Epithelial Organization Research Articles

The people behind the papers - Mingxi Deng and Yan Yan.

Drosophila wing discs are epithelial sac-like organs and a powerful model for investigating the link between proliferation and patterning. Of particular interest is the question of how single cells in the disc integrate information regarding position and growth control, as morphogens that pattern an axis can also regulate cell division. A new Techniques and Resources article in Development reports the application of single cell sequencing technologies to dissociated discs in an effort to understand these problems. We caught up with first author Mingxi Deng and his supervisor Yan Yan, Assistant Professor at The Hong Kong University of Science and Technology (HKUST), to hear more about the story.

Effect of Tff3 Deficiency and ER Stress in the Liver.

Endoplasmic reticulum (ER) stress, a cellular condition caused by the accumulation of unfolded proteins inside the ER, has been recognized as a major pathological mechanism in a variety of conditions, including cancer, metabolic and neurodegenerative diseases. Trefoil factor family (TFFs) peptides are present in different epithelial organs, blood supply, neural tissues, as well as in the liver, and their deficiency has been linked to the ER function. Complete ablation of Tff3 expression is observed in steatosis, and as the most prominent change in the early phase of diabetes in multigenic mouse models of diabesity. To elucidate the role of Tff3 deficiency on different pathologically relevant pathways, we have developed a new congenic mouse model Tff3−/−/C57BL6/N from a mixed background strain (C57BL6/N /SV129) by using a speed congenics approach. Acute ER stress was evoked by tunicamycin treatment, and mice were sacrificed after 24 h. Afterwards the effect of Tff3 deficiency was evaluated with regard to the expression of relevant oxidative and ER stress genes, relevant proinflammatory cytokines/chemokines, and the global protein content. The most dramatic change was noticed at the level of inflammation-related genes, while markers for unfolded protein response were not significantly affected. Ultrastructural analysis confirmed that the size of lipid vacuoles was affected as well. Since the liver acts as an important metabolic and immunological organ, the influence of Tff3 deficiency and physiological function possibly reflects on the whole organism.

EpiGraph: an open-source platform to quantify epithelial organization.

SummaryHere we present EpiGraph, an image analysis tool that quantifies epithelial organization. Our method combines computational geometry and graph theory to measure the degree of order of any packed tissue. EpiGraph goes beyond the traditional polygon distribution analysis, capturing other organizational traits that improve the characterization of epithelia. EpiGraph can objectively compare the rearrangements of epithelial cells during development and homeostasis to quantify how the global ensemble is affected. Importantly, it has been implemented in the open-access platform Fiji. This makes EpiGraph very user friendly, with no programming skills required.Availability and implementationEpiGraph is available at https://imagej.net/EpiGraph and the code is accessible (https://github.com/ComplexOrganizationOfLivingMatter/Epigraph) under GPLv3 license.Supplementary information Supplementary data are available at Bioinformatics online.

573 Hair follicles employ a 2-dimensional healing strategy to repair 3-dimensional injuries

Epithelial organs vary from 2-dimensional sheets to 3-dimensional folded architectures. While wounds in 2-dimensional sheets are healed through collective movement along the basement membrane (BM), how injuries in 3-dimensional architecture are repaired remains elusive. Using post-radiation anagen hair follicle repair (AHFR), we studied this process through multiphoton intravital imaging. Before radiation, the growth of hair follicles (HFs) is primarily supported by proliferative germinative/matrix cells surrounding dermal papilla at the base. Upon radiation, these cells became apoptotic. In response to cell loss, basal outer root sheath cells/lower proximal cup cells on the cylindrical wall that were normally immotile became motile for internal structure regeneration. Interestingly, they did not take the shortest route by penetrating through the internal layers but moved along the outer cylindrical surface plane toward the injured zone. Additionally, unlike the healing on planar sheets, these cells did not move collectively, but rather independently and stochastically along the preserved BM. Through topological transformation, we found that the observed 3-dimensional regeneration process could still be mapped to 2-dimensional motion on a virtual plane, resembling the conventional 2-dimensional healing process. Mathematical modeling showed that this strategy allowed for long-range cell communication to replenish stochastic cell loss while maintaining the 3-dimensional organ architecture. Thus, this simplified healing strategy of AHFR enables timely regeneration in 3-dimensional HFs to resume the same anagen and bypasses the need for hair cycle resetting.

AMBN mutations causing hypoplastic amelogenesis imperfecta and Ambn knockout-NLS-lacZ knockin mice exhibiting failed amelogenesis and Ambn tissue-specificity.

BackgroundAmeloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel‐specific with respect to its essential functions. Biallelic AMBN defects cause non‐syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin.MethodsWe characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS‐lacZ (Ambn lacZ/lacZ) knockin mice.ResultsNo AMBN protein was detected using immunohistochemistry in null mice. ß‐galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. Ambn lacZ/lacZ 7‐week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the Ambn lacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the Ambn lacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS‐lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X‐gal.ConclusionsAmeloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

Cell death induced autophagy contributes to terminal differentiation of skin and skin appendages

ABSTRACTIn the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide evidence that macroautophagy/autophagy has a dual role in the skin. In addition to its known catabolic protective role as an evolutionary conserved upstream regulator of lysosomal degradation, we show that autophagy induced cell death (CDA) occurs in epithelial lineage-derived organs, such as the inter-follicular epidermis, the sebaceous- and the Harderian gland. By utilizing GFP-LC3 transgenic and ATG7-deficient mice, we show that CDA is initiated during terminal differentiation at a stage when the cells have become highly resistant to apoptosis. In these transitional cells, the Golgi compartment expands, which accounts for the formation of primary lysosomes, and the nucleus starts to condense. During CDA a burst of autophagosome formation is observed, first the endoplasmic reticulum (ER) is phagocytosed followed by autophagy of the nucleus. By this selective form of cell death, most of the cytoplasmic organelles are degraded, but structural proteins remain intact. In the absence of autophagy, consequently, parts of the ER, ribosomes, and chromatin remain. A burst of autophagy was stochastically observed in single cells of the epidermis and collectively in larger areas of ductal cells, arguing for a coordinated induction. We conclude that autophagy is an integral part of cell death in keratinocyte lineage cells and participates in their terminal cell fate.Abbreviations: Atg7: autophagy related 7; BECN1: beclin 1; CDA: cell death-induced autophagy; Cre: Cre-recombinase; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmatic reticulum; GFP: green fluorescent protein; HaGl: haderian gland; IVL: involucrin; KRT14: keratin 14; LD: lipid droplet; LSM: laser scanning microscope; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PN: perinuclear space; RB: residual body; rER: rough endoplasmatic reticulum; SB: sebum; SG-SC: stratum granulosum – stratum corneum; SGl: sebaceous gland; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix.

Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. Here, we demonstrate that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. We further demonstrate that integrin localization changes dynamically during the rotation process. Our data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90° rotation movement.

Perfusion culture maintained with an air-liquid interface to stimulate epithelial cell organization in renal organoids in vitro

BackgroundOrganoids derived from induced pluripotent stem (iPS) or embryonic stem (ES) cells have been evaluated as in vitro models of development and disease. However, maintaining these cells under long-term static culture conditions is difficult because of nutrition shortages and waste accumulation. To overcome these issues, perfusion culture systems are required for organoid technology. A system with a stable microenvironment, nutrient availability, and waste removal will accelerate organoid generation. The aim of this study was to develop a novel perfusion system for renal organoids by maintaining the air-liquid interface with a device fabricated using a 3D printer.ResultsOur results revealed slow flow at the organoid cultivation area based on microbead movement on the membrane, which depended on the perfusion rate under the membrane. Moreover, the perfused culture medium below the organoids via a porous membrane diffused throughout the organoids, maintaining the air-liquid interface. The diffusion rates within organoids were increased according to the flow rate of the culture medium under the membrane. The perfused culture medium also stimulated cytoskeletal and basement membrane re-organization associated with promotion tubular formation under 2.5 μL/min flow culture. In contrast, tubules in organoids were diminished at a flow rate of 10 μL/min.ConclusionsOur liquid-air interface perfusion system accelerated organization of the renal organoids. These results suggest that suitable perfusion conditions can accelerate organization of epithelial cells and tissues in renal organoids in vitro.

Yorkie controls tube length and apical barrier integrity during airway development.

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity

Tubulogenesis in epithelial organs often initiates with the acquisition of apicobasal polarity, giving rise to the formation of small lumens that expand and fuse to generate a single opened cavity. In this study, we present a micropattern-based device engineered to generate epithelial tubes through a process that recapitulates in vivo tubule morphogenesis. Interestingly, tubulogenesis in this device is dependent on microenvironmental cues such as cell confinement, extracellular matrix composition, and substrate stiffness, and our set-up specifically allows the control of these extracellular conditions. Additionally, proximal tubule cell lines growing on micropatterns express higher levels of drug transporters and are more sensitive to nephrotoxicity. These tubes display specific morphological defects that can be linked to nephrotoxicity, which would be helpful to predict potential toxicity when developing new compounds. This device, with the ability to recapitulate tube formation in vitro, has emerged as a powerful tool to study the molecular mechanisms involved in organogenesis and, by being more physiologically relevant than existing cellular models, becomes an innovative platform to conduct drug discovery assays.

Abstract 4636: Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment

Abstract The “seed and soil” hypothesis proposed by Stephen Paget (1889) highlights the importance of tissue microenvironment for secondary tumor formation. The microenvironment that promotes primary tumor origination, however, remains largely unclear. Transition zones, where two types of epithelial tissue meet, have been characterized as high-risk sites for tumorigenesis. Here we show that the Drosophila salivary gland (SG) imaginal ring (ImR) can be used as a model to study tumorigenesis in transition zones. We found that constitutive activation of Notch signaling during the third larval instar drives overproliferation and tumor formation in the SG ImR. Interestingly, tumorigenesis always occurs at the posterior end of the ImR, which is a transitional area between the polyploid giant cells and diploid ImR cells. These Notch-induced tumors display disrupted epithelial organization, grow continuously and can metastasize once transplanted into the abdomen of adult flies, suggesting that they have the characteristics of malignant neoplasms. Further analyses revealed that the SG ImR transition zone possesses endogenously high levels of Janus kinase/signal transducers and activators of transcription (JAK-STAT) and c-Jun amino-terminal kinases (JNK), both of which are necessary for tumor growth. In this region, JNK signaling induces the expression of matrix metalloproteinase 1 (MMP1), which is also required for tumor formation in this model system. Furthermore, we found that ectopic MMP1 expression can transform the anterior end of the SG ImR, which is normally refractory to oncogenic Notch-induced tumorigenesis, into a tumor hotspot. Together, these studies reveal that local endogenous activation of JNK and JAK-STAT signaling creates a niche-like tissue microenvironment that is susceptible to oncogene-induced neoplastic tumor formation. Citation Format: Wu-Min Deng, Sheng-An Yang. Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4636.

Porcine keratin 75 in developing enamel

ObjectiveTo provide in vivo biochemical evidence for the isolation, identification, and characterization of porcine keratin 75 (K75) in developing enamel. MethodsImmunolocalization of K75 was observed in mandibles from mice at postnatal days 5 and 11. K75 gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction using enamel organ epithelium (EOE) of incisors from pigs at 5 months of age. Enamel protein was extracted and isolated from both immature and mature enamel of second molars from 5-month-old pigs, and the K75 antibody-positive fraction was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). In vitro protease digestion of K75-antibody-positive fraction was carried out using porcine kallikrein 4 (pKLK4) or recombinant human enamelysin (rhMMP20) and their degradation patterns were characterized by both SDS-PAGE and western blotting. ResultsSpecific immunostaining for K75 was restricted to the layers of stratum intermedium and the enamel side of ameloblasts in mice at postnatal day 5, and to the papillary layer at postnatal day 11. Porcine K75 was expressed throughout enamel formation, but its transcript levels were significantly higher in the transition EOE than in the secretory- and maturation-stage EOE. Porcine K75 was extracted from the neutral soluble fraction from both immature and mature enamel. It was identified by LC-MS/MS analysis, and was found not to be degraded by either pKLK4 or rhMMP20. ConclusionWe propose that K75 is present in the developing enamel and undergoes different processing/degradation compared to other enamel proteins.

Vitamin D3 constrains estrogen\u2019s effects and influences mammary epithelial organization in 3D cultures

Vitamin D3 (vitD3) and its active metabolite, calcitriol (1,25-(OH)2D3), affect multiple tissue types by interacting with the vitamin D receptor (VDR). Although vitD3 deficiency has been correlated with increased incidence of breast cancer and less favorable outcomes, randomized clinical trials have yet to provide conclusive evidence on the efficacy of vitD3 in preventing or treating breast cancer. Additionally, experimental studies are needed to assess the biological plausibility of these outcomes. The mammary gland of VDR KO mice shows a florid phenotype revealing alterations of developmental processes that are largely regulated by mammotropic hormones. However, most research conducted on vitD3’s effects used 2D cell cultures and supra-physiological doses of vitD3, conditions that spare the microenvironment in which morphogenesis takes place. We investigated the role of vitD3 in mammary epithelial morphogenesis using two 3D culture models. VitD3 interfered with estrogen’s actions on T47D human breast cancer cells in 3D differently at different doses, and recapitulated what is observed in vivo. Also, vitD3 can act autonomously and affected the organization of estrogen-insensitive MCF10A cells in 3D collagen matrix by influencing collagen fiber organization. Thus, vitD3 modulates mammary tissue organization independent of its effects on cell proliferation.

Neuroendocrine neoplasms of gastrointestinal tract and secondary primary synchronous tumors: A systematic review of case reports. Casualty or causality?

BackgroundNeuroendocrine neoplasms (NENs) can arise in most of the epithelial organs of the body and are not a rare condition in the gastrointestinal tract (GIT). The presence of NENs in GIT associated with other secondary primary malignancies (SPM) has been considered an exotic event. This study aims to describe the case reports of NENs accompanied by synchronous primary tumors.Methods and findingsWe performed a systematic literature search of the databases Scopus, PubMed, Scielo and LILACS to identify case reports that described the presence of NENs in GIT with SPM. 78 case reports were included. The mean of age of the cases was 60.2 years. 60% were male. 95.4% were NENs G1. 17 cases of NENs had metastasis. 80% of SPM were recognized in the GIT (36% in stomach, 27% in large intestine, 11.2% in small intestine, and 5.6% in esophagus). The most common type of SPM was adenocarcinoma (49.4%), followed by GIST (13.5%), other NENs in different GIT segment (7.9%), lymphoma (6.8%), and squamous cell carcinoma (4.5%). The most common tumor in GIT was adenocarcinoma (97.6%) and the presence of adenocarcinoma in the same segment of GIT was found in 68.4% of the cases. Association between adenocarcinomas and NENs in GIT (p:<0.0001) and adenocarcinoma and tumor in the same segment of GIT location were observed (p<0.001).ConclusionThese results demonstrate that NENs with SPM are not a rare condition. Several theories have been proposed to explain this association; one of these is the ability of NENs to generate synchronous tumors by autocrine and paracrine effect. We observed an association between adenocarcinomas and NENs in the same segment of GIT.

VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland

The VANGL family of planar cell polarity proteins is implicated in breast cancer however its function in mammary gland biology is unknown. Here, we utilized a panel of Vang1 and Vangl2 mouse alleles to examine the requirement of VANGL family members in the murine mammary gland. We show that Vang1CKOΔ/Δ glands display normal branching while Vangl2flox/flox and Vangl2Lp/Lp tissue exhibit several phenotypes. In MMTV-Cre;Vangl2flox/flox glands, cell turnover is reduced and lumens are narrowed. A Vangl2 missense mutation in the Vangl2Lp/Lp tissue leads to mammary anlage sprouting defects and deficient outgrowth with transplantation of anlage or secondary tissue fragments. In successful Vangl2Lp/Lp outgrowths, three morphological phenotypes are observed: distended ducts, supernumerary end buds, and ectopic acini. Layer specific defects are observed with loss of Vangl2 selectively in either basal or luminal layers of mammary cysts. Loss in the basal compartment inhibits cyst formation, but has the opposite effect in the luminal compartment. Candidate gene analysis on MMTV-Cre;Vangl2flox/flox and Vangl2Lp/Lp tissue reveals a significant reduction in Bmi1 expression, with overexpression of Bmi1 rescuing defects in Vangl2 knockdown cysts. Our results demonstrate that VANGL2 is necessary for normal mammary gland development and indicate differential functional requirements in basal versus luminal mammary compartments.

Paxillin-dependent regulation of apical-basal polarity in mammary gland morphogenesis.

Establishing apical-basal epithelial cell polarity is fundamental for mammary gland duct morphogenesis during mammalian development. While the focal adhesion adapter protein paxillin is a well-characterized regulator of mesenchymal cell adhesion signaling, F-actin cytoskeleton remodeling and single cell migration, its role in epithelial tissue organization and mammary gland morphogenesis in vivo has not been investigated. Here, using a newly developed paxillin conditional knockout mouse model with targeted ablation in the mammary epithelium, in combination with ex vivo three-dimensional organoid and acini cultures, we identify new roles for paxillin in the establishment of apical-basal epithelial cell polarity and lumen formation, as well as mammary gland duct diameter and branching. Paxillin is shown to be required for the integrity and apical positioning of the Golgi network, Par complex and the Rab11/MyoVb trafficking machinery. Paxillin depletion also resulted in reduced levels of apical acetylated microtubules, and rescue experiments with the HDAC6 inhibitor tubacin highlight the central role for paxillin-dependent regulation of HDAC6 activity and associated microtubule acetylation in controlling epithelial cell apical-basal polarity and tissue branching morphogenesis.

Establishment and characterization of immortalized mouse ameloblast-like cell lines.

Enamel organ epithelium (EOE) gives rise to the major epithelial-derived cell types of tooth including the ameloblasts. The formation of enamel, termed amelogenesis, occurs through the cytodifferentiation of ameloblasts, ultimately leading to apoptosis and necrosis of these cells with eruption. Therefore, studies regarding enamel matrix formation and bioengineering have been limited. In this study, we establish and characterize two mouse immortalized ameloblast-like cell lines using human papillomavirus 16 (HPV16) E6/E7 oncogenes for the first time. Two mouse EOE dental cell lines (EOE-2M and EOE-3M). Isolated EOE primary cells were used to establish clonal cell lines and immortalized using the HPV16 E6/E7 gene platform. Two established cell lines were characterized by growth rate (Cell Proliferation Assay, MTS), gene (quantitative RT-PCR) and protein (immunocytochemistry) expression profiles, and mineralization potential (in situ alkaline phosphatase (ALP) histochemistry and Xylene Orange staining) in media supplemented with ascorbic acid and β-glycerophosphate. Gene and protein expression analyses included specific enamel matrix and ameloblast cell markers: Amel, Ambn, Enam, Amtn, ODAM, MMP20, Krt14 and DLX3. Both cell lines were maintained in excess of 30 passages, with EOE-3M cells proliferating at a slightly higher rate. The cell lines expressed all tested enamel matrix markers and produced a mineralized ECM demonstrating an ameloblast-like profile. Two mouse ameloblasts-like immortalized cell lines have been characterized that will be useful tool for studies regarding enamel bioengineering.

Dipotassium Glycyrrhizate Improves Intestinal Mucosal Healing by Modulating Extracellular Matrix Remodeling Genes and Restoring Epithelial Barrier Functions.

Gut mucosal healing (MH) is considered a key therapeutic target and prognostic parameter in the management of inflammatory bowel disease (IBD). The dipotassium glycyrrhizate (DPG), a salt of the glycoconjugated triterpene glycyrrhizin, has been shown to inhibit the High Mobility Group Box 1 (HMGB1) protein, an allarmin strongly implicated in the pathogenesis of most inflammatory and auto-immune disorders. Here we discuss new insights on how DPG acts on MH comparing the acute phase and the recovery phase from experimental colitis in mice. We found that DPG strongly accelerates MH by differently regulating pro-inflammatory (CXCL1, CXCL3, CXCL5, PTGS2, IL-1β, IL-6, CCL12, CCL7) and wound healing (COL3A1, MMP9, VTN, PLAUR, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1) genes as observed only during the recovery phase of colitis. Relevant issue is the identification of extracellular matrix (ECM) remodeling genes, VTN, and PLAUR, as crucial genes to achieve MH during DPG treatment. Furthermore, a noticeable recovery of intestinal epithelial barrier structural organization, wound repair ability, and functionality is observed in two human colorectal adenocarcinoma cell lines exposed to DPG during inflammation. Thus, our study identifies DPG as a potent tool for controlling intestinal inflammation and improving MH.

Centriolar satellites are required for efficient ciliogenesis and ciliary content regulation.

Centriolar satellites are ubiquitous in vertebrate cells. They have recently emerged as key regulators of centrosome/cilium biogenesis, and their mutations are linked to ciliopathies. However, their precise functions and mechanisms of action remain poorly understood. Here, we generated a kidney epithelial cell line (IMCD3) lacking satellites by CRISPR/Cas9‐mediated PCM1 deletion and investigated the cellular and molecular consequences of satellite loss. Cells lacking satellites still formed full‐length cilia but at significantly lower numbers, with changes in the centrosomal and cellular levels of key ciliogenesis factors. Using these cells, we identified new ciliary functions of satellites such as regulation of ciliary content, Hedgehog signaling, and epithelial cell organization in three‐dimensional cultures. However, other functions of satellites, namely proliferation, cell cycle progression, and centriole duplication, were unaffected in these cells. Quantitative transcriptomic and proteomic profiling revealed that loss of satellites affects transcription scarcely, but significantly alters the proteome. Importantly, the centrosome proteome mostly remains unaltered in the cells lacking satellites. Together, our findings identify centriolar satellites as regulators of efficient cilium assembly and function and provide insight into disease mechanisms of ciliopathies.

Cellular and Molecular Dynamics of Vertebrate Eye Morphogenesis: How Neural Crest Shapes the Embryonic Eye

Precise and stereotyped 3‐dimensional tissue structure, established during the process of morphogenesis, is essential for proper organ function. In the visual system, aberrant structural development of the embryonic eye can lead to visual impairment and blindness. Despite increasing knowledge of pathways controlling the differentiation of many cell types in the eye, we still lack a basic understanding of the mechanisms controlling its morphogenesis. Using a combination of 4‐dimensional timelapse imaging, computational approaches, and zebrafish genetics, we seek to unravel the cellular and molecular basis, including both intrinsic and extrinsic cues, for the morphogenesis of the initial embryonic eye structure, the optic cup. One particular extrinsic cell population of interest is the neural crest: migratory mesenchymal cells are essential for the development and morphogenesis of many epithelial organs. In mouse, eye defects have been described in a neural crest mutant, but the mechanisms underlying aberrant optic cup development are unknown. Using zebrafish, we asked when and how neural crest contacts the developing eye, and how optic cup defects arise.We find that at the earliest stages of optic vesicle formation, neural crest cells are in contact with the optic vesicle; by the end of optic cup morphogenesis, these neural crest derived cells encapsulate the optic stalk and optic cup, except for its distal, lens‐facing side. To test the role of neural crest, we utilize the zebrafish tfap2ats213;foxd3zdf10 double mutant: in the absence of neural crest, optic cup invagination is defective, and using 4D cell tracking, we pinpointed the cell movements disrupted by loss of neural crest.What molecule is neural crest providing to control optic cup morphogenesis? Strikingly, using electron microscopy, we directly observe loss of basement membrane assembly in the neural crest mutant. Nidogen, a laminin‐collagen crosslinking protein expressed by neural crest and essential for optic cup morphogenesis in embryonic stem cells, appears to be a crucial factor: dominant negative nidogen disrupts wild type optic cup morphogenesis without affecting neural crest migration, and overexpression of wild type nidogen partially rescues optic cup morphogenesis in neural crest mutants. There are four nidogen genes in zebrafish (nid1a, nid1b, nid2a, and nid2b), and we are now generating quadruple mutants to examine loss‐of‐function phenotypes.Our data suggest that neural crest provides crucial extracellular matrix factors to regulate optic cup morphogenesis, and highlight diverse roles mesenchyme can play in epithelial organ development and morphogenesis.Support or Funding InformationNIH/NEI R01EY025378, R01EY025780 to KMK. CDB was supported by the University of Utah T32 Developmental Biology Training Grant (NIH T32HD007491).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.