In seahorses (family Syngnathidae), the male brood pouch provides a protected environment for developing embryos. During pregnancy, embryos develop within an enclosed brood pouch, requiring a physiological mechanism to eliminate the nitrogenous waste excreted by the embryos. This study investigated the expression and localization of rhesus (Rh) glycoproteins, expected to function as ammonia transporters, in the brood pouch of the pot-bellied seahorse Hippocampus abdominalis. The expression of the RhAG, RhBG, and RhCG2 genes in the pouch was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). In situ hybridization and immunohistochemistry revealed that RhBG and RhCG2 are localized in the inner epithelium of the pouch, while RhAG is present in erythrocytes and the endothelium of the blood vessels. Based on these findings, we propose the following waste removal pathway. Ammonia, presumably released from the embryo, would be transported from the lumen of the pouch through apical RhCG2 and basolateral RhBG of inner epithelial cells into the connective tissue beneath the inner epithelium. In the connective tissue, ammonia passes through RhAG-expressing endothelial cells into the vascular lumen and is likely taken up by RhAG-expressing erythrocytes, which then travel to the gills via the bloodstream. Then, ammonia is released from the RhAG-expressing erythrocytes through RhAG-expressing pillar cells, basolateral RhBG- and apical RhCG2-expressingpavement cells, finally into the surrounding seawater. This study provides a first molecular and cellular basis for a proposed ammonia removal mechanism within the seahorse brood pouch, highlighting physiological adaptations associated with male pregnancy in syngnathid fish.
The zebrafish is widely used to study glucose homeostasis, diabetes, and endocrine cell plasticity, yet a definitive ultrastructural characterization of adult pancreatic endocrine cell types has remained incomplete. Here, we combined transmission electron microscopy with hormone-specific immunogold labeling to define the ultrastructural characteristics of the major endocrine cell populations in adult zebrafish pancreatic islets. Three principal endocrine cell types were distinguished based on secretory granule morphology, electron density, and intracellular organization. Glucagon-positive α-cells contained electron-dense granules with a characteristic halo and eccentric dense core, insulin-positive β-cells displayed larger moderately electron-dense granules, and somatostatin-positive δ-cells possessed smaller, predominantly electron-dense granules of heterogeneous shape. Immunogold labeling for glucagon, insulin, and somatostatin linked granule ultrastructure to endocrine cell identity. To independently validate β-cell assignment, we performed nitroreductase-mediated β-cell ablation in juvenile zebrafish and observed a marked depletion of cells containing the characteristic medium electron-dense granules. Comparative analysis indicates that zebrafish endocrine granule ultrastructure differs in several respects from mammalian islets and exhibits similarities to selected teleost species, highlighting evolutionary diversity in endocrine granule organization. This study provides a foundational ultrastructural reference for the three major endocrine cell types of the adult zebrafish endocrine pancreas and establishes criteria for identifying these populations in studies of pancreatic disease, regeneration, and cell plasticity while also identifying an additional unassigned endocrine population that warrants further characterization.
The main olfactory bulb (MOB) hosts diverse γ-aminobutyric acid (GABA)ergic interneurons that play critical roles in sensory processing. Previous studies have reported variability in GABA immunoreactivity among cells in the granule cell layer (GCL) of the mouse MOB. However, the degree of intracellular GABA content across MOB layers and its relationship with molecular subtypes remain unclear. In this study, we conducted a quantitative analysis of GABA immunoreactivity in individual cells across all MOB layers using confocal laser scanning and electron microscopy. We found striking variability in the GABA immunoreactivity intensity among individual neurons across all layers, not limited to the GCL. Electron tomography with immunogold labeling confirmed more precisely substantial differences in intracellular GABA content, particularly in the glomerular layer and GCL. Mitral cells exhibited minimal GABA immunoreactivity, consistent with their excitatory phenotype. Co-labeling of molecular markers, including tyrosine hydroxylase, calbindin, calretinin, parvalbumin, and secretagogin, revealed that variability in GABA levels is common across all interneuron subtypes. Furthermore, a comparative analysis of the depth-dependent distribution of cells within the GCL and their GABA immunoreactivity demonstrated that more superficial regions contain stronger immunoreactive cells. This likely reflects the function of interneurons that regulate the olfactory output of tufted cells, which are projection neurons located in the superficial layers. To investigate the contribution of GABA-synthesizing enzymes, we examined the expression of glutamate decarboxylase 67 (GAD67) using GAD67-GFP knock-in mice. Although GFP intensity, reflecting GAD67 promoter activity, varied among cells throughout the MOB, it did not correlate clearly with GABA immunoreactivity. This finding suggests that GAD67 expression alone does not fully explain the observed intracellular GABA variability. Our findings show the molecular and cellular heterogeneity of GABAergic neurons in the MOB. Further integrative studies are needed to elucidate the mechanisms regulating GABA content and their functional relevance.
Temperature is a key environmental driver of hepatic physiology in ectotherms, and three-dimensional (3D) fish liver models may serve as an ethically advantageous platform to investigate warming effects under controlled conditions while reducing the need for experimental animals. Despite their increasing use in toxicology, their application to assess climate-relevant temperature effects on liver function remains limited. This study investigated the temporal effects of a warming scenario on primary hepatocyte spheroids from juvenile brown trout (Salmo trutta), a bioindicator species. The study explores how a + 3 °C increase can affect spheroid development and maintenance, as well as its impact on metabolic activity, cell proliferation and death, and morphology over time. Spheroids were maintained at 18 °C and 21 °C for 25 days and analysed at five time points using metabolic, morphometric, immunocytochemical, and ultrastructural approaches. Mitochondrial metabolic activity, assessed by resazurin reduction, showed no significant temperature-related differences. In contrast, warming accelerated spheroid formation and produced larger spheroids. Proliferative activity, assessed by proliferating cell nuclear antigen (PCNA) immunostaining, was significantly reduced at 21 °C, while caspase-3 levels remained unchanged, indicating no increase in apoptosis. The autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3A/B) showed lower immunoreactivity at 21 °C, with no temporal variation. Ultrastructural analysis revealed preserved hepatocyte integrity at both temperatures and abundant cytoplasmic dense bodies consistent with autolysosomal structures, which increased over time. Overall, a realistic warming scenario altered growth dynamics and cellular morphology. Further, this data reinforces that 3D fish liver models are viable alternative systems for assessing climate-driven effects.
CEACAM1 (CC1) is an important mediator of cell proliferation and adhesion and serves as an angiogenic factor through interaction with VEGF. Although the role of CC1 has been extensively studied in organs such as the heart, liver and lung in which CC1 is considered an important regulator of various pathological conditions, little is known about the function of CEACAM1 in the eye. In this study, we investigated the expression and function of CC1 in the retina and choroid of healthy adult mice using immunohistochemistry, fluorescence activated cell sorting (FACS) and RNA sequencing. We found CC1 to be expressed in endothelial and myeloid cells of the retina and the choroid. However, deletion of CC1 did not result in vascular abnormalities of the retina and choroid or changes in retinal myeloid cell morphology and number. Furthermore, the retinal architecture was not affected and morphometric measurements of the thickness of the inner and outer retinal layers were not altered by deletion of CC1. Accordingly, we did not observe structural or transcriptomic changes in the choroid. Our data suggest that the role of CC1 in the adult eye during steady state is attenuated or can be compensated by other molecular mediators. However, one could speculate that CC1 may become functionally relevant during pathological conditions, such as in neovascular eye diseases like proliferative diabetic retinopathy or neovascular age-related macular degeneration.
Left atrial remodeling (LAR) critically contributes to the progression of heart failure (HF) and the development of atrial fibrillation (AF) following myocardial infarction (MI). The protein α2δ1, primarily known for its role in neuropathic pain, is abundantly expressed in atrial tissue, but its involvement in post-MI LAR remains unclear. Here, LAR models were established in rats post-MI, and atrial hypertrophy was induced in HL-1 cells using angiotensin II (AngII). The role of α2δ1 in atrial hypertrophy was examined through treatment with either the α2δ1 inhibitor gabapentin or a C-terminal interfering peptide (α2δ1 CT-pep). A significant upregulation of α2δ1 expression was observed in the left atrium (LA) of MI rats and in AngII-treated HL-1 cells. Western blot analysis revealed increased α2δ1 levels in membrane fractions and decreased levels in the cytoplasmic fractions compared to controls. Both gabapentin and α2δ1 CT-pep treatment significantly reduced HL-1 cell hypertrophy and inhibited CAMKII and HDAC4 phosphorylation. Co-immunoprecipitation assays demonstrated an interaction between α2δ1 and GluN1, which was enhanced by AngII stimulation. Inhibition of α2δ1 attenuated the α2δ1-GluN1 interaction and reduced GluN1 translocation to the plasma membrane. In MI-induced HF rats, gabapentin treatment diminished atrial hypertrophy, suppressed AF inducibility and duration, and decreased membrane-associated α2δ1 and GluN1 levels. These findings suggest that the C-terminal domain of α2δ1 may contribute to left atrial hypertrophy in chronic ischemic heart failure and is associated with altered membrane GluN1 abundance and p-CAMKII/p-HDAC4 signaling. α2δ1 may therefore represent a potential therapeutic target for left atrial remodeling in ischemic heart failure.
The Magadi tilapia thrives in arguably the most extreme aquatic environment on earth for fish, the hot springs of Lake Magadi in Kenya with its severe water chemistry: pH 10, alkalinity 300 mEq·L−1. This fish is 100
The heart of Phasmida (stick insects) remains poorly understood, particularly regarding its overall physiology and associated cells. Here, we integrate the morphology of the heart and associated cells in Cladomorphus phyllinus with transcriptomic profiling of corresponding structures in Cladomorphus trimariensis. The heart is a tubular structure composed of cardiomyocytes physically associated with alary muscles, with a location and structure similar to that described in other insects. Numerous pericardial cells are organized in cord-like aggregations around the heart, displaying abundant membrane invaginations and an extensive endocytic and lysosomal machinery, consistent with their role in hemolymph filtration. Given their abundance, these cells likely represent the main source of the numerous transcripts related to endocytosis, vesicular trafficking and lysosome-mediated digestion. Hemocytes are also present around the heart, often forming aggregations indicative of immune responses. The transcriptomic analysis indicates immune activity, including melanization and antibacterial defense, predominantly driven by the hemocytes, with the pericardial cells providing a supportive role. Our results confirm that heart and associated cells act not only in hemolymph propulsion, but also as a site involved in maintaining hemolymph homeostasis and immune defense. This work provides an integrated morphological and functional framework for the circulatory system of Phasmida, expanding comparative perspectives across insects.
The urinary bladder is innervated by two anatomically distinct populations of sensory neurons originating from dorsal root ganglia (DRG): one from the thoracolumbar (TL) and another from the lumbosacral (LS) spinal region. While we previously characterized the morphology and distribution of sensory endings arising from LS DRG, the terminals of TL afferents remain undefined. In this study, we employed anterograde neuronal tracing to map these endings in mice. Following bilateral injection of dextran biotin into T12–L3 DRG and a 7–9-day recovery, whole bladders were processed to visualize spinal afferent axons and their calcitonin gene-related peptide (CGRP) immunoreactivity. We identified four morphological types of sensory endings, similar to those described for LS DRG: simple and complex types in both the sub/urothelium and the detrusor muscle. However, their distribution was fundamentally different. Most TL afferents (79
Osteoarthritis (OA) is a degenerative joint disease closely linked to iron dysregulation. While the role of iron in OA is recognized, the precise mechanism of cell death remains unclear. This study aimed to elucidate how chronic low-dose iron exposure influences chondrocyte survival, iron metabolism, and ferroptosis susceptibility. The 32-day exposure effects of low-dose (20 µM) ferric ammonium citrate (FAC) treatment on the immortalized human chondrocyte cell line (C-20/A4) in terms of survival and susceptibility to ferroptosis were investigated. Cell viability, morphology, and expression of key iron regulatory genes (IRGs) and ferroptosis-related stress genes were assessed. At day-32 of FAC treatment, chondrocyte proliferation and glutathione peroxidase 4 (GPX4) expression increased relative to controls (p < 0.0001). Inhibition of GPX4 with RSL3 in FAC-treated cells resulted in significantly increased cell death (p < 0.001), indicating a possible protective role of GPX4. Iron chelation with deferoxamine conferred protection against ferroptosis. Dynamic changes in IRGs and cell cycle regulators, consistent with an adaptive strategy to resist iron overload and support survival, were observed. In-silico analysis of OA cartilage transcriptomic data identified 170 differentially expressed genes. Functional enrichment showed upregulation of ubiquitin-dependent catabolic processes and downregulation of mitotic pathways, supporting altered stress responses and cell cycle disruptions in iron-exposed chondrocytes. Chronic low-dose iron exposure induced an adaptive upregulation of GPX4, enhancing chondrocyte resistance to cell death by ferroptosis by maintaining lipid peroxide reduction. Therapeutic modulation of GPX4 and iron homeostasis may offer novel strategies for OA management where cartilage iron imbalance is implicated. Chronic low-dose iron exposure induces adaptive GPX4 upregulation in chondrocytes, enhancing resistance to ferroptosis. Inhibition of GPX4 unmasks vulnerability, promoting ferroptotic cell death, highlighting GPX4’s pivotal role in osteoarthritis pathogenesis and its potential as a therapeutic target.
Bone regeneration remains constrained by incomplete osteogenic commitment of mesenchymal stem cells (MSCs), underscoring the need for precise lineage control. CRISPR/Cas-based epigenome editing provides programmable access to chromatin regulators without altering the DNA sequence, and catalytically inactive Cas9 (dCas9) fused to transcriptional activators, repressors, or chromatin modifiers enables locus-specific modulation of key osteogenic networks, including RUNX2, OSX, and BMP2, while suppressing inhibitory loci such as PPARG, SOST, and DKK1. Multiplex strategies further allow the concurrent activation of osteogenic genes and repression of adipogenic or Wnt antagonists, reshaping lineage allocation in vitro and in vivo. Delivery innovations—from AAV vectors and lipid nanoparticles to biomaterial scaffolds and extracellular vesicles—support local and systemic applications with increasing precision, while whole-genome chromatin profiling and high-fidelity Cas variants reduce off-target risk, and CRISPRoff/on platforms provide reversible and heritable control of transcriptional states. Proof-of-concept studies in small animals demonstrate bone repair in preclinical models, with emerging large-animal data highlighting translational potential. Remaining challenges include payload size, immunogenicity, durability of epigenetic states, GMP-grade manufacturing, and regulatory classification. Looking ahead, advances such as AI-guided gRNA libraries, mechano-responsive scaffolds, and long-term tracking of epigenetic memory may yield durable “smart” osteo-epigenetic therapies. Collectively, CRISPR/dCas9-based epigenome editing is progressing from mechanistic exploration toward clinically viable strategies for skeletal regeneration.
Temporomandibular joint osteoarthritis (TMJOA) is a degenerative joint disease characterized by the progressive deterioration of the condylar cartilage, compensatory remodeling of subchondral bone, and persistent synovial inflammation. The onset of TMJOA is accompanied by the activation of cytokines, which exacerbate the imbalance in joint homeostasis and accelerate the destruction and even irreversible degradation of joint structures. FGF19, as an important multifunctional fibroblast growth factor, has been reported to be substantially up-regulated in the cavity of osteoarthritis joint. However, its role in TMJOA progression remains unclear. In this study, we investigated its role in the condyle cartilage in TMJOA. We first established a mouse TMJOA model by unilateral anterior crossbite (UAC) and achieved overexpression of FGF19 by direct injection of adeno-associated virus carrying the Fgf19 gene. We found that FGF19 exacerbated the progressive deterioration of cartilage-subchondral bone unit in the progression of TMJOA. FGF19 accelerated the phenotypic transformation from mature cartilage to hypertrophic cartilage by reducing the expression of collagen type II (COL2A1) and aggrecan, enhancing the expression of collagen type X (COL10A1), matrix metallopeptidase 13 (MMP13), and a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5), and exhibiting a fibrocartilage-associated change in the cartilage layer by increasing the expression of collagen type I alpha 1 and 2 (COL1A1 and COL1A2). Furthermore, FGF19 induces calcification progression of cartilage hypertrophic layer by up-regulating the expression of dentin matrix acidic phosphoprotein 1 (DMP1) via transcription factor osterix (OSX). These data provide evidence of the importance of FGF19 in the progression of TMJOA and show potential cues for interventions of cartilage disease.
The posterior lobe (PL) of the pituitary contains a specialized neurovascular junction, where neurosecretory axon terminals closely associate with fenestrated capillaries to enable efficient hormone release into the circulation. The neurosecretory axon terminals and the fenestrated capillaries are supported by an outer basement membrane (BM) and an inner BM, respectively. Although BMs are critical structural components of this junction, their molecular composition and regulatory mechanisms remain poorly understood. We previously demonstrated that COL15A1, the α1 chain of collagen XV, localizes to the BMs surrounding fenestrated capillaries in the anterior lobe of the rat pituitary. In the present study, we examined the localization and regulation of COL15A1 in the rat PL. Immunohistochemical analyses revealed that COL15A1 is localized to the inner BMs of fenestrated capillaries. Combined in situ hybridization and immunohistochemistry demonstrated that Col15a1 is predominantly expressed in NG2-positive pericytes. Water deprivation, which induces arginine vasopressin (AVP) secretion, significantly decreased Col15a1 expression in the PL, whereas AVP stimulation did not affect its expression in primary cultured posterior and intermediate lobe (PIL) cells. We also found that TGFβ2 is produced by S100β-positive pituicytes and TGFβ receptor II is localized to NG2-positive pericytes in the PL. Furthermore, TGFβ2 induces Col15a1 expression in primary cultured PIL cells via TGFβ receptor II and SMAD2 signaling. Collectively, our results identify collagen XV as a key component of the inner BMs surrounding fenestrated capillaries and demonstrate that pericytes orchestrate the structural and functional organization of the neurovascular junction in the PL.
Chimeras (Holocephali) occupy a pivotal phylogenetic position, being the oldest jawed vertebrate, for understanding the evolution of hypothalamic-pituitary organization in vertebrates. However, the structural organization of the hypothalamic-pituitary axis and the anatomical relationships with the buccal lobe (BL), a unique gland thought to play a role in reproduction, remains poorly characterized. We present an anatomo-histological analysis of the chimaera Callorhinchus callorynchus using classical histology, immunohistochemistry, and RT-PCR, focus on key neuropeptides related to the control of the reproductive activity in other vertebrates: gonadotropin-releasing hormone (GnRH), secretoneurin (SN), cholecystokinin (CCK), and gonadotropic hormones. The pituitary gland displays clear regionalization into the rostral pars distalis, proximal pars distalis, and neurointermediate lobe. The BL is connected to the posterior telencephalon by paired canals containing a prominent blood vessel and bundles of neuropeptidergic fibers. Gonadotropin-releasing hormone-immunoreactive nerve cell bodies and fibers were identified in the posterior telencephalon using two antisera, with fibers projecting to the proximal pars distalis of the pituitary and the BL. Secretoneurin-immunoreactive neuronal somata and fibers were observed in the posterior telencephalon and hypothalamus, and SN-immunoreactive cells were also present within the pituitary and BL. Cholecystokinin-immunoreactive neuronal elements were detected in the hypothalamus, while CCK-immunoreactive cells were present in the pituitary, and a subset of fibers extending into the BL. RT-PCR analyses revealed the presence of Fshb and Fhb transcripts in the pituitary and BL, with a stronger apparent signal in the BL. These observations provide anatomical evidence for direct neuropeptidergic innervation of both the pituitary and BL and indicate that the BL is associated with gonadotropin subunit gene expression in C. callorynchus. This new anatomical framework of the hypothalamus-pituitary-BL axis identifies two new pathways: direct GnRH and CCK innervation, and local SN-mediated autocrine/paracrine signaling.
Proximal tubule in vitro systems are important tools in pre-clinical drug development, providing insights into drug disposition and safety. Cells are often cultured in medium containing fetal calf serum (FCS); however, FCS addition is not physiologically relevant for cells of human origin. Here, in vitro cultures of human kidney proximal tubule cells were transitioned to FCS-free medium. The impact of FCS-free medium on maturation and maintenance for multiple passages of commonly used conditionally immortalized proximal tubule epithelial cells overexpressing the pharmacologically-relevant organic anion transporter 1 (ciPTEC-OAT1) was evaluated. This transport protein is lost upon isolation and in vitro culture of tubule cells, and due to its clinical relevance, an OAT1 expressing cell line was chosen. Supplementation of FCS-free medium with human platelet lysates supported longer-term cell growth. OAT1-mediated fluorescein transport was sensitive to probenecid inhibition under both conditions, although intracellular fluorescein accumulation was reduced in FCS-free cultures. KEGG and GO enrichment analysis of differentially expressed genes revealed that inflammatory response and HIF-1 signaling were downregulated, whereas cell adhesion and ATP-binding cassette transporter genes were upregulated in FCS-free cultures. Interestingly, an increase in bioenergetic profile was observed for ciPTEC-OAT1 cultured in optimized FCS-free medium. Furthermore, in 3D bioengineered kidney tubule cultures, supplementation with an increased concentration of human platelet lysates lead to improved cell viability and fiber coverage. To conclude, we highlighted significant efforts and strategies in transitioning FCS-containing to FCS-free medium for ciPTEC-OAT1 cultures.
Precise positioning and alignment of specific cell types, such as those in the central nervous system and the muscular system, is essential for their functional integration, their migration, and proliferation in vivo. Cell alignment in physiologically relevant tissue models and constructs is challenging to reproduce in vitro unless a three-dimensional scaffold is used. This study demonstrates that cell alignment can be guided quickly, inexpensively, and efficiently using polymeric micro-hollow fiber membranes. These micro-hollow fiber membranes are fabricated via single orifice wet spinning from biocompatible polymers—polystyrene and polycaprolactone. The physicochemical characterization of the micro-hollow fiber membranes confirmed their unique architecture, presenting a special patterning on their outer surface. To establish their potential as a platform for cell alignment via contact guidance, the viability, and degree of alignment of relevant cell lines were evaluated when cultured on the micro-hollow fiber membranes. NG108-15, olfactory ensheathing cells and SH-SY5Y cells were used with the aim to simulate the microspatial distribution of cells within the spinal cord, and C2C12 myoblasts were selected to mimic the highly organized structure seen in muscle tissue. Moreover, differentiation of SH-SY5Y cells was successfully induced while cells remained aligned with respect to the micro-HFM’s axis. The degree of alignment in all cases was quantified via image analysis in combination with the Fast Fourier Transform algorithm method. This work establishes a platform with very particular micro-topographical features that can be employed to direct growth, orientation, and even differentiation of various cell types for tissue engineering and in vitro modelling.
Among all known repair enzymes, methionine sulfoxide reductase A (MsrA) plays a key role in protecting against oxidative stress and its damaging effects. The enzyme is ubiquitously expressed in eukaryotic cells and different splice variants of the MsrA gene have been shown to permit the specific prevalence of the protein in different cell organelles. Here, we present novel insights in the intracellular localization of MsrA in selected samples from mouse and human tissue. Using immunocytochemical techniques at both the light and electron microscopic levels, we show incidence of MsrA in human oral mucosa cells, predominantly in the cell nucleus. In addition, the enzyme has been localized in mitochondria of mouse liver and kidney cells, in the cytoplasm of retina cells, and in the endoplasmic reticulum of glandular cells. Overall, the results indicate a correlation between the generation of reactive oxygen species (ROS) and the expression level of MsrA in subcellular compartments of the tissues examined.
Triple-negative breast cancer (TNBC) lacks targeted therapies and is driven by dysregulated signaling networks that promote migration, invasion, and survival. Connexin43 (Cx43), a gap junction protein essential for maintaining normal mammary epithelial homeostasis, becomes aberrantly phosphorylated and mislocalized in breast cancer, contributing to disease progression. Because the tyrosine kinases Pyk2 and Src regulate Cx43 and multiple pro-tumorigenic pathways, we investigated whether their combined inhibition could suppress malignant behaviors in TNBC. In MDA-MB-231 cells, the Pyk2 inhibitor PF4618433 and Src inhibitor Saracatinib modestly reduced metabolic activity at high concentrations; however dual treatment produced a dose-dependent and synergistic reduction in viability. In migration and invasion assays, each inhibitor reduced motility, however dual inhibition produced the strongest suppression. Cx43 knockdown impaired baseline migration and invasion and altered the response to Pyk2/Src inhibition, indicating that Cx43 modulates sensitivity to these agents. PF4618433 increased Cx43 plaque formation without changing total protein levels. Mechanistically, Pyk2 inhibition reduced phosphorylation of Cx43 at Y265 and decreased levels of TAZ, p-Erk1/2, p130Cas, and Notch1, whereas Src inhibition only reduced p-Erk1/2. Dual treatment did not further decrease these signaling nodes but nonetheless produced stronger functional inhibition of viability and motility, and the shared regulation of p-Erk1/2 by Pyk2 and Src may help explain how compensatory Pyk2 activation limits the effectiveness of Src-targeted therapies. Together, these findings show that coordinated Pyk2 and Src inhibition restores Cx43 organization and disrupts multiple malignant traits in TNBC cells, supporting this combination as a promising therapeutic strategy.
Antennae of insects are essential mechanosensory organs that facilitate active tactile exploration and spatial navigation. Hair plate sensilla at the base of the antenna flagellum provide proprioceptive inputs to detect the antennae’s position. In hemimetabolous insects, such as crickets, the first instar immediately after hatching also possesses antennae; however, the developmental dynamics and spatial organization of antennal hair plates remain poorly understood. We present a comprehensive three-dimensional analysis of the antennal hair plates in crickets (Gryllus bimaculatus) across developmental stages, from the first instar to the adult stage. We demonstrated that hair plate sensilla were present from the first instar and maintained a highly stereotyped spatial arrangement throughout development. Three-dimensional quantification revealed that new sensilla added during molting were formed at specific sites within the hair plate clusters that had existed at the previous stage, maintaining the spatial pattern despite the substantial growth of the antenna. Multidimensional analyses indicated that the spatial arrangement of sensilla was consistent across individuals, suggesting that their organization was under genetic control. Anterograde labeling of sensory afferents showed that sensory neurons in the hair plates converged their axons, extended axon collaterals into the ipsilateral region of the antennal mechanosensory and motor center, and ultimately projected to the subesophageal ganglion. There was no apparent difference in the projection sites among the hair plates, suggesting no clear evidence of topographic organization. Our findings highlight the conserved spatial organization of hair plate sensilla in crickets, suggesting a robust proprioceptive system that provides reliable feedback on antennal position throughout development.
