bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stereocilin Paralogs Have Distinct Roles in Zebrafish Inner Ear Development
Authors: Kramer Kenneth L , Inserra Christopher J 
Published: November 2023

In the vertebrate inner ear, sound and movement are transduced into electrical signals by specialized bundles of cilia on the apical surface of sensory hair cells. Each hair bundle is composed of a single Tubulin‐filled kinocilium attached to multiple rows of Actin‐packed stereocilia. Cilia are linked to each other and to an overlying membrane by a complex of proteins that includes Stereocilin (STRC), a putative GPI‐linked protein that is expressed in both stereocilia and kinocilia in mouse auditory and vestibular hair cells. Since loss of STRC in mouse results in defects in the attachments both between neighboring cilia as well as between cilia and the overlying tectorial membrane, we sought to determine if STRC is similarly required in zebrafish inner ear development. We generated antibodies that specifically distinguish the two zebrafish STRC paralogs and found that STRCa and STRCb are both expressed during hair cell formation but with slightly different expression patterns. STRCb is found on the distal tip of kinocilia when the hair cells first start tethering to the seeding otoliths, while STRCa is found a few hours later in a few punctate locations along the distal half of the kinocilium. Knockdown of strcb by injecting antisense morpholinos or eliminating strcb in CRISPR/Cas9‐generated mutant embryos blocked the ability of kinocilia to tether otoliths to hair cells. In contrast, otolith attachment appeared to be normal in strca morphants and mutant embryos. Kinocilia in strcb morphant and mutant embryos were normal in length, yet proteins in the unattached otolith were disorganized compared to otoliths in normal and strca‐deficient zebrafish. We conclude that STRCb is uniquely required for zebrafish otolith attachment and organization.Support or Funding InformationThe work was supported by grants from the National Institutes of Health (8P20GM103471) and the Creighton Health Science Strategic Investment Fund.
Cell‐Specific Relations between Vasopressin and Renin‐Angiotensin‐Aldosterone Systems
Authors: Giesecke Yvonne , Giesecke Torsten , Kawahara Katsumasa , Koshimizu Taka Aki , Gimber Niclas , Schmoranzer Jan , Bachmann Sebastian , Mutig Kerim 
Published: November 2023

Vasopressin (AVP) is crucial for body water and electrolyte homeostasis. Its antidiuretic effects are mediated chiefly via renal V2 vasopressin receptors (V2R). Parallel activation of V1a and V1b receptors (V1aR and V1bR) may modulate the renin‐angiotensin‐aldosterone system (RAAS). Despite a large body of functional evidence for a crosstalk between AVP and RAAS, little is known about distribution of AVP receptors in RAAS‐related signalling. In this study we have comparatively analyzed V1aR, V1bR, and V2R distribution in the kidney, adrenal glands, and brain with focus on their potential role in modulation of RAAS.Custom antibodies to individual AVP receptors were generated and characterized using respective mouse knockout tissues. Distribution of the receptors was studied by immunofluorescence, immunocytochemistry and super resolution imaging using rodent and human tissues.The anti‐V1aR antibody produced signal in macula densa cells of the kidney, all three cortical zones of the adrenal glands, and suprachiasmatic and supraoptic nuclei of the hypothalamus. V1bR was detected in the mark of adrenal gland and hypothalamic nuclei but not in the kidney. V2R signal was observed along the distal nephron and principal cells of collecting duct cells of the kidney, but not in the adrenal glands or hypothalamus.In summary, our results document presence of V1aR and V1bR in hypothalamic nuclei and adrenal cortex which suggest their involvement in the release of adrenocorticotropic hormone and aldosterone. In addition, expression of V1aR in macula densa cells may affect renin release in a paracrine way.
Primary Sertoli Cell Cultures from 30‐Day Old Rats Form Cords with a Morphologically Differentiated Phenotype
Authors: Sriram Aarati , Huynh Hong Doan Nghi , Shadarevian John , Djaksigulova Diana , Vogl A. Wayne 
Published: November 2023

The seminiferous epithelium lines the seminiferous tubules of the mammalian testes and is comprised of Sertoli cells and germ cells. Sertoli cells are tall columnar cells that extend from the base of the seminiferous epithelium to the tubule lumen. They play a key role in spermatogenesis by providing support and nourishment for the developing spermatogenic cells. We have been working to establish a primary culture system for studying junction turnover in morphologically differentiated Sertoli cells. We have found that when the age of the rats used for isolating Sertoli cells are older than 20 days, the cells respond much differently to the same culture conditions than cells from younger animals. The purpose of this study is to characterize the differences in growth and morphology between these two populations of cells. Sertoli cells were enzymatically dissociated from the testes of 20‐day or older rats and were plated at a density of 1.3×106 cells /0.2 mL on Transwell culture inserts coated with undiluted Matrigel. Sertoli cells were cultured in serum‐free defined medium (SFDM) containing hormones (FSH and testosterone), vitamins and antibiotics and incubated at 34°C in 5% CO2 for either 9 days or 2 weeks. The cultured cells were treated with a brief hypotonic wash after two days to lyse any contaminating germ cells. Finally, the cells were fixed and processed for immunofluorescence microscopy or transmission electron microscopy. Sertoli cells isolated from 20‐day old rats formed a confluent layer within 24–48 hours with cells having a cuboidal to columnar shape. Cells formed a monolayer interrupted by areas where they formed focal clumps. The cells generally were polarized with nuclei and junction networks basally located. Sertoli cells isolated from 25–30 day old rats rapidly reorganized to form a condensed mass from which cylindrical cords of cells projected into the surrounding remodeled Matrigel. Sertoli cells in the cords were columnar and their irregularly shaped nuclei were located basally around the periphery of the cords. Intercellular junctions appeared better formed at the base of Sertoli cells than in cultures from 20‐day old animals. Claudin‐11 staining confirmed the presence of numerous tight junctions within ectoplasmic specializations. All the primary cultures were predominantly comprised of Sertoli cells with few contaminating germ and myoid cells. This study presents an improved primary Sertoli cell culture system that more closely resembles the in vivo phenotype and may be a more effective model for studying junction turnover.Support or Funding InformationSupported by a NSERC Discovery Grant (#155397‐2013) to AWV.
Expansion of Hepatic Progenitor Cell Population in Liver Fibrosis of Elderly Cadavers
Authors: Chiu Sophia , Mak Ki M. 
Published: November 2023

Human hepatic progenitor cells (HPC) normally reside in a quiescent state in the canals of Hering located at the interface between the portal tract and the periportal parenchyma of the liver lobule. In chronic liver disease such as alcoholic liver disease, nonalcoholic fatty liver disease, viral hepatitis, chronic cholestasis, hereditary liver disorders, and neoplasia, HPC become activated and proliferate. Being bipotential progenitors, HPC can differentiate into cholangiocytes or hepatocytes. Expansion of HPC population in the periportal parenchyma, which constitutes the HPC compartment, is termed ductular reaction or HPC response. Preliminary studies revealed ductular reaction associated with liver fibrosis of elderly cadavers with diverse causes of death (FASEB 2016; 30:1044.4; 1044.5). The present study assessed fibrosis‐associated HPC expansion in progressive stages of fibrosis of the aged liver. Paraffin liver sections of embalmed cadavers were immunoperoxidase stained using monoclonal cytokeratin 7 (CK7) antibody. Sections were post‐stained with Sirius red stain for assessment of fibrosis. HPC are small oval‐shaped cells containing an ovoid nucleus and scant cytoplasm that stained positively for CK7, a phenotypic marker of HPC. In livers with minimal fibrotic changes, CK7 + HPC lined the canals of Hering, formed reactive ductules in the form of anastomotic strings of cuboidal cells in the periportal HPC compartment, and presented as isolated cells in the liver parenchyma at variable distance from the portal tract. In septal fibrosis, in addition to the abovementioned lobular distribution, CK7 + HPC were observed at the septal‐parenchymal border of developing septa and bridging septa, and around perisinusoidal fibrotic lesions. Counts of HPC from 11 liver samples in each stage of fibrosis showed that the number of cells per HPC compartment increased from 7.93 ± 1.32 in minimum fibrosis to 17.18 ± 2.96 (P = 0.01) in septal fibrosis, and 21.83 ± 3.69 (P = 0.002) in bridging fibrosis. In the mid‐pericentral lobular areas, HPC counts increased from 2.72 ± 0.96 cells/field in minimum fibrosis to 6.92 ± 1.39 cells/field (P = 0.02) in septal fibrosis, and 11.15 ± 1.99 cells/field (P = 0.001) in bridging fibrosis.ConclusionsExpansion of HPC population in the periportal HPC compartment in advanced stages of fibrosis represents a HPC response to liver fibrogenesis. Increased intralobular distribution of HPC likely resulted from periportal to lobular parenchyma migration, possibly relating to hepatic regeneration consequent to liver injury caused by fibrosis. Elderly cadavers provide a valuable source of liver specimens for assessing HPC expansion associated with liver fibrosis progression in the aged liver.
Cell Migration and Palatal Fusion
Authors: Logan Shaun , Benson Douglas 
Published: November 2023

Cleft palate, one of the most common birth defects, occurs as a result of the developing secondary palatal shelves' failure to grow to midline, degrade the midline epithelial seam (MES), and fuse to leave a confluent shelf. The mechanism of MES degradation is largely unknown, but the role of ephrin signaling in palatal fusion suggests an induction of epithelial to mesenchymal transition (EMT). Our goal is to determine the role of ephrin signaling in MES degradation. We dissected palatal shelves from embryonic day 14.5 Ephrin‐B2/GFP mice, in which a nuclear‐localized green fluorescent protein (GFP) under control of the ephrin‐B2 (EB2) gene labels 100% of medial edge epithelia (MEE). The adhered shelves were placed oral‐side down in a glass‐bottomed dish, encased in low‐melt agarose, and submerged in culture medium. Palates were imaged same day for a total of 48 hours, using a Zeiss 880 laser scanning confocal with a humidified chamber. Displacement of Ephrin‐B2/GFP labeled cells was tracked using Imaris Software. After imaging, palates were sectioned and stained with hematoxylin and eosin. Fluorescently labeled MEE cells moved as an aggregate/sheet and not individually. Histology confirmed that the imaged palates fused. Cell expression of EB2/GFP was visible throughout the 48‐hour experiment. The data indicate that MES degradation involves MEE cell migration and suggest that elements of the EMT program are a part of palatal fusion. Continued expression of EB2/GFP throughout fusion suggests that MEE cells do not undergo apoptosis prior to seam degradation.Support or Funding InformationNIDCR R01DE022804
In Vivo Inhibition of microRNA (miR) Using a Novel miR Inhibitor System Reveals a Role for miR‐17‐92 in Palate Elevation and/or Extension
Authors: Ries Ryan J. , Yu Wenjie , Eliason Steven L , Cao Huojun , Amendt Brad A. 
Published: November 2023

MicroRNAs (miRs) are short non‐coding RNA molecules approximately 22 nucleotides long. miRs bind to complementary targets on the 3′ untranslated region (UTR) of messenger RNAs (mRNAs), attenuating mRNA translation via either mRNA strand degradation or sequestration. Given their redundancy throughout the genome, conventional knockout strategies are difficult to employ when studying miRs. To circumvent this difficulty, we have developed a novel method of miR inhibition, the Plasmid‐Based miRNA Inhibition System (PMIS), to allow for the simultaneous knockdown of homologous miRs. Short hairpin structures that flank the anti‐sense miR target site on the PMIS inhibitor complex (PMIS‐IC) dramatically increase miR inhibition by associating with RISC complex factors. The PMIS‐IC is composed of native, unmodified nucleic acids and can be stably integrated into the genome. In this study, we employ this strategy to analyze the effects of inhibition of mature miRs in the miR‐17‐92 cluster. Here we show how the PMIS system can be used to inhibit miRs from the miR‐17‐92 cluster and associated family members in stable cell lines and transgenic mice. We also show how inhibition of the miR‐17‐92 cluster can lead to differences in the point of palate formation arrest in mice carrying PMIS‐ICs for miR‐17‐92. These results demonstrate the effectiveness of this novel miR inhibition strategy and indicate new roles for miR‐17‐92 in palatogenesis.Support or Funding InformationUniversity of Iowa Carver College of MedicineUniversity of Iowa College of DentistryUniversity of Iowa Research FoundationNational Institutes of Health (National Institute for Dental and Craniofacial Research)
Gene expression of angiogenesis markers in mouse mandibular condyle during development
Authors: Sato Iwao , Maeda Yuuki , Miwa Yoko , Sunohara Masataka 
Published: November 2023

Angiogenesis and osteogenesis markers are an important factor during development in relation between mandibular condyle and Meckel's cartilage. However, it has many functions in addition to the formation of mandible. We need to clearly the relationship between mandibular condyle and Meckel's cartilage to angiogenesis and osteogenesis markers during formation in mouse fetal and early postnatal stages. Therefore we try to investigate the correlation between these markers during the development of the mouse mandible. We analyzed the expression and localization of VEGF by in‐situ hybridization in the developing mouse mandibular condyle from embryonic days 12.5 to days 5. We also detected the mRNA abundance of mandible with various markers from embryonic and early postnatal stages by real‐time RT‐PCR. Our results was performed for mouse mandible in different postnatal stages and after birth and the expression of the following some mRNA markers (CD31, LYVE‐1, VEGF, collagen I, II, osteopontin, and osteocalcin). The antisense probe for VEGF was detected in mesenchymal cells surrounding around MC and bone matrices area from E17.5, and clearly localization was seen at E18.5 in the embryonic mouse mandible by in‐situ hybridization. These data suggested that angiogenesis markers were effected by development stages after or before birth in morphogenesis of mandible. VEGF also affects the other morphological markers in these specific stages terminating the differentiation in mouse mandibular condyle development.Support or Funding InformationNone
A Sox2‐Lef‐1 Protein Interaction Inhibits Lef‐1 Transcriptional Activity and WNT Signaling During Odontogenesis
Authors: Sweat Mason E , Yu Wenjie , Eliason Steve , Amendt Brad A 
Published: November 2023

Tooth development proceeds through a series of steps wherein epithelial and mesenchymal tissue layers cooperate to form a tooth bud, and cells in the tooth bud are organized and differentiated into a mature tooth. Throughout this process, the changes in gene expression that are required for tooth formation are prompted by transcription factors. Lef‐1 is a transcription factor and a major effector of WNT signaling, and the loss of Lef‐1 expression or an ectopic increase in Lef‐1 results in dramatic dental anomalies in mice. The transcriptional mechanisms responsible for maintaining appropriate levels of Lef‐1 expression and downstream targets are not well understood. We show that Sox2, a major regulator of dental stem cells inhibits Lef‐1 transcriptional activation. Endogenous Sox2 directly binds to Lef‐1 and we have identified specific motifs in the Sox2 and Lef‐1 proteins regulating this interaction. By immobilizing different GST‐labeled Sox2 domains we show that the HMG domain of Sox2 is required for the Sox2‐Lef1 protein interaction. Functionally, Sox2 is capable of disrupting the association of Lef‐1 protein with its consensus DNA binding sequence. Taken together, these data suggest a novel mechanism wherein the Sox2 protein functions to negatively regulate the expression of Lef‐1 and Lef‐1 target genes during odontogenisis. These new mechanisms may also regulate other epithelial organs during development.Support or Funding InformationIowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA.
Do Secretory‐Stage Ameloblasts Divide? Experimental Evidence
Authors: Hogg Russell , Smith Timothy , Ward Nikolai , Gografe Sylvia 
Published: November 2023

Our objective is to test the dictum that secretory ameloblasts cannot divide. This idea is often mentioned in dental biology textbooks without citation or evidence; its source seems to be studies of rodent incisors from the 1960's. However, rodent incisors are ever‐growing, and not acceptable models for enamel epithelia function in other mammals. Also, there are two reasons to expect that mature ameloblasts divide. First, recent studies show that tensile forces induce mitosis in epithelia, and it is likely that the enamel epithelium undergoes tension as enamel is deposited and the surface area of the growing crown expands. Second, we now know that ameloblasts are subject to the same biorhythms that synchronize proliferation of osteoblasts. If ameloblast division is similarly coordinated by the nervous system and occurs at night, it is unlikely that it will have been observed since most experimental protocols are carried out during daylight hours.To test the hypothesis, we employed two methods. First, 3–6 day old outbred ICR mouse pups (Crl:CD1) used for another research purpose were euthanized at 4 hour intervals that spanned the entire day/night cycle. Specimens euthanized during the day were utilized as controls, while those specimens euthanized at night were considered as experimental tissues for the purpose of the study. Whole heads were thin‐sectioned in a coronal plane and stained using standard H&E protocols to identify mitotic figures in secretory‐stage ameloblast cells. Second, we labeled juvenile primate specimens for immunoreactivity to Proliferating Cell Nuclear Antigen (PCNA), which is expressed in cells that are undergoing DNA repair and/or replication in preparation for mitosis. Animal protocols were approved by respective Institutional Animal Care and Use Committees (IACUC) at Slippery Rock University and Florida Atlantic University.In our mouse specimens, we have not found signs of mitotic figures; however in one juvenile collared lemur we did see signs of strong PCNA expression in cervical loop ameloblasts that had just entered the secretory stage. This weakly supports our hypothesis, and warrants searches for other patterns of gene expression and mitosis.
Distribution of Neural/Stem Progenitor Cells in the Brain of the Leopard Gecko (Eublepharis macularius)
Authors: McDonald Rebecca Paula , Bradley Stefanie Simone , Vickaryous Matthew Kenneth 
Published: November 2023

Endogenous neural stem/progenitor cells (NSPCs) have been identified across vertebrate species. Among adult mammals, NSPCs are spatially restricted to just two neurogenic regions: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In other species – including teleost fish and urodeles – NSPCs are known to have a much broader distribution. For reptiles, less is known. Here we identify NSPCs along the rostral caudal axis of the brain of the leopard gecko (Eublepharis macularius; hereafter, ‘gecko’). The ventricular system of the gecko brain is bordered by a distinct neurogenic niche, the ventricular zone (VZ). All VZ cells (including those of the lateral, third and fourth ventricles) constitutively express the hallmark transcription factor of stemness, SOX 2. Additionally, VZ cells have a radial‐like morphology and pattern of protein expression (including immunostaining for the intermediate filaments Vimentin and glial fibrillary acid protein [GFAP]) closely comparable to mammalian embryonic radial glial cells. At the level of the cerebellum, putative NSPCs (SOX2+ and GFAP+) were also identified independent of the ventricular system. Proliferating cells (identified by phosphorylated histone H3 expression) are most abundant in the VZ of the lateral ventricle and cerebellar parenchyma. The VZ of the third ventricle demonstrates a comparatively modest level of proliferation, while the VZ of the fourth ventricle is largely quiescent. The presence of NSPCs, both dormant and constitutively active, across the lizard brain offers compelling evidence for its neurogenic and possibly regenerative capacity.Support or Funding InformationNatural Sciences and Engineering Research Council Discovery Grant 400358
NEURON‐SPECIFIC TRANSGENE EXPRESSION OF IMP2 MEDIATED BY SYNAPSIN PROMOTER‐DRIVEN AAV9
Authors: Park Danielle , Blizard Sarah , Austin Scarlett , Harman Joshua , Haraszti Samantha , Xu Mei 
Published: November 2023

Insulin‐like growth factor‐II (IGF‐II) mRNA‐binding protein‐2 (IMP2) is one of the three homologues (IMP1‐3) that play an important role in the post‐transcriptional regulation of gene expression during development in several tissues including the nervous system. An alternative splice product of IMP2 lacking exon 10 aberrantly expresses in human hepatocellular carcinoma and is identified as HCC. Several lines of studies have demonstrated that IMP1/ZBP1 (zipcode binding protein) is critical in axon guidance and regeneration by regulating localization and translation of specific mRNAs. However, a role of IMP2 in the neural tissue is largely unknown. Previously, we used a custom made IMP2‐specific antibody to locate the endogenous IMP2 expression in mouse neurons and glial cells at different developmental stages. Our pilot study shows that IMP2 expression is sustained throughout life and it may play a role to facilitate axon regeneration. In this study, we used the synapsin promoter‐driven adeno‐associated viral (AAV) 9 constructs to express YFP‐IMP2 and –HCC specifically in neurons both in vitro and in vivo. We applied AAV9.hSyn.YFP.PolyA, AAV9.hSyn.YFP‐hIMP2.PolyA and AAV9.hSyn.YFP‐hHCC.PolyA in the primary dorsal root ganglion mouse neuron culture as an in vitro model. We also injected the viral vectors into the crushed mouse sciatic nerve as an in vivo model. Our results demonstrate the moderate neuron‐specific transgene expression of IMP2/HCC in both models. The data indicate that the synapsin promoter‐driven AAV9 can be used as an important tool for further study of the role of IMP2 in the nervous system.Support or Funding InformationThis work is supported by the Department of Bio‐Medical Sciences, PCOM and Health Research Grant, Pennsylvania Department of Health.