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Kidney development is initiated by the outgrowth of an epithelial ureteric bud into a population of mesenchymal cells. Reciprocal morphogenetic responses between these two populations generate a highly branched epithelial ureteric tree with the mesenchyme differentiating into nephrons, the functional units of the kidney. While we understand some of the mechanisms involved, current knowledge fails to explain the variability of organ sizes and nephron endowment in mice and humans. Here we present a spatially-averaged mathematical model of kidney morphogenesis in which the growth of the two key populations is described by a system of time-dependant ordinary differential equations. We assume that branching is symmetric and is invoked when the number of epithelial cells per tip reaches a threshold value. This process continues until the number of mesenchymal cells falls below a critical value that triggers cessation of branching. The mathematical model and its predictions are validated against experimentally quantified C57Bl6 mouse embryonic kidneys. Numerical simulations are performed to determine how the final number of branches changes as key system parameters are varied (such as the growth rate of tip cells, mesenchyme cells, or component cell population exit rate). Our results predict that the developing kidney responds differently to loss of cap and tip cells. They also indicate that the final number of kidney branches is less sensitive to changes in the growth rate of the ureteric tip cells than to changes in the growth rate of the mesenchymal cells. By inference, increasing the growth rate of mesenchymal cells should maximise branch number. Our model also provides a framework for predicting the branching outcome when ureteric tip or mesenchyme cells change behaviour in response to different genetic or environmental developmental stresses. [ABSTRACT FROM AUTHOR]

Many mammalian organs undergo branching morphogenesis to create highly arborized structures with maximized surface area for specialized organ function. Cooperative cell–cell and cell–matrix adhesions that sculpt the emerging tissue architecture are guided by dynamic basement membranes. Properties of the basement membrane are reciprocally controlled by the interacting epithelial and mesenchymal cell populations. Here we discuss how basement membrane remodeling is required for branching morphogenesis to regulate cell–matrix and cell–cell adhesions that are required for cell patterning during morphogenesis and how basement membrane impacts morphogenesis by stimulation of cell patterning, force generation, and mechanotransduction. We suggest that in addition to creating mature epithelial architecture, remodeling of the epithelial basement membrane during branching morphogenesis is also essential to promote maturation of the stromal mesenchyme to create mature organ structure. Recapitulation of developmental cell–matrix and cell–cell interactions are of critical importance in tissue engineering and regeneration strategies that seek to restore organ function. [ABSTRACT FROM AUTHOR]

Mechanical properties of the microenvironment regulate cell morphology and differentiation within complex organs. However, methods to restore morphogenesis and differentiation in organs in which compliance is suboptimal are poorly understood. We used mechanosensitive mouse salivary gland organ explants grown at different compliance levels together with deoxycholate extraction and immunocytochemistry of the intact, assembled matrices to examine the compliance-dependent assembly and distribution of the extracellular matrix and basement membrane in explants grown at permissive or non-permissive compliance. Extracellular matrix and basement membrane assembly were disrupted in the glands grown at low compliance compared to those grown at high compliance, correlating with defective morphogenesis and decreased myoepithelial cell differentiation. Extracellular matrix and basement membrane assembly as well as myoepithelial differentiation were restored by addition of TGFβ1 and by mechanical rescue, and mechanical rescue was prevented by inhibition of TGFβ signaling during the rescue. We detected a basal accumulation of active integrin β1 in the differentiating myoepithelial cells that formed a continuous peripheral localization around the proacini and in clefts within active sites of morphogenesis in explants that were grown at high compliance. The pattern and levels of integrin β1 activation together with myoepithelial differentiation were interrupted in explants grown at low compliance but were restored upon mechanical rescue or with application of exogenous TGFβ1. These data suggest that therapeutic application of TGFβ1 to tissues disrupted by mechanical signaling should be examined as a method to promote organ remodeling and regeneration. [ABSTRACT FROM AUTHOR]

Branching morphogenesis depends on the precise temporal and spatial control of epithelial dynamics. In the vertebrate head, endodermal branches, called pharyngeal pouches, form through the transient stratification, collective migration and reorganization of epithelial cells into bilayers. Here, we report novel requirements for the EphrinB ligands B2a and B3b, the Ephb4a receptor and the Pak2a kinase in the development of pouches and the posterior facial skeleton that depends on pouches for its segmentation. Time-lapse imaging in zebrafish shows that EphB-Pak2a signaling is required to stabilize pouch epithelial cells at the end of branching morphogenesis. Transgenic rescue experiments further demonstrate that endodermal Eph-ephrin signaling promotes pouch integrity by targeting Pak2a to the plasma membrane, where subsequent activation by Wnt4a-Cdc42 signaling increases junctional E-cadherin in maturing pouches. Integration of Eph-ephrin and Wnt4a signaling through Pak2a thus signals the end of branching morphogenesis by increasing intercellular adhesion that blocks further epithelial rearrangements. [ABSTRACT FROM AUTHOR]

Histone deacetylases (HDACs) regulate a broad range of biological processes through removal of acetyl groups from histones as well as non-histone proteins. Our previous studies showed that Hdac1 and Hdac2 are bound to promoters of key renal developmental regulators and that HDAC activity is required for embryonic kidney gene expression. However, the existence of many HDAC isoforms in embryonic kidneys raises questions concerning the possible specificity or redundancy of their functions. We report here that targeted deletion of both the Hdac1 and Hdac2 genes from the ureteric bud (UB) cell lineage of mice causes bilateral renal hypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient to sustain normal renal development. In addition to defective cell proliferation and survival, genome-wide transcriptional profiling revealed that the canonical Wnt signaling pathway is specifically impaired in UBHdac1,2-/- kidneys. Our results also demonstrate that loss of Hdac1 and Hdac2 in the UB epithelium leads to marked hyperacetylation of the tumor suppressor protein p53 on lysine 370, 379 and 383; these post-translational modifications are known to boost p53 stability and transcriptional activity. Genetic deletion of p53 partially rescues the development of UBHdac1,2-/- kidneys. Together, these data indicate that Hdac1 and Hdac2 are crucial for kidney development. They perform redundant, yet essential, cell lineage-autonomous functions via p53-dependent and -independent pathways. [ABSTRACT FROM AUTHOR]

Vertebrate airway has a tree-like-branched structure. This structure is generated by repeated tip splitting, which is called branching morphogenesis. Although this phenomenon is extensively studied in developmental biology, the mechanism of the pattern formation is not well understood. Conversely, there are many tree-like structures in purely physical or chemical systems, and their pattern formation mechanisms are well-understood using mathematical models. Recent studies correlate these biological observations and mathematical models to understand lung branching morphogenesis. These models use slightly different mechanisms. In this article, we will review recent progress in modelling lung branching morphogenesis, and future directions to experimentally verify the models. [ABSTRACT FROM PUBLISHER]

The intracellular protein p120 catenin aids in maintenance of cell–cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120 f/f pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development. [ABSTRACT FROM AUTHOR]

This report presents a novel mechanism for remodelling a branched epithelial tree. The mouse renal collecting duct develops by growth and repeated branching of an initially unbranched ureteric bud: this mechanism initially produces an almost fractal form with young branches connected to the centre of the kidney via a sequence of nodes (branch points) distributed widely throughout the developing organ. The collecting ducts of a mature kidney have a different form: from the nephrons in the renal cortex, long, straight lengths of collecting duct run almost parallel to one another through the renal medulla, and open together to the renal pelvis. Here we present time-lapse studies of E11.5 kidneys growing in culture: after about 5 days, the collecting duct trees show evidence of 'node retraction', in which the node of a 'Y'-shaped branch moves downwards, shortening the stalk of the 'Y', lengthening its arms and narrowing their divergence angle so that the 'Y' becomes a 'V'. Computer simulation suggests that node retraction can transform a spread tree, like that of an early kidney, into one with long, almost-parallel medullary rays similar to those seen in a mature real kidney. [ABSTRACT FROM AUTHOR]

Integrin-dependent interactions between cells and extracellular matrix regulate lung development; however, specific roles for β1-containing integrins in individual cell types, including epithelial cells, remain incompletely understood. In this study, the functional importance of β1 integrin in lung epithelium during mouse lung development was investigated by deleting the integrin from E10.5 onwards using surfactant protein C promoter-driven Cre. Thesemutant mice appeared normal at birth but failed to gain weight appropriately and died by 4 months of age with severe hypoxemia. Defects in airway branching morphogenesis in association with impaired epithelial cell adhesion and migration, aswell asalveolarizationdefectsandpersistentmacrophagemediated inflammation were identified. Using an inducible system to delete β1 integrin after completion of airway branching, we showed that alveolarization defects, characterizedby disrupted secondary septation, abnormal alveolar epithelial cell differentiation, excessive collagen I and elastin deposition, and hypercellularity of the mesenchyme occurred independently of airway branching defects. By depleting macrophages using liposomal clodronate, we found that alveolarization defects were secondary to persistent alveolar inflammation. β1 integrin-deficient alveolar epithelial cells produced excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting a direct role for β1 integrin in regulating alveolar homeostasis. Taken together, these studies define distinct functions of epithelial β1 integrin during both early and late lung development that affect airway branchingmorphogenesis, epithelial cell differentiation, alveolar septation and regulation of alveolar homeostasis. [ABSTRACT FROM AUTHOR]

Early branching events during lung development are stereotyped. Although key regulatory components have been defined, the branching mechanism remains elusive. We have now used a developmental series of 3D geometric datasets of mouse embryonic lungs as well as time-lapse movies of cultured lungs to obtain physiological geometries and displacement fields.We find that only a ligand-receptor-based Turing model in combination with a particular geometry effect that arises from the distinct expression domains of ligands and receptors successfully predicts the embryonic areas of outgrowth and supports robust branch outgrowth. The geometry effect alone does not support bifurcating outgrowth, while the Turing mechanism alone is not robust to noisy initial conditions. The negative feedback between the individual Turing modules formed by fibroblast growth factor 10 (FGF10) and sonic hedgehog (SHH) enlarges the parameter space for which the embryonic growth field is reproduced.We therefore propose that a signaling mechanism based on FGF10 and SHH directs outgrowth of the lung bud via a ligand-receptor-based Turing mechanism and a geometry effect. [ABSTRACT FROM AUTHOR]

The human kidney is composed of an arborized network of collecting ducts, calyces and urinary pelvis that facilitate urine excretion and regulate urine composition. The renal collecting system is formed in utero, completed by the 34th week of gestation in humans, and dictates final nephron complement. The renal collecting system arises from the ureteric bud, a derivative of the intermediate-mesoderm derived nephric duct that responds to inductive signals from adjacent tissues via a process termed ureteric induction. The ureteric bud subsequently undergoes a series of iterative branching and remodeling events in a process called renal branching morphogenesis. Altered signaling that disrupts patterning of the nephric duct, ureteric induction, or renal branching morphogenesis leads to varied malformations of the renal collecting system collectively known as congenital anomalies of the kidney and urinary tract (CAKUT) and is the most frequently detected congenital renal aberration in infants. Here, we describe critical morphogenetic and cellular events that govern nephric duct specification, ureteric bud induction, renal branching morphogenesis, and cessation of renal branching morphogenesis. We also highlight salient molecular signaling pathways that govern these processes, and the investigative techniques used to interrogate them. [ABSTRACT FROM AUTHOR]

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling. [ABSTRACT FROM AUTHOR]

Most epithelial tissues rapidly become complex during embryonic development while being surrounded by the basement membrane (BM). Thus, the BM shape is thought to change dramatically as the epithelium grows, but the underlying mechanism is not yet clear. Nidogen-1 is ubiquitous in the BM and binds to various other BM components, including laminin and type IV collagen. To elucidate the behavior of the BM during epithelial morphogenesis, we attempted to live-label the developing BM with recombinant human nidogen-1 fused to an enhanced green fluorescent protein (hNid1-EGFP). Submandibular glands of mouse embryos were cultured in glass-bottomed dishes and incubated in media containing hNid1-EGFP. Subsequent confocal microscopy clearly visualized the BMs surrounding the epithelial end buds. On three-dimensional reconstruction from Z-series confocal sections, the epithelial BM was observed as a thin sheet that expanded continuously around the entire epithelial basal surface. Because the explants continued to grow well in the presence of hNid1-EGFP, time-lapse confocal microscopy was performed to follow the dynamics of the BM. We found that the epithelial BM is an adaptive structure that deforms in accordance with the rapid shape changes of the developing epithelium. Furthermore, hNid1-EGFP was found to be incorporated differently into the epithelial BM compared with that reported for fibronectin or type IV collagen, suggesting that individual BM components assemble in different ways to form the BM., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many epithelial tissues expand rapidly during embryonic development while remaining surrounded by a basement membrane. Remodeling of the basement membrane is assumed to occur during branching morphogenesis to accommodate epithelial growth, but how such remodeling occurs is not yet clear. We report that the basement membrane is highly dynamic during branching of the salivary gland, exhibiting both local and global remodeling. At the tip of the epithelial end bud, the basement membrane becomes perforated by hundreds of well-defined microscopic holes at regions of rapid expansion. Locally, this results in a distensible, mesh-like basement membrane for controlled epithelial expansion while maintaining tissue integrity. Globally, the basement membrane translocates rearward as a whole, accumulating around the forming secondary ducts, helping to stabilize them during branching. Both local and global dynamics of the basement membrane require protease and myosin II activity. Our findings suggest that the basement membrane is rendered distensible by proteolytic degradation to allow it to be moved and remodeled by cells through actomyosin contractility to support branching morphogenesis. [ABSTRACT FROM AUTHOR]

Background Glandular organs require the development of a correctly patterned epithelial tree. These arise by iterative branching: early branches have a stereotyped anatomy, while subsequent branching is more flexible, branches spacing out to avoid entanglement. Previous studies have suggested different genetic programs are responsible for these two classes of branches. Results Here, working with the urinary collecting duct tree of mouse kidneys, we show that the transition from the initial, stereotyped, wide branching to narrower later branching is independent from previous branching events but depends instead on the proximity of other branch tips. A simple computer model suggests that a repelling molecule secreted by branches can in principle generate a well-spaced tree that switches automatically from wide initial branch angles to narrower subsequent ones, and that co-cultured trees would distort their normal shapes rather than colliding. We confirm this collision-avoidance experimentally using organ cultures, and identify BMP7 as the repelling molecule, Conclusions We propose that self-avoidance, an intrinsically error-correcting mechanism, may be an important patterning mechanism in collecting duct branching, operating along with alreadyknown mesenchyme-derived paracrine factors. [ABSTRACT FROM AUTHOR]

FGF signaling is essential for mammary gland development, yet the mechanisms by which different members of the FGF family control stem cell function and epithelial morphogenesis in this tissue are not well understood. Here, we have examined the requirement of Fgfr2 in mouse mammary gland morphogenesis using a postnatal organ regeneration model. We found that tissue regeneration from basal stem cells is a multistep event, including luminal differentiation and subsequent epithelial branching morphogenesis. Basal cells lacking Fgfr2 did not generate an epithelial network owing to a failure in luminal differentiation. Moreover, Fgfr2 null epithelium was unable to undergo ductal branch initiation and elongation due to a deficiency in directional migration. We identified FGF10 and FGF2 as stromal ligands that control distinct aspects of mammary ductal branching. FGF10 regulates branch initiation, which depends on directional epithelial migration. By contrast, FGF2 controls ductal elongation, requiring cell proliferation and epithelial expansion. Together, our data highlight a pleiotropic role of Fgfr2 in stem cell differentiation and branch initiation, and reveal that different FGF ligands regulate distinct aspects of epithelial behavior. [ABSTRACT FROM AUTHOR]

Branching epithelial morphogenesis is closely linked to epithelial-to-mesenchymal transition (EMT), a process important in normal development and cancer progression. The miR-200 family regulates epithelial morphogenesis and EMT through a negative feedback loop with the ZEB1 and ZEB2 transcription factors. miR-200 inhibits expression of ZEB1/2 mRNA, which in turn can down-regulate the miR-200 family that further results in down-regulation of E-cadherin and induction of a mesenchymal phenotype. Recent studies show that the expression of miR-200 genes is high during late pregnancy and lactation, thereby indicating that these miRs are important for breast epithelial morphogenesis and differentiation. miR-200 genes have been studied intensively in relation to breast cancer progression and metastasis, where it has been shown that miR-200 members are down-regulated in basal-like breast cancer where the EMT phenotype is prominent. There is growing evidence that the miR-200 family is up-regulated in distal breast metastasis indicating that these miRs are important for colonization of metastatic breast cancer cells through induction of mesenchymal to epithelial transition. The dual role of miR-200 in primary and metastatic breast cancer is of interest for future therapeutic interventions, making it important to understand its role and interacting partners in more detail. [ABSTRACT FROM AUTHOR]

Highlights: [•] The common architecture of tubes comprises a lumen lined by polarized cells. [•] The common architecture of tubes can be mediated by biologically distinct processes. [•] In vitro 3D models recapitulate many aspects of tube formation. [•] Simple 3D in vitro models are reviewed with emphasis on ECM-specific behaviors. [Copyright &y& Elsevier]

Highlights: [•] Mammary epithelium transitions between simple and stratified organization during development. [•] Terminal end buds (TEBs) elongate mammary tubes during puberty without ECM directed protrusions. [•] TEBs are stratified, have low apico-basal polarity, and high proliferation. [•] Stratification is initiated by an asymmetric cell division with concomitant loss of polarity. [•] Normal cells are protrusive and migratory within the epithelium but do not disseminate. [Copyright &y& Elsevier]

Highlights: [•] Single tip cells or groups of leading cells develop at the forefront of growing tissues. [•] Tip cells regulate tubule growth and morphogenesis. [•] Tip cells develop distinctive patterns of gene expression and specialised characteristics. [•] Tip cells are required for health and may be involved in the progression of cancer. [Copyright &y& Elsevier]

Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans. [ABSTRACT FROM AUTHOR]

Background: The laying hen model of spontaneous epithelial ovarian cancer (EOC) is unique in that it is the only model that enables observations of early events in disease progression and is therefore also uniquely suited for chemoprevention trials. Previous studies on the effect of dietary flaxseed in laying hens have revealed the potential for both amelioration and prevention of ovarian cancer. The objective of this study was to assess the effect of flaxseed on genes and pathways that are dysregulated in tumors. We have used a bioinformatics approach to identify these genes, followed by qPCR validation, immunohistochemical localization, and in situ hybridization to visualize expression in normal ovaries and tumors from animals fed a control diet or a diet containing 10% flaxseed. Results: Bioinformatic analysis of ovarian tumors in hens led to the identification of a group of highly up-regulated genes that are involved in the embryonic process of branching morphogenesis. Expression of these genes coincides with expression of E-cadherin in the tumor epithelium. Levels of expression of these genes in tumors from flax-fed animals are reduced 40-60%. E-cadherin and miR200 are both up-regulated in tumors from control-fed hens, whereas their expression is decreased 60-75% in tumors from flax-fed hens. This does not appear to be due to an increase in ZEB1 as mRNA levels are increased five-fold in tumors, with no significant difference between control-fed and flax-fed hens. Conclusions: We suggest that nutritional intervention with flaxseed targets the pathways regulating branching morphogenesis and thereby alters the progression of ovarian cancer. [ABSTRACT FROM AUTHOR]

Recent experimental work in lung morphogenesis has described an elegant pattern of branching phenomena. Two primary forms of branching have been identified: side branching and tip splitting. In our previous study of lung branching morphogenesis, we used a 4 variable partial differential equation (PDE), due to Meinhardt, as our mathematical model to describe the reaction and diffusion of morphogens creating those branched patterns. By altering key parameters in the model, we were able to reproduce all the branching styles and the switch between branching modes. Here, we attempt to explain the branching phenomena described above, as growing out of two fundamental instabilities, one in the longitudinal (growth) direction and the other in the transverse direction. We begin by decoupling the original branching process into two semi-independent sub-processes, 1) a classic activator/inhibitor system along the growing stalk, and 2) the spatial growth of the stalk. We then reduced the full branching model into an activator/inhibitor model that embeds growth of the stalk as a controllable parameter, to explore the mechanisms that determine different branching patterns. We found that, in this model, 1) side branching results from a pattern-formation instability of the activator/inhibitor subsystem in the longitudinal direction. This instability is far from equilibrium, requiring a large inhomogeneity in the initial conditions. It successively creates periodic activator peaks along the growing stalk, each of which later on migrates out and forms a side branch; 2) tip splitting is due to a Turing-style instability along the transversal direction, that creates the spatial splitting of the activator peak into 2 simultaneously-formed peaks at the growing tip, the occurrence of which requires the widening of the growing stalk. Tip splitting is abolished when transversal stalk widening is prevented; 3) when both instabilities are satisfied, tip bifurcation occurs together with side branching. [ABSTRACT FROM AUTHOR]

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor ( HGF) is a key factor in epithelial-stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial-stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. [ABSTRACT FROM AUTHOR]

Abstract: Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles. [Copyright &y& Elsevier]

Abstract: Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increase surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs at the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find that the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes. [Copyright &y& Elsevier]

The collecting duct network and the urothelium of the ureter of the metanephric kidney are derived from the ureteric bud epithelium, initially an outgrowth from the caudal end of the Wolffian duct at the onset of the metanephric kidney development. The tips of the ureteric bud epithelium undergo reiterative branching morphogenesis, which generates more tips and trunks, whereas the ureteric trunks grow and differentiate into principal cells and intercalated cells of the collecting ducts that regulate body water and acid-base homeostasis. microRNAs (miRNAs) are a family of small non-coding RNAs that regulate a diversity of biological processes including organogenesis, mostly by negatively regulating their target gene expression. In this review, I will summarize the current knowledge on the critical roles of miRNAs expressed in the ureteric bud epithelium in ureteric bud morphogenesis and differentiation, including ureteric bud branching morphogenesis, collecting duct terminal differentiation, cystogenesis of the collecting ducts, and ureter development. [ABSTRACT FROM AUTHOR]

The development of branched epithelial organs, such as the kidney, mammary gland, lung, pancreas, and salivary gland, is dependent upon the involvement and interaction of multiple regulatory/modulatory molecules, including soluble growth factors, extracellular matrix components, and their receptors. How the function of these molecules is coordinated to bring about the morphogenetic events that regulate iterative tip-stalk generation (ITSG) during organ development remains to be fully elucidated. A common link to many growth factor-dependent morphogenetic pathways is the involvement of variably sulfated heparan sulfates (HS), the glycosaminoglycan backbone of heparan sulfate proteoglycans (HSPG) on extracellular surfaces. Genetic deletions of HS biosynthetic enzymes (e.g., C5-epimerase, Hs2st), as well as considerable in vitro data, indicate that variably sulfated HS are essential for kidney development, particularly in Wolffian duct budding and early ureteric bud (UB) branching. A role for selective HS modifications by enzymes (e.g., Ext, Ndst, Hs2st) in stages of branching morphogenesis is also strongly supported for mammary gland ductal branching, which is dependent upon a set of growth factors similar to those involved in UB branching. Taken together, these studies provide support for the notion that the specific spatio-temporal HS binding of growth factors during the development of branched epithelial organs (such as the kidney, mammary gland, lung and salivary gland) regulates these complex processes by potentially acting as 'morphogenetic switches' during the various stages of budding, branching, and other developmental events central to epithelial organogenesis. It may be that two or more growth factor-selective HS interactions constitute a functionally equivalent morphogenetic switch; this may help to explain the paucity of severe branching phenotypes with individual growth factor knockouts. [ABSTRACT FROM AUTHOR]

Background: The Matrigel-embedded epithelium of the mouse submandibular gland undergoes branching morphogenesis when cultured in medium supplemented with fibroblast growth factor 7 (FGF7) and lysophosphatidic acid (LPA), whereas it elongates a stalk with limited branching in medium with only FGF7. Because LPA is a well-known activator of epidermal growth factor (EGF) signaling, we hypothesized the involvement of autocrine EGF family growth factors in the branching morphogenesis. Results: Reverse transcriptase polymerase chain reaction studies showed that three members, Tgfa, Hbegf,and Nrg1 of the EGF family were expressed in the epithelium cultured with FGF7 + LPA as well as in the epithelium freshly isolated from the rudiments. All the growth factors induced extensive branching morphogenesis in the Matrigel-embedded epithelium in the presence of LPA. Tyrphostin AG112, an inhibitor of EGF signaling, severely impaired branching morphogenesis induced by FGF7 + LPA without exogenous addition of EGF family growth factors to the culture medium. The shaking cultures, which were expected to decrease the concentration of autocrine growth factors near the epithelium by promoting their diffusion, significantly reduced branching morphogenesis induced by FGF7 + LPA. Conclusions: Autocrine EGF family growth factors are involved in epithelial branching morphogenesis induced by FGF7 + LPA. Developmental Dynamics 243:552-559, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2-/-). Results: We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2-/- mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression. Conclusions: Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/ progenitor cells during lung regeneration in both homeostatic turnover and repair after injury. [ABSTRACT FROM AUTHOR]

Background: Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to determine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to identify the target cell for VEGF action. Methods: Timed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were treated ± Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody or GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence studies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies of lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF. Results: Heparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced epithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required NRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated that the VEGF-mediated anti-apoptotic effects were dependent on PKC activation. Conclusions: Vegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine signal that in turn inhibits epithelial cell apoptosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Salivary glands develop by branching morphogenesis. [•] Development involves interaction among epithelial, mesenchymal, neuronal and endothelial cells. [•] The developing nervous system instructs epithelial progenitor cells. [•] There is dynamic interaction among multiple epithelial progenitor cells. [•] Regeneration strategies involve gene- and cell-therapy and tissue bioengineering. [ABSTRACT FROM AUTHOR]