Nfatc1’s Role in Mammary Epithelial Morphogenesis and Basal Stem/progenitor Cell Self-renewal

Mammary gland is an outstanding system to study the regulatory mechanisms governing adult epithelial stem cell activity. Stem cells in the basal layer of the mammary gland fuel the morphogenesis and regeneration of a complex epithelial network during development and upon transplantation. The self-renewal of basal stem/progenitor cells is subjected to regulation by both cell-intrinsic and extrinsic mechanisms. Nfatc1 is a transcription factor that regulates breast tumorigenesis and metastasis, but its role in mammary epithelial development and stem cell function has not been investigated. Here we show that Nfatc1 is expressed in a small subset of mammary basal epithelial cells and its epithelial-specific deletion results in mild defects in side branching and basal-luminal cell balance. Moreover, Nfatc1-deficient basal cells exhibit reduced colony forming ability in vitro and somewhat compromised regenerative potential upon transplantation. Thus, our study provides evidence for a detectable yet non-essential role of Nfatc1 in mammary epithelial morphogenesis and basal stem/progenitor cell self-renewal.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish

Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

Multifaceted control of E-cadherin dynamics by the Adaptor Protein Complex 1 during epithelial morphogenesis

Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargos. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1, which localizes at the subapical Adherens Junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumour-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion.

Cooperation of membrane-translocated syntaxin4 and basement membrane for dynamic mammary epithelial morphogenesis

Mammary epithelia undergo dramatic morphogenesis after puberty. During pregnancy, luminal epithelial cells in ductal trees are arranged to form well-polarized cystic structures surrounded by a myoepithelial cell layer, an active supplier of the basement membrane (BM). Here, we identified a novel regulatory mechanism in this process by using a reconstituted BM-based three-dimensional culture and aggregates of a model cell line EpH4, which had been manipulated for inducible expression of a t-SNARE protein syntaxin4, either in an intact or signal peptide-connected form, and those genetically deficient in syntaxin4. We found that cells extruded syntaxin4 upon stimulation with the lactogenic hormone, prolactin, which in turn accelerated the turnover of E-cadherin. In response to extracellular expression of syntaxin4, cell populations that were less affected by BM actively migrated and integrated into the BM-faced cell layer. Concurrently, the BM-faced cells, which were simultaneously stimulated with syntaxin4 and BM, acquired unique epithelial characteristics to undergo dramatic cellular arrangement for cyst formation. These results highlight the importance of the concerted action of extracellular syntaxin4 extruded by the lactogenic hormone and BM components in epithelial morphogenesis.

Programmed Cell Death Not as Sledgehammer but as Chisel: Apoptosis in Normal and Abnormal Craniofacial Patterning and Development

Coordination of craniofacial development involves an complex, intricate, genetically controlled and tightly regulated spatiotemporal series of reciprocal inductive and responsive interactions among the embryonic cephalic epithelia (both endodermal and ectodermal) and the cephalic mesenchyme — particularly the cranial neural crest (CNC). The coordinated regulation of these interactions is critical both ontogenetically and evolutionarily, and the clinical importance and mechanistic sensitivity to perturbation of this developmental system is reflected by the fact that one-third of all human congenital malformations affect the head and face. Here, we focus on one element of this elaborate process, apoptotic cell death, and its role in normal and abnormal craniofacial development. We highlight four themes in the temporospatial elaboration of craniofacial apoptosis during development, namely its occurrence at (1) positions of epithelial-epithelial apposition, (2) within intra-epithelial morphogenesis, (3) during epithelial compartmentalization, and (4) with CNC metameric organization. Using the genetic perturbation of Satb2, Pbx1/2, Fgf8, and Foxg1 as exemplars, we examine the role of apoptosis in the elaboration of jaw modules, the evolution and elaboration of the lambdoidal junction, the developmental integration at the mandibular arch hinge, and the control of upper jaw identity, patterning and development. Lastly, we posit that apoptosis uniquely acts during craniofacial development to control patterning cues emanating from core organizing centres.

FAK Shutdown: Consequences on Epithelial Morphogenesis and Biomarker Expression Involving an Innovative Biomaterial for Tissue Regeneration

By employing an innovative biohybrid membrane, the present study aimed at elucidating the mechanistic role of the focal adhesion kinase (FAK) in epithelial morphogenesis in vitro over 4, 7, and 10 days. The consequences of siRNA-mediated FAK knockdown on epithelial morphogenesis were monitored by quantifying cell layers and detecting the expression of biomarkers of epithelial differentiation and homeostasis. Histologic examination of FAK-depleted samples showed a significant increase in cell layers resembling epithelial hyperplasia. Semiquantitative fluorescence imaging (SQFI) revealed tissue homeostatic disturbances by significantly increased involucrin expression over time, persistence of yes-associated protein (YAP) and an increase of keratin (K) 1 at day 4. The dysbalanced involucrin pattern was underscored by ROCK-IISer1366 activity at day 7 and 10. SQFI data were confirmed by quantitative PCR and Western blot analysis, thereby corroborating the FAK shutdown-related expression changes. The artificial FAK shutdown was also associated with a significantly higher expression of filaggrin at day 10, sustained keratinocyte proliferation, and the dysregulated expression of K19 and vimentin. These siRNA-induced consequences indicate the mechanistic role of FAK in epithelial morphogenesis by simultaneously considering prospective biomaterial-based epithelial regenerative approaches.

Evolution of mechanisms controlling epithelial morphogenesis across animals: new insights from dissociation-reaggregation experiments in the sponge Oscarella lobularis

Background The ancestral presence of epithelia in Metazoa is no longer debated. Porifera seem to be one of the best candidates to be the sister group to all other Metazoa. This makes them a key taxon to explore cell-adhesion evolution on animals. For this reason, several transcriptomic, genomic, histological, physiological and biochemical studies focused on sponge epithelia. Nevertheless, the complete and precise protein composition of cell–cell junctions and mechanisms that regulate epithelial morphogenetic processes still remain at the center of attention. Results To get insights into the early evolution of epithelial morphogenesis, we focused on morphogenic characteristics of the homoscleromorph sponge Oscarella lobularis. Homoscleromorpha are a sponge class with a typical basement membrane and adhaerens-like junctions unknown in other sponge classes. We took advantage of the dynamic context provided by cell dissociation-reaggregation experiments to explore morphogenetic processes in epithelial cells in a non-bilaterian lineage by combining fluorescent and electron microscopy observations and RNA sequencing approaches at key time-points of the dissociation and reaggregation processes. Conclusions Our results show that part of the molecular toolkit involved in the loss and restoration of epithelial features such as cell–cell and cell–matrix adhesion is conserved between Homoscleromorpha and Bilateria, suggesting their common role in the last common ancestor of animals. In addition, sponge-specific genes are differently expressed during the dissociation and reaggregation processes, calling for future functional characterization of these genes.

Germline soma communication mediated by gap junction proteins regulates epithelial morphogenesis

Gap junction (GJ) proteins, the primary constituents of GJ channels, are conserved determinants of patterning. Canonically, a GJ channel, made up of two hemi-channels contributed by the neighboring cells, facilitates transport of metabolites/ions. Here we demonstrate the involvement of GJ proteins during cuboidal to squamous epithelial transition displayed by the anterior follicle cells (AFCs) from Drosophila ovaries. Somatically derived AFCs stretch and flatten when the adjacent germline cells start increasing in size. GJ proteins, Innexin2 (Inx2) and Innexin4 (Inx4), functioning in the AFCs and germline respectively, promote the shape transformation by modulating calcium levels in the AFCs. Our observations suggest that alterations in calcium flux potentiate STAT activity to influence actomyosin-based cytoskeleton, possibly resulting in disassembly of adherens junctions. Our data have uncovered sequential molecular events underlying the cuboidal to squamous shape transition and offer unique insight into how GJ proteins expressed in the neighboring cells contribute to morphogenetic processes.