Disruption of extracellular pH and proton-sensing can profoundly impact cellular and protein functions, leading to developmental defects. To visualize changes in extracellular pH in the developing embryo, we generated a...Disruption of extracellular pH and proton-sensing can profoundly impact cellular and protein functions, leading to developmental defects. To visualize changes in extracellular pH in the developing embryo, we generated a zebrafish transgenic line that ubiquitously expresses the ratiometric pH-sensitive fluorescent protein pHluorin2, tethered to the extracellular face of the plasma membrane using a glycosylphosphatidylinositol (GPI) anchor. Monitoring of pHluorin2 with ratiometric fluorescence revealed dynamic and discrete domains of extracellular acidification over the first 72 h of embryonic development. These included acidification of the notochord intercalations, transient acidification of the otic placode, and persistent acidification of the extracellular space of the myotome at distinctly different pH from that within the T-tubules. Knockdown of centronuclear myopathy genes Bin1b (OMIM: 255200) and MTM1 (OMIM: 310400), which disrupt T-tubule formation, also disrupted myotome acidification. In this study we visualize extracellular acidic microdomains in the tissues of whole live animals. This real-time reporter line for directly measuring changes in extracellular pH can be used to illuminate the role of extracellular pH in normal physiological development and disease states.
BACKGROUND: Previous studies with Gfi1-mutated lines have shown that Gfi1 is essential for hair cell maturation and survival. RESULTS: We analyzed the phenotype of another Gfi1-mutated line Gfi1 in the inner ears of neon...BACKGROUND: Previous studies with Gfi1-mutated lines have shown that Gfi1 is essential for hair cell maturation and survival. RESULTS: We analyzed the phenotype of another Gfi1-mutated line Gfi1 in the inner ears of neonates at P5-7 and found that the cochlea phenotypically differed from the vestibule in the Gfi1 mouse. Specifically, there was a marked reduction in hair cells in the cochlea, which was characterized by greater reductions in the outer hair cells but far less reductions (mainly in the basal turn) in the inner hair cells, whereas the vestibular hair cells remained unaffected. These results were consistent with findings from previous studies. Unexpectedly, the number of cochlear non-sensory supporting cells significantly decreased. However, the vestibular supporting cells did not demonstrate any abnormalities in number. CONCLUSION: Gfi1 exhibits different functions in the cochlea and vestibule during inner ear development.
BACKGROUND: Insights into the development and evolution of asymmetrical jaws will require an understanding of the gene regulatory networks that underpin the differential morphogenesis of the maxillary and mandibular doma...BACKGROUND: Insights into the development and evolution of asymmetrical jaws will require an understanding of the gene regulatory networks that underpin the differential morphogenesis of the maxillary and mandibular domains of the first pharyngeal arch in a variety of gnathostomes. While a robust relationship has been demonstrated between jaw patterning and the Endothelin-Dlx gene axis, much less is known of the next level of genes in the jaw patterning hierarchy. RESULTS: Several genes, whose expression depends on Dlx5 and/or Dlx6, have been identified in mice. Here, we examined the expression patterns of the chick orthologues of some of those genes, namely GSC, PITX1, HAND2, and GBX2, and tested their dependence on endothelin signaling to assess whether there is a conserved regulatory relationship between those genes in the chick embryo. To further validate these genes as direct DLX targets, we identified conserved non-coding sequences containing candidate DLX binding motifs and demonstrated DLX-responsiveness in vitro. CONCLUSIONS: The evidence presented in this study combines to support the hypothesis that these four genes are direct targets of DLX transcription factors in the lower jaw-forming tissue.
BACKGROUND: The FOXOs regulate the transcription of many genes, including ones directly linked to pathways required for lens development. However, this transcription factor family has rarely been studied in the context o...BACKGROUND: The FOXOs regulate the transcription of many genes, including ones directly linked to pathways required for lens development. However, this transcription factor family has rarely been studied in the context of development, including the development of the lens. FOXO expression, regulation, and function during lens development remained unexplored. RESULTS: In studies of the embryonic lens, we showed that both FOXO1 and FOXO4, which share many downstream targets, are expressed in a differentiation-state-specific manner, most highly in lens epithelial and differentiating cortical fiber cells. Their expression patterns and subcellular distributions suggest both shared and distinct functions. Stabilization of FOXO cytoplasmic pools involved their binding to the chaperone protein 14-3-3. FOXO association with β-catenin linked this transcription complex to fiber cell-specific gene activation. Inhibition of PI3K/Akt signaling promoted FOXO1/FOXO4 nuclear localization in lens epithelial and fiber cells and expression of the CDKi p27 in the lens epithelium where it has been linked to lens cell withdrawal from the cell cycle and initiation of the lens differentiation program. We showed that FOXO1 transcriptional activation is required for the induction of p27 when Akt signaling is blocked, demonstrating the linearity of the PI3K/Akt/FOXO1/p27 pathway. CONCLUSIONS: PI3K/Akt signaling regulates FOXO-dependent lens cell differentiation.
BACKGROUND: Perfluoroalkyl substances (PFAS) are persistent environmental contaminants previously used for industrial purposes as a non-stick coating and flame retardant. The stability of these molecules prevents their b...BACKGROUND: Perfluoroalkyl substances (PFAS) are persistent environmental contaminants previously used for industrial purposes as a non-stick coating and flame retardant. The stability of these molecules prevents their breakdown, which results in ground water contamination across the globe. Perfluoroalkyl substances molecules are known to bioaccumulate in various organisms. However, the health consequences remain unclear due to the large number of molecules in the PFAS family and different effects on various tissues. Here, we use the frog Xenopus laevis to investigate the developmental consequences of exposure to the PFAS molecule perfluoro-octanoic sulfonate (PFOS). RESULTS: We find that exposure to high levels of PFOS results in significant axial shortening of developing tadpoles. Further, we find that PFOS exposure results in a dose-dependent formation of a cellular mass in the dorsal fin. Unexpectedly, we found that these developmental phenotypes are exacerbated upon co-exposure with commonly used antibiotics. Specifically, PFOS and gentamicin co-treatment results in increased apoptosis, loss of cellular integrity, and increased overall lethality. CONCLUSIONS: Our results suggest a mechanism whereby gentamicin reaches levels that are toxic to mitochondria only in the presence of PFOS. These findings add to our understanding of PFOS exposure to vertebrate development and present an added concern with potential interactions with antibiotics.
Morozumi R, Okamoto K, Enomoto E
… +13 more, Tsukamoto Y, Kyakuno M, Suzuki N, Tazawa I, Furuno N, Ogino H, Kamei Y, Matsunami M, Shigenobu S, Suzuki K, Uemasu H, Namba N, Hayashi T
BACKGROUND: The pancreas exhibits diverse structures and roles across vertebrates. The pancreas has evolved to include both endocrine and exocrine cells, a change that occurred during the transition from fish to amphibia...BACKGROUND: The pancreas exhibits diverse structures and roles across vertebrates. The pancreas has evolved to include both endocrine and exocrine cells, a change that occurred during the transition from fish to amphibian. This event emphasizes the evolutionary significance of amphibians. However, research has focused predominantly on anuran amphibians, with urodeles, such as newts, remaining underexplored. In this study, we investigated the development of the pancreas using Pleurodeles waltl as a model species of urodele. RESULTS: The newt pancreas consists of a single organ with exocrine tissue characterized by acinar structures and endocrine tissue forming islets. Notably, the newt possesses unique pancreas-like tissues on their intestines. We found that disruption of the newt Pancreatic and Duodenal Homeobox (Pdx) 1 gene resulted in an underdeveloped pancreas. Conversely, disruption of the Pdx2 paralog in newt had no significant impact on pancreatic development. CONCLUSION: The newt pancreas shows a morphology similar to that of the mammalian pancreas, which includes both exocrine and endocrine tissues. These results highlight the intermediate evolutionary position of the newt in the context of the evolution of pancreatic development. Our findings indicate that characterization of the newt pancreas will be crucial for understanding the evolutionary progression of pancreatic function in vertebrates.
The rising incidence of Alzheimer's disease (AD) and the associated economic impacts has prompted a global focus in the field. In recent years, there has been a growing understanding of the pathogenic mechanisms of AD, i...The rising incidence of Alzheimer's disease (AD) and the associated economic impacts has prompted a global focus in the field. In recent years, there has been a growing understanding of the pathogenic mechanisms of AD, including the aggregation of β-amyloid, hyperphosphorylated tau, and neuroinflammation. These processes collectively lead to neurodegeneration and cognitive decline, which ultimately results in the loss of autonomy in patients. Currently, there are three main types of AD treatments: clinical tools, pharmacological treatment, and material interventions. This review provides a comprehensive analysis of the underlying etiology and pathogenesis of AD, as well as an overview of the current prevalence of AD treatments. We believe this article can help deepen our understanding of the AD mechanism, and facilitate the clinical translation of scientific research or therapies, to address this global problem of AD.
BACKGROUND: Neural crest cells (NCCs) are migratory embryonic stem cells that give rise to a diverse set of cell types. Here we describe the dynamic distribution of NCCs in developing embryos of the common wall lizard Po...BACKGROUND: Neural crest cells (NCCs) are migratory embryonic stem cells that give rise to a diverse set of cell types. Here we describe the dynamic distribution of NCCs in developing embryos of the common wall lizard Podarcis muralis inferred from 10 markers. Our aim is to provide insights into the NCC development of lacertid lizards and to infer evolutionary modifications by comparisons to other tetrapods. RESULTS: NCC migration is ongoing at oviposition, following three streams in the head and multiple in the trunk. From 21ss, we observe expression patterns indicating the beginning of differentiation toward mesenchymal and neuronal fates. By 35ss, migration is restricted to caudal levels, and fully differentiated chromaffin cells are observed. CONCLUSIONS: We find that some markers show patterns that differ from other tetrapods. For example, the antibody HNK-1 labels three NCC streams from the hindbrain while some comparable reptile studies describe four. However, the information emerging from all markers combined shows that the overall spatiotemporal distribution of NCCs in the common wall lizard is largely conserved with that of other tetrapods. Our study highlights the dynamic nature of seemingly canonical marker genes and provides the first description of spatiotemporal NCC dynamics in a lacertid lizard.
BACKGROUND: RhoA GTPases play critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, adhesion, migration and changes in cell shape, al...BACKGROUND: RhoA GTPases play critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, adhesion, migration and changes in cell shape, all required for cutaneous wound healing. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in skin keratinocytes and is decreased in the absence of interferon regulator factor 6, a critical regulator of cell proliferation, migration, and wound healing. However, the role for ARHGAP29 in keratinocyte biology is unknown. RESULTS: We generated ARHGAP29 knockdown keratinocyte cell lines and show they displayed increased filamentous actin, phospho-myosin regulatory light chain, cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single and collective cell migration conditions; these delays were rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. In vivo, however, Arhgap29 heterozygotes or keratinocyte-specific knockouts showed on-time wound healing. CONCLUSIONS: These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration in vitro but is dispensable during wound healing in vivo.
BACKGROUND: Technical advances in whole tissue imaging and clearing have allowed 3D reconstruction of exocrine uterine glands deep-seated in the endometrium. However, there are limited gland structure analysis platforms...BACKGROUND: Technical advances in whole tissue imaging and clearing have allowed 3D reconstruction of exocrine uterine glands deep-seated in the endometrium. However, there are limited gland structure analysis platforms to analyze these imaging data sets. Here, we present a pipeline for segmenting and analyzing uterine gland shape. RESULTS: Using our segmentation methodology, we derive metrics to describe gland length, shape, and branching patterns. We then quantify gland behavior with respect to organization around the embryo and proximity of each gland to the uterine lumen. We apply this image analysis pipeline to uterine glands at the peri-implantation time points of a mouse pregnancy. Our analysis reveals that at the time of embryo or egg entry into the uterus, glands show changes in length, tortuosity, and proximity to the uterine lumen while gland branch number stays the same. Eventually, these shape changes aid in reorganization of the glands around the embryo implantation site. We further apply our analysis pipeline to human and guinea pig uterine glands, extending feasibility to other mammalian species. CONCLUSION: This work serves as a resource for researchers to extract quantitative and reproducible morphological features from three-dimensional uterine gland images to reveal insights about functional and structural patterns.
BACKGROUND: The branchial epithelium is one of the main tissues in which histone H3K4 trimethylation (H3K4me3) occurs in the budding tunicate, Polyandrocarpa misakiensis. It contains proliferating and undifferentiated ce...BACKGROUND: The branchial epithelium is one of the main tissues in which histone H3K4 trimethylation (H3K4me3) occurs in the budding tunicate, Polyandrocarpa misakiensis. It contains proliferating and undifferentiated cell aggregates at the bottom of each pharyngeal cleft, providing the nest for the adult stem cell niche. We examined the sustainable mechanism enabling epigenetic histone methylation in adult stem cells. RESULTS: Histone H3K4 demethylase (PmisKdm5) was not co-expressed in vivo with the transcription factor, signal transduction and activator of transcription (PmisStat) in the same cells. PmisStat mRNA, when electroporated into zooids, suppressed the gene expression of PmisKdm5 and facilitated the trimethylation of H3K4. A STAT5 inhibitor blocked the nuclear localization of PmisStat. It stimulated PmisKdm5 gene expression irrespective of PmisStat mRNA. The KDM5 inhibitor, CPI-455, stimulated H3K4me3 similarly to PmisStat mRNA. PmisStat mRNA and CPI-455 both induced the gene expression of PmisAp2 and PmisSp8, which were recently identified as budding/regeneration-related genes. When zooid tissues were treated with both CPI-455 and the STAT5 inhibitor, CPI-455 overwhelmed the effects of the STAT inhibitor on PmisAp2 and PmisSp8. CONCLUSION: PmisStat is involved in epigenetic histone methylation at H3K4 through the inhibition of PmisKdm5. H3K4me3 affects downstream gene expression more directly and strongly than PmisStat.
BACKGROUND: The eustachian tube (ET), a critical conduit connecting the middle ear and nasopharynx, is essential for normal middle ear function. However, it remains one of the least understood anatomical structures due t...BACKGROUND: The eustachian tube (ET), a critical conduit connecting the middle ear and nasopharynx, is essential for normal middle ear function. However, it remains one of the least understood anatomical structures due to its complexity and the challenges of in vitro manipulation. Historically, these challenges have hindered research into the morphology and function development of the ET. This study elucidates the spatiotemporal relationship of ET morpho-functional maturation in mice, identifying key periods and factors that lay the theoretical foundation for exploring the molecular mechanisms of ET-related diseases. RESULTS: We comprehensively characterized the ET development in C57BL/6 mice from embryonic day (E) 12.5 to postnatal day (P) 30, focusing on the development of cilia, secretory cells, surrounding glands, and macrophages. Immunostaining identified the localization and secretion patterns of the mucins Muc5b and Muc5ac within the ET. Additionally, using improved ET function assessment tools, we evaluated the developmental features of ET mucociliary clearance and ventilation functions. CONCLUSIONS: In C57BL/6 mice, E16.5 marks a critical period for middle ear cavity and ET formation. Muc5b plays a foundational role during early stages, while Muc5ac enhances function in later stages. During P7-11, despite morphological maturity, ET function remains underdeveloped but continues to improve with growth.
BACKGROUND: Eph receptors and ephrin ligands, the transmembrane proteins, function as a mechanism of communication between cells. Therefore, we intended to explore the expression array of EphB2 and EphB4 receptors and ep...BACKGROUND: Eph receptors and ephrin ligands, the transmembrane proteins, function as a mechanism of communication between cells. Therefore, we intended to explore the expression array of EphB2 and EphB4 receptors and ephrin-B1 ligand in postnatal developing mouse epididymis during 1 day to 8 weeks using RT-PCR amplification and immunofluorescence staining. RESULTS: RT-PCR analysis indicated that the expression levels of EphB2, EphB4, and ephrin-B1 in the epididymis declined with the advancement of age during the initial phases of postnatal development and stayed relatively near to adult levels until 4 weeks. We discovered that the predominant compartments expressing EphB2/B4 and ephrin-B1 emerged in the excurrent duct epithelia of postnatal developing epididymis until 3 weeks. Consequently, even before spermatozoa reach the excurrent duct in epididymis, at the age of 3 weeks, the epididymal excurrent duct system exhibits characteristics similar to those of an adult in terms of expression of EphB2/B4 and ephrin-B1. Moreover, ephrin-B1 was expressed in epididymal epithelial cells throughout the development and EphB4 was expressed only in early postnatal stages while basal cells expressed EphB4 throughout the postnatal development. CONCLUSION: The study represents the first expression analysis of ephrin-B1, EphB2, and EphB4 in the normal mouse epididymis during the postnatal development.
BACKGROUND: The trigeminal nerve is the largest cranial nerve and functions in somatosensation. Cell bodies of this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neural crest and p...BACKGROUND: The trigeminal nerve is the largest cranial nerve and functions in somatosensation. Cell bodies of this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neural crest and placode cells. While this dual cellular origin has been known for decades, the molecular mechanisms controlling trigeminal ganglion development remain obscure. We performed RNA sequencing on the forming chick trigeminal ganglion and identified Elongator acetyltransferase complex subunit 1 (Elp1) for further study. Mutations in ELP1 cause familial dysautonomia (FD), a fatal disorder characterized by the presence of smaller trigeminal nerves and sensory deficits. While Elp1 has established roles in neurogenesis, its function in placode cells during trigeminal gangliogenesis has not been investigated. RESULTS: To this end, we used morpholinos to deplete Elp1 from chick trigeminal placode cells. Elp1 knockdown decreased trigeminal ganglion size and led to aberrant innervation of the eye by placode-derived neurons. Trigeminal nerve branches also appeared to exhibit reduced axon outgrowth to target tissues. CONCLUSIONS: These findings reveal a new role for Elp1 in placode-derived neurons during chick trigeminal ganglion development. These results have potential high significance to provide new insights into trigeminal ganglion development and the etiology of FD.
Breaking radial symmetry for anterior-posterior axis formation is one of the key developmental steps of vertebrate gastrulation and is established through a succession of transient domains defined by morphology or gene e...Breaking radial symmetry for anterior-posterior axis formation is one of the key developmental steps of vertebrate gastrulation and is established through a succession of transient domains defined by morphology or gene expression. Three such domains were interpreted recently in the rabbit to be part of a "three-anchor-point model" for axis formation. To answer the question as to whether the model is generally applicable to mammals, the dynamic expression patterns of four marker genes were analyzed in the pig, where gastrulating epiblast forms from half the inner cell mass: EOMES and PKDCC transcripts display decreasing expression intensities in the anterior hypoblast and-together with WNT3-increasing intensity in the anterior streak domain and the node; TBX6 expression changes from an initial central expression to exclusive expression in the posterior extremity of the primitive streak. The anterior streak domain has thus a molecular footprint similar to the one in the rabbit, the end node shares TBX6 between the species, while the anterior hypoblast-mirroring specific porcine epiblast derivation and fate-is marked by PKDCC instead of WNT3. The molecular similarities in transient domains point to conserved mechanisms for establishing the mammalian anterior-posterior axis and, possibly, breaking radial symmetry.
Earthworms are a highly abundant species in nature, with nearly 7000 different species being discovered. Despite the similarities in morphology among earthworm species, their regeneration capabilities vary based on the c...Earthworms are a highly abundant species in nature, with nearly 7000 different species being discovered. Despite the similarities in morphology among earthworm species, their regeneration capabilities vary based on the clitellum. The clitellum plays a crucial role in the clitellum-dependent worms, as it is involved in the processes of regeneration and reproduction in earthworms. The fascinating characteristic of the clitellum, which serves as a hub for stem cells in clitellum-dependent worms, plays a crucial role in various biological processes that require further exploration. This review focuses on the overall physiological functions and uncovers the lesser-known roles of the clitellum that have been documented in various research articles. In recent times, numerous studies have been conducted using the earthworm model to explore various areas. In that regard, the clitellum's different roles in regulating and controlling stem cells, the regeneration process, regulation of organogenesis, stress response, aging, autotomy, and various features have been briefly discussed. Ultimately, we emphasized the unique and versatile role of the clitellum in the animal model, making it an ideal choice for studying development, regeneration, stem cells, organogenesis, toxicology, autotomy, and aging response.
BACKGROUND: Life cycle evolution includes ecological transitions and shifts in the timing of somatic and reproductive development (heterochrony). However, heterochronic changes can be tissue-specific, ultimately leading...BACKGROUND: Life cycle evolution includes ecological transitions and shifts in the timing of somatic and reproductive development (heterochrony). However, heterochronic changes can be tissue-specific, ultimately leading to the differential diversification of traits. Salamanders exhibit alternative life cycle polymorphisms involving either an aquatic to terrestrial metamorphosis (biphasic) or retention of aquatic larval traits into adulthood (paedomorphic). In this study, we used gene expression and histology to evaluate how life cycle evolution impacts temporal reproductive patterns in males of a polymorphic salamander. RESULTS: We found that heterochrony shifts the distribution of androgen signaling in the integument, which is correlated with significant differences in seasonal reproductive gland development and pheromone gene expression. In the testes, androgen receptor (ar) expression does not significantly vary between morphs or across seasons. We found significant differences in the onset of spermatogenesis, but by peak breeding season the testes were the same with respect to both histology and gene expression. CONCLUSION: This study provides an example of how seasonal heterochronic shifts in tissue-specific ar gene expression can disparately impact seasonal development and expression patterns across tissues, providing a potential mechanism for differential diversification of reproductive traits.