directly or indirectly connects to the actin bundle promoting polymerization. Alternatively, the Cad99C extracellular domain might stabilize the plasma membrane of a microvillus. This may be important because to lengthen a cellular protrusion, the force created by actin polymerization has to overcome the counteracting force caused by tension in the plasma membrane envelope while it is pushed outward. Stabilization of the plasma membrane might alleviate the tension, allowing actin polymerization to proceed. The cadherin domains of Cad99C could stabilize the plasma membrane either by binding to the extracellular matrix or by self-assembling into an extracellular meshwork that forms a supporting scaffold surrounding the microvillus. The striking conservation of the Cad99C/PCDH15 extracellular domains may reflect the geometric constraints imposed by such a scaffold. The latter model in particular would be consistent with the concentration dependency of Cad99C activity and with its effect on the size and shape of microvilli (D'Alterio, 2005).

Cad99C is expressed in follicle cells surrounding the oocyte and is component of follicle cell microvilli

To identify novel regulators of cell and tissue morphogenesis, the expression patterns of uncharacterized members of the cadherin gene superfamily were studied during D. melanogaster oogenesis, when follicle cells undergo a series of well-described morphogenetic movements. Cad99C, a cadherin gene that is named after its chromosomal map position, is transcribed in cells of the follicular epithelium, but not in the germline cyst. Changes in the Cad99C expression level largely coincide with morphogenetically active phases of follicle cells. Cad99C mRNA was found in anterior and posterior follicle cells at stages 2-5 but is restricted to posterior follicle cells at late stage 6. Follicle cells that move over the oocyte at stage 9 and form a columnar epithelium at stage 10a express very high levels of Cad99C transcript. By stage 10b, high levels of expression are retained only in centripetal follicle cells that migrate inward to envelope the oocyte anteriorly. Expression levels peak again in all follicle cells when they flatten to accommodate the growth of the oocyte. During late oogenesis, some follicle cells form tubelike structures -- the micropyle and dorsal appendages. This is a process accompanied by a local increase of Cad99C expression. This dynamic expression profile suggests that Cad99C makes important contributions to follicle cell development. Consistent with its mRNA distribution, Cad99C protein is first seen during stages 2–5 of oogenesis in anterior and posterior terminal follicle cells. Beginning with stage 6, Cad99C is detected only in follicle cells that are in contact with the oocyte, a distribution that persists for the rest of oogenesis. At all stages, Cad99C is located on the apical plasma membrane of follicle cells, which faces the oocyte (D'Alterio. 2005).

To address the function of Cad99C in the follicular epithelium, the subcellular localization of this cadherin was examined and it was found to be confined to the apical microvillus brush border. Cad99C is located apical to DE-cadherin, which marks the adherens junctions. In contrast to DE-cadherin, Cad99C is not seen at the apicolateral and lateral plasma membrane, where follicle cells are in contact with one another. Cad99C shows a spiky pattern in a side view and a carpetlike pattern in a front view of the apical cell surface that is consistent with a localization to microvilli. Several observations support the idea that Cad99C is specific for follicle cell microvilli and not found in oocyte microvilli, with which they make contact: (1) Cad99C expression was not detected in the germline; (2) the pattern of Cad99C-positive microvilli reflects the honeycomb pattern of follicle cells, whereas oocyte microvilli form a continuous lawn; (3) Cad99C does not overlap with Yolkless, a marker that labels the oocyte cortex but colocalizes with a marker specifically expressed in follicle cells (CD8-GFP). It is inferred that Cad99C does not mediate homophilic adhesion between follicle cells or between microvilli of follicle cells and those of the oocyte (D'Alterio. 2005).

Search PubMed for articles about Drosophila Cad96Ca

D'Alterio, C., et al. (2005). Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli. J. Cell Biol. 171(3): 549-58. 16260500

Chen, C., Sun, H., Zhong, T., Liu, D., Lao, J., Zhang, Y., Shi, Z., Chen, J., Shen, M., Ma, S., Jia, L. (2025). Effect of receptor tyrosine kinase family member CAD96CA on hormone signalling and ontogeny of silkworm. Insect Mol Biol, PubMed ID: 41273026

Choi, B. J., Chen, Y. C., Desplan, C. (2025). Retinal calcium waves coordinate uniform tissue patterning of the Drosophila eye. Science, 390(6775):eady5541 PubMed ID: 41264707

D'Alterio, C., et al. (2005). Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli. J. Cell Biol. 171(3): 549-58. 16260500

Fung, S., Wang, F., Chase, M., Godt, D., Hartenstein, V. (2008). Expression profile of the cadherin family in the developing Drosophila brain. J Comp Neurol, 506(3):469-488 PubMed ID: 18041774

Li, Y. X., Kang, X. L., Li, Y. L., Wang, X. P., Yan, Q., Wang, J. X., Zhao, X. F. (2025). Receptor tyrosine kinases CAD96CA and FGFR1 function as the cell membrane receptors of insect juvenile hormone. Elife, 13 PubMed ID: 40085503

O'Farrell, F., Wang, S., Katheder, N., Rusten, T. E., Samakovlis, C. (2013). Two-tiered control of epithelial growth and autophagy by the insulin receptor and the ret-like receptor, stitcher. PLoS Biol, 11(7):e1001612 PubMed ID: 23935447

Schlichting, K., Demontis, F. and Dahmann, C. (2005). Cadherin Cad99C is regulated by Hedgehog signaling in Drosophila. Dev. Biol. 279(1): 142-54. 15708564

Schlichting, K., Wilsch-Brauninger, M., Demontis, F. and Dahmann, C. (2006). Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells. J. Cell Sci. 119: 1184-95. 16507588

Tsarouhas, V., Yao, L., Samakovlis, C. (2014). Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila. J Cell Sci, 127(Pt 8):1829-1839 PubMed ID: 24522188

Wang, S., Tsarouhas, V., Xylourgidis, N., Sabri, N., Tiklova, K., Nautiyal, N., Gallio, M., Samakovlis, C. (2009). The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila. Nat Cell Biol, 11(7):890-895 PubMed ID: 19525935 Biological Overview

date revised: 14 March 2026

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