Vascular and Liver Homeostasis in Juvenile Mice Require Endothelial Cyclic AMP-Dependent Protein Kinase A.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

Ciliary Hedgehog signaling patterns the digestive system to generate mechanical forces driving elongation.

How tubular organs elongate is poorly understood. We found that attenuated ciliary Hedgehog signaling in the gut wall impaired patterning of the circumferential smooth muscle and inhibited proliferation and elongation of developing intestine and esophagus. Similarly, ablation of gut-wall smooth muscle cells reduced lengthening. Disruption of ciliary Hedgehog signaling or removal of smooth muscle reduced residual stress within the gut wall and decreased activity of the mechanotransductive effector YAP. Removing YAP in the mesenchyme also reduced proliferation and elongation, but without affecting smooth muscle formation, suggesting that YAP interprets the smooth muscle-generated force to promote longitudinal growth. Additionally, we developed an intestinal culture system that recapitulates the requirements for cilia and mechanical forces in elongation. Pharmacologically activating YAP in this system restored elongation of cilia-deficient intestines. Thus, our results reveal that ciliary Hedgehog signaling patterns the circumferential smooth muscle to generate radial mechanical forces that activate YAP and elongate the gut.

Intussusceptive Angiogenesis in Human Metastatic Malignant Melanoma.

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, in vitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation in vitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.

A Qualitative Change in the Transcriptome Occurs after the First Cell Cycle and Coincides with Lumen Establishment during MDCKII Cystogenesis.

Madin-Darby canine kidney II (MDCKII) cells are widely used to study epithelial morphogenesis. To better understand this process, we performed time course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome that occurs after the first cell cycle and coincides with lumen establishment. This change appears to be linked to translocation of ß-catenin, supported by analyses with AVL9- and DENND5A-knockdown clones, and regulation by HNF1B, supported by ATAC-seq study. These findings indicate a qualitative change model for transcriptome remodeling during epithelial morphogenesis, leading to cell proliferation decrease and cell polarity establishment. Furthermore, our study reveals that active mitochondria are retained and chromatin accessibility decreases in 3D cysts but not in 2D polarized cells. This indicates that 3D culture is a better model than 2D culture for studying epithelial morphogenesis.

Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Simple Rules Determine Distinct Patterns of Branching Morphogenesis.

Many metazoan organs are comprised of branching trees of epithelial tubes; how patterning occurs in these trees is a fundamental problem of development. Commonly, branch tips fill the volume of the organ approximately uniformly, e.g., in mammalian lung, airway branch tips are dispersed roughly uniformly throughout the volume of the lung. In contrast, in the developing metanephric kidney, the tips of the ureteric bud tree are located close to the outer surface of the kidney rather than filling the kidney. Here, we describe a simple alteration in the branching rules that accounts for the difference between the kidney pattern that leads to tips near the organ surface versus previously known patterns that lead to the branch tips being dispersed throughout the organ. We further use a simple toy model to deduce from first principles how this rule change accounts for the differences in the two types of trees.

The phospholipid PI(3,4)P2 is an apical identity determinant.

Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P2 localization and function is distinct from the basolateral determinant PI(3,4,5)P3. PI(3,4)P2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P2 is a determinant of apical membrane identity.

Fibroblast-derived HGF drives acinar lung cancer cell polarization through integrin-dependent RhoA-ROCK1 inhibition.

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate ß1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.

Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity.

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens.

cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior.

Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex.

Exosomes, 40-150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively.

p114RhoGEF governs cell motility and lumen formation during tubulogenesis through a ROCK-myosin-II pathway.

Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here, we utilized tubulogenesis induced by hepatocyte growth factor (HGF) in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of p114RhoGEF (also known as ARHGEF18), a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by HGF. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho kinase (ROCK) or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live-cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a new pathway that regulates directed cell migration and formation of a single lumen during epithelial tube morphogenesis.

A molecular switch for the orientation of epithelial cell polarization.

The formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a ß1-integrin/FAK/p190RhoGAP complex to downregulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCßII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.

Parasympathetic innervation regulates tubulogenesis in the developing salivary gland.

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.

Host cell polarity proteins participate in innate immunity to Pseudomonas aeruginosa infection.

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses.

Intercellular transfer of GPRC5B via exosomes drives HGF-mediated outward growth.

How cells communicate during development and regeneration is a critical question. One mechanism of intercellular communication is via exosomes, extracellular vesicles that originate by the fusion of multivesicular endosomes with the plasma membrane [1-8]. To model exosome-based intercellular communication, we used Madin-Darby canine kidney (MDCK) cell cysts grown in 3D gels of extracellular matrix, which form tubules in response to hepatocyte growth factor (HGF). We report that GPRC5B, an orphan G protein coupled receptor, is in exosomes produced by HGF-treated cysts and released into the cyst lumen. Exosomal GPRC5B is taken up by nearby cells and together with HGF promotes extracellular signal-regulated kinase 1/2 (ERK1/2) activation and tubulogenesis, even under conditions where tubulogenesis would otherwise not occur. Recovery from injury, such as acute kidney injury (AKI), often recapitulates developmental processes. Here, we show that GPRC5B is elevated in urinary exosomes from patients with AKI. Our results elucidate how GPRC5B is carried by exosomes and augments HGF-induced morphogenesis. The unexpected role of exosomes in transporting GPRC5B between cells during morphogenesis and the ability of GPRC5B to predict the disease state of AKI elucidate a novel mechanism for intercellular communication during development and repair.

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures.

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell-cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell-cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis.