Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion.

Many embryonic organs undergo epithelial morphogenesis to form tree-like hierarchical structures. However, it remains unclear what drives the budding and branching of stratified epithelia, such as in the embryonic salivary gland and pancreas. Here, we performed live-organ imaging of mouse embryonic salivary glands at single-cell resolution to reveal that budding morphogenesis is driven by expansion and folding of a distinct epithelial surface cell sheet characterized by strong cell-matrix adhesions and weak cell-cell adhesions. Profiling of single-cell transcriptomes of this epithelium revealed spatial patterns of transcription underlying these cell adhesion differences. We then synthetically reconstituted budding morphogenesis by experimentally suppressing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells, which required ß1-integrin-mediated cell-matrix adhesion for successful budding. Thus, stratified epithelial budding, the key first step of branching morphogenesis, is driven by an overall combination of strong cell-matrix adhesion and weak cell-cell adhesion by peripheral epithelial cells.

The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

A Unifying Theory of Branching Morphogenesis.

The morphogenesis of branched organs remains a subject of abiding interest. Although much is known about the underlying signaling pathways, it remains unclear how macroscopic features of branched organs, including their size, network topology, and spatial patterning, are encoded. Here, we show that, in mouse mammary gland, kidney, and human prostate, these features can be explained quantitatively within a single unifying framework of branching and annihilating random walks. Based on quantitative analyses of large-scale organ reconstructions and proliferation kinetics measurements, we propose that morphogenesis follows from the proliferative activity of equipotent tips that stochastically branch and randomly explore their environment but compete neutrally for space, becoming proliferatively inactive when in proximity with neighboring ducts. These results show that complex branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple but generic rule, without recourse to a rigid and deterministic sequence of genetically programmed events.

Lung endoderm morphogenesis: gasping for form and function.

The respiratory endoderm develops from a small cluster of cells located on the ventral anterior foregut. This population of progenitors generates the myriad epithelial lineages required for proper lung function in adults through a complex and delicately balanced series of developmental events controlled by many critical signaling and transcription factor pathways. In the past decade, understanding of this process has grown enormously, helped in part by cell lineage fate analysis and deep sequencing of the transcriptomes of various progenitors and differentiated cell types. This review explores how these new techniques, coupled with more traditional approaches, have provided a detailed picture of development of the epithelial lineages in the lung and insight into how aberrant development can lead to lung disease.

Spatially distinct epithelial and mesenchymal cell subsets along progressive lineage restriction in the branching embryonic mammary gland.

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis.

Mechanism of branching morphogenesis inspired by diatom silica formation.

The silica-based cell walls of diatoms are prime examples of genetically controlled, species-specific mineral architectures. The physical principles underlying morphogenesis of their hierarchically structured silica patterns are not understood, yet such insight could indicate novel routes toward synthesizing functional inorganic materials. Recent advances in imaging nascent diatom silica allow rationalizing possible mechanisms of their pattern formation. Here, we combine theory and experiments on the model diatom Thalassiosira pseudonana to put forward a minimal model of branched rib patterns-a fundamental feature of the silica cell wall. We quantitatively recapitulate the time course of rib pattern morphogenesis by accounting for silica biochemistry with autocatalytic formation of diffusible silica precursors followed by conversion into solid silica. We propose that silica deposition releases an inhibitor that slows down up-stream precursor conversion, thereby implementing a self-replicating reaction-diffusion system different from a classical Turing mechanism. The proposed mechanism highlights the role of geometrical cues for guided self-organization, rationalizing the instructive role for the single initial pattern seed known as the primary silicification site. The mechanism of branching morphogenesis that we characterize here is possibly generic and may apply also in other biological systems.

FGF signaling regulates salivary gland branching morphogenesis by modulating cell adhesion.

Loss of FGF signaling leads to defects in salivary gland branching, but the mechanisms underlying this phenotype remain largely unknown. We disrupted expression of Fgfr1 and Fgfr2 in salivary gland epithelial cells and found that both receptors function coordinately in regulating branching. Strikingly, branching morphogenesis in double knockouts is restored by Fgfr1 and Fgfr2 (Fgfr1/2) knock-in alleles incapable of engaging canonical RTK signaling, suggesting that additional FGF-dependent mechanisms play a role in salivary gland branching. Fgfr1/2 conditional null mutants showed defective cell-cell and cell-matrix adhesion, both of which have been shown to play instructive roles in salivary gland branching. Loss of FGF signaling led to disordered cell-basement membrane interactions in vivo as well as in organ culture. This was partially restored upon introducing Fgfr1/2 wild-type or signaling alleles that are incapable of eliciting canonical intracellular signaling. Together, our results identify non-canonical FGF signaling mechanisms that regulate branching morphogenesis through cell-adhesion processes.

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Collective cell migration is spatiotemporally regulated during mammary epithelial bifurcation.

Branched epithelial networks are generated through an iterative process of elongation and bifurcation. We sought to understand bifurcation of the mammary epithelium. To visualize this process, we utilized three-dimensional (3D) organotypic culture and time-lapse confocal microscopy. We tracked cell migration during bifurcation and observed local reductions in cell speed at the nascent bifurcation cleft. This effect was proximity dependent, as individual cells approaching the cleft reduced speed, whereas cells exiting the cleft increased speed. As the cells slow down, they orient both migration and protrusions towards the nascent cleft, while cells in the adjacent branches orient towards the elongating tips. We next tested the hypothesis that TGF-ß signaling controls mammary branching by regulating cell migration. We first validated that addition of TGF-ß1 (TGFB1) protein increased cleft number, whereas inhibition of TGF-ß signaling reduced cleft number. Then, consistent with our hypothesis, we observed that pharmacological inhibition of TGF-ß1 signaling acutely decreased epithelial migration speed. Our data suggest a model for mammary epithelial bifurcation in which TGF-ß signaling regulates cell migration to determine the local sites of bifurcation and the global pattern of the tubular network.

Exocrine gland structure-function relationships.

Fluid secretion by exocrine glandular organs is essential to the survival of mammals. Each glandular unit within the body is uniquely organized to carry out its own specific functions, with failure to establish these specialized structures resulting in impaired organ function. Here, we review glandular organs in terms of shared and divergent architecture. We first describe the structural organization of the diverse glandular secretory units (the end-pieces) and their fluid transporting systems (the ducts) within the mammalian system, focusing on how tissue architecture corresponds to functional output. We then highlight how defects in development of end-piece and ductal architecture impacts secretory function. Finally, we discuss how knowledge of exocrine gland structure-function relationships can be applied to the development of new diagnostics, regenerative approaches and tissue regeneration.

Repetitive accumulation of interstitial cells generates the branched structure of Cladonema medusa tentacles.

The shaping of tissues and organs in many animals relies on interactions between the epithelial cell layer and its underlying mesoderm-derived tissues. Inductive signals, such as receptor tyrosine kinase (RTK) signaling emanating from mesoderm, act on cells of the epithelium to initiate three-dimensional changes. However, how tissues are shaped in a diploblastic animal with no mesoderm remains largely unknown. In this study, the jellyfish Cladonema pacificum was used to investigate branch formation. The tentacles on its medusa stage undergo branching, which increases the epithelial surface area available for carrying nematocytes, thereby maximizing prey capture. Pharmacological and cellular analyses of the branching process suggest a two-step model for tentacle branch formation, in which mitogen-activated protein kinase kinase signaling accumulates interstitial cells in the future branch-forming region, and fibroblast growth factor signaling regulates branch elongation. This study highlights an essential role for these pluripotent stem cells in the tissue-shaping morphogenesis of a diploblastic animal. In addition, it identifies a mechanism involving RTK signaling and cell proliferative activity at the branch tip for branching morphogenesis that is apparently conserved across the animal kingdom.

A mechanogenetic role for the actomyosin complex in branching morphogenesis of epithelial organs.

The actomyosin complex plays crucial roles in various life processes by balancing the forces generated by cellular components. In addition to its physical function, the actomyosin complex participates in mechanotransduction. However, the exact role of actomyosin contractility in force transmission and the related transcriptional changes during morphogenesis are not fully understood. Here, we report a mechanogenetic role of the actomyosin complex in branching morphogenesis using an organotypic culture system of mouse embryonic submandibular glands. We dissected the physical factors arranged by characteristic actin structures in developing epithelial buds and identified the spatial distribution of forces that is essential for buckling mechanism to promote the branching process. Moreover, the crucial genes required for the distribution of epithelial progenitor cells were regulated by YAP and TAZ through a mechanotransduction process in epithelial organs. These findings are important for our understanding of the physical processes involved in the development of epithelial organs and provide a theoretical background for developing new approaches for organ regeneration.

Ret signaling in ureteric bud epithelial cells controls cell movements, cell clustering and bud formation.

Ret signaling promotes branching morphogenesis during kidney development, but the underlying cellular mechanisms remain unclear. While Ret-expressing progenitor cells proliferate at the ureteric bud tips, some of these cells exit the tips to generate the elongating collecting ducts, and in the process turn off Ret. Genetic ablation of Ret in tip cells promotes their exit, suggesting that Ret is required for cell rearrangements that maintain the tip compartments. Here, we examine the behaviors of ureteric bud cells that are genetically forced to maintain Ret expression. These cells move to the nascent tips, and remain there during many cycles of branching; this tip-seeking behavior may require positional signals from the mesenchyme, as it occurs in whole kidneys but not in epithelial ureteric bud organoids. In organoids, cells forced to express Ret display a striking self-organizing behavior, attracting each other to form dense clusters within the epithelium, which then evaginate to form new buds. The ability of forced Ret expression to promote these events suggests that similar Ret-dependent cell behaviors play an important role in normal branching morphogenesis.

Defective mesothelium and limited physical space are drivers of dysregulated lung development in a genetic model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a developmental disorder associated with diaphragm defects and lung hypoplasia. The etiology of CDH is complex and its clinical presentation is variable. We investigated the role of the pulmonary mesothelium in dysregulated lung growth noted in the Wt1 knockout mouse model of CDH. Loss of WT1 leads to intrafetal effusions, altered lung growth, and branching defects prior to normal closure of the diaphragm. We found significant differences in key genes; however, when Wt1 null lungs were cultured ex vivo, growth and branching were indistinguishable from wild-type littermates. Micro-CT imaging of embryos in situ within the uterus revealed a near absence of space in the dorsal chest cavity, but no difference in total chest cavity volume in Wt1 null embryos, indicating a redistribution of pleural space. The altered space and normal ex vivo growth suggest that physical constraints are contributing to the CDH lung phenotype observed in this mouse model. These studies emphasize the importance of examining the mesothelium and chest cavity as a whole, rather than focusing on single organs in isolation to understand early CDH etiology.

Branching morphogenesis of the mouse mammary gland after exposure to benzophenone-3.

Pubertal mammary branching morphogenesis is a hormone-regulated process susceptible to exposure to chemicals with endocrine disruptive capacity, such as the UV-filter benzophenone-3 (BP3). Our aim was to assess whether intrauterine or in vitro exposure to BP3 modified the branching morphogenesis of the female mouse mammary gland. For this, pregnant mice were dermally exposed to BP3 (0.15 or 50 mg/kg/day) from gestation day (GD) 8.5 to GD18.5. Sesame oil treatment served as control. Changes of the mammary glands of the offspring were studied on postnatal day 45. Further, mammary organoids from untreated mice were cultured under branching induction conditions and exposed for 9 days to BP3 (1 × 10-6 M, 1 × 10-9 M, or 1 × 10-12 M with 0.01% ethanol as control) to evaluate the branching progression. Mice that were exposed to BP3 in utero showed decreased mRNA levels of progesterone receptor (PR) and WNT4. However, estradiol and progesterone serum levels, mammary histomorphology, proliferation, and protein expression of estrogen receptor alpha (ESR1) and PR were not significantly altered. Interestingly, direct exposure to BP3 in vitro also decreased the mRNA levels of PR, RANKL, and amphiregulin without affecting the branching progression. Most effects were found after exposure to 50 mg/kg/day or 1 × 10-6 M of BP3, both related to sunscreen application in humans. In conclusion, exposure to BP3 does not impair mammary branching morphogenesis in our models. However, BP3 affects PR transcriptional expression and its downstream mediators, suggesting that exposure to BP3 might affect other developmental stages of the mammary gland.

ß-Catenin in the kidney stroma modulates pathways and genes to regulate kidney development.

BACKGROUND: Kidney development is regulated by cellular interactions between the ureteric epithelium, mesenchyme, and stroma. Previous studies demonstrate essential roles for stromal ß-catenin in kidney development. However, how stromal ß-catenin regulates kidney development is not known. We hypothesize that stromal ß-catenin modulates pathways and genes that facilitate communications with neighboring cell populations to regulate kidney development. RESULTS: We isolated purified stromal cells with wild type, deficient, and overexpressed ß-catenin by fluorescence-activated cell sorting and conducted RNA Sequencing. A Gene Ontology network analysis demonstrated that stromal ß-catenin modulates key kidney developmental processes, including branching morphogenesis, nephrogenesis and vascular formation. Specific stromal ß-catenin candidate target genes that may mediate these effects included secreted, cell-surface and transcriptional factors that regulate branching morphogenesis and nephrogenesis (Wnts, Bmp, Fgfr, Tcf/Lef) and secreted vascular guidance cues (Angpt1, VEGF, Sema3a). We validated established ß-catenin targets including Lef1 and novel candidate ß-catenin targets including Sema3e which have unknown roles in kidney development. CONCLUSIONS: These studies advance our understanding of gene and biological pathway dysregulation in the context of stromal ß-catenin misexpression during kidney development. Our findings suggest that during normal kidney development, stromal ß-catenin may regulate secreted and cell-surface proteins to communicate with adjacent cell populations.

Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development.

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

Chromatin remodeler Znhit1 controls bone morphogenetic protein signaling in embryonic lung tissue branching.

Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1 (zinc finger HIT-type containing 1), acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional KO mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the chromatin immunoprecipitation assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein (BMP) 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.

FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Claudin-3 regulates luminal fluid accumulation in the developing chick lung.

Claudins are a family of tight junction proteins expressed in epithelial tissues during development and in postnatal life. We hypothesized that claudins are required for branching morphogenesis in the developing chick lung. To test this hypothesis, we exposed cultured chick lung explants at embryonic day 5 to a truncated non-toxic form of the Clostridium perfringens enterotoxin known as C-CPE that removes C-CPE-sensitive claudins from tight junctions. Using in situ hybridization and immunofluorescence studies, we established that only one C-CPE-sensitive claudin, Claudin-3, was expressed in the chick lung at this stage. C-CPE treated lung explants did not exhibit any defect in lung branching compared to controls. However, they did exhibit a significantly smaller lumen area, suggesting that paracellular permeability was perturbed. The decrease in lumen area was associated with a loss of Claudin-3 expression within tight junctions of the respiratory epithelium and an increase in permeability of the respiratory epithelium. When C-CPE-treated lung explants were treated with forskolin, lumen area was restored. In summary, removal of a sealing claudin, Claudin-3, from tight junctions in embryonic lung epithelium results in a decrease in lumen area and in hydrostatic pressure needed for lung development.