/ 4/1 Branching Morphogenesis David Hipfner Epithelial Cell Biology Research Unit IRCM What is morphogenesis? Gastrulation and neurulation in Xenopus laevis (15 h elapsed time) http://faculty.virginia.edu/shook/showmovies/xenopus_gastrulation.mov 1
/ 4/1 What is morphogenesis? Dorsal closure and head involution in Drosophila http://www.celldynamics.org/celldynamics/gallery/livelabel.html What is morphogenesis? Imaginal disc eversion in Drosophila Ward, R.E. et al. (2003). Dev. Biol., 256: 389-402. 2
/ 4/1 What is branching morphogenesis? The restructuring of epithelial tissues to complex but highly organized tubular networks that transport gases and/or fluids Affolter et al., 2003 - the most common structural design for organs is a branched tubular network Many organs are composed of branching tubes Lungs Kidney Vasculature Salivary gland Mammary glands Drosophila trachea 3
/ 4/1 Why branch? Branching morphogenesis is a fundamental evolutionary adaptation to increasing organism size: - enables targetted delivery of nutrients (e.g. vasculature, trachea) - allows efficient packing into a minimal volume (e.g. secretory glands) - maximizes surface area for metabolic exchange (e.g. vasculature, lung, kidney) Example - the lung Without branching: ~0.5 m 2 surface area for gas exchange with the blood With branching (300 million alveoli): ~100 m 2 surface area for gas exchange 4
/ 4/1 Goal of today s lecture o o Provide general background on how epithelial cells and tissues are shaped Illustrate key conserved principles of branching morphogenesis, using two well characterized models:! Drosophila trachea! mammalian kidney o At the end, you should be able to answer questions like:! What mechanisms are used to shape epithelial cells?! How do epithelial cells form tubes?! What aspects of branching morphogenesis are conserved across systems/species?! What aspects are different?! What role does signalling from mesenchyme to epithelia play?! What role does signalling from epithelia to mesenchyme play?! How are different types of branches formed by reiterative use of one signalling pathway? Epithelial versus mesenchymal cells Mesenchyme: - connective tissue - derives from embryonic mesoderm - consists of loosesly packed, unspecialized cells embedded in extracellular matrix - e.g. fibroblasts Epithelia: - line nearly all surfaces - most derives from embryonic ectoderm or endoderm - consists of highly organized layers of tightly adherent cells with a shared apical-basal polarity - rest on basement membrane 5
/ 4/1 Epithelial cell have apical-basal polarity Drosophila hindgut epithelium APICAL BASOLATERAL JUNCTIONS http://mcb.berkeley.edu/labs/bilder/polarity.html Adherens junctions - provide cell-cell adhesion; provide anchor point for actin cytoskeleton; join cytoskeletons of neighbouring cells, allowing contractility across the epithelial layer from Alberts et al., Molecular Biology of the Cell 6
4/1 Epithelial and mesenchymal cells can be interconverted MESENCHYMAL CELL mesenchymal-to-epithelial transition (MET) epithelial-to-mesenchymal transition (EMT) EPITHELIAL CELL What kinds of (cellular) processes drive branching morphogenesis? Time-lapse video of developing embryo with tracheal cells expressing GFP 7
/ 4/1 Identifying genes required for morphogenesis A. GENETIC SCREENS Identifying genes required for morphogenesis Wild-type punt (Type II TGF-β receptor) canoe (Afadin) kayak (Fos) 8
/ 4/1 Morphogenesis relies upon a conserved signaling cascade +ve regulators Rho GTPase ROCK Myosin II TENSION -ve regulators Quintin et al. (2008). Trends in Genetics 24:221-230 Actomyosin-based contractility muscle Myosin II Vale and Milligan, Science (2000) 9
/ 4/1 Regulation of non-muscle Myosin II by RLC phosphorylation Essential light chain Heavy chain rod domain Regulatory light chain Heavy chain head domain adapted from Vicente-Manzanares et al., Nature Reviews (2000) RLC phosphorylation promotes Myosin antiparallel filament formation Actin bundling adapted from Vicente-Manzanares et al., Nature Reviews (2000) 10
/ 4/1 RLC phosphorylation stimulates actinactivated ATPase activity adapted from Vicente-Manzanares et al., Nature Reviews (2000) Rho GTPases act as molecular switches to control actin cytoskeleton dynamics OFF ON GAP = GTPase activating protein GEF = GDP:GTP exchange factor 11
/ 4/1 Rho-GTPase signaling regulates MRLC through the protein kinase ROCK Epithelial cells can change their SHAPE and ARRANGEMENT Quintin et al. (2008). Trends in Genetics 24:221-230 12
/ 4/1 How do epithelial cells form tubes? Tube formation is driven by: CELL MIGRATION CELL REARRANGEMENT CELL SHAPE CHANGE * In mammals, CELL PROLIFERATION is also essential for forming branched organs* Shaping epithelial cells into tubes Multicellular tubes e.g. largest primary trachea branches, larger blood vessels, most branched vertebrate organs from Uv et al., 2003 13
/04/18 How are tubular cell shapes generated? Multicellular tubes are usually formed from existing epithelia from Lubarsky and Krasnow, 2003 Tube formation by apical constriction shrink or constrict apical membrane surface area both involve apical constriction to generate bud 14
Shaping epithelial cells into tubes Unicellular tubes e.g. smaller primary trachea branches, smaller blood vessels from Uv et al., 2003 How are tubular cell shapes generated? Unicellular tubes are formed from multicellular tubes Cells reach around each other to form autocellular junction from Kerman et al., 2006 15
Shaping epithelial cells into tubes Intracellular terminal tubes e.g. tracheal terminal branches from Uv et al., 2003 How are tubular cell shapes generated? De novo formation of intracellular terminal tubes - intracellular tubes can be created by synthesis of apical membrane vesicles followed by targetted vesicle fusion from Lubarsky and Krasnow, 2003 16
Early studies of the branching process 1950s-1970s mesenchyme epithelial primordium +.45µm filter Inductive signals for branching morphogenesis - epithelial branching process is induced by signals from neighbouring mesenchyme embryonic lung epithelium stripped of mesenchyme and grown in culture on.45µm filter embryonic lung epithelium stripped of mesenchyme and grown in culture on.45µm filter opposite a piece of lung mesenchyme Shannon, J.M. and Hyatt, B.A. (2004). Annu. Rev. Physiol. 66:625-45 17
Inductive signals for branching morphogenesis - mesenchyme responds to inductive signals from neighbouring epithelia Taderera, J.V. (1967). Dev Bio16:489-512 Complex Epithelial - Mesodermal Signaling MESODERM EPITHELIUM - mesoderm signals to epithelium to induce budding and branching - triggers signals within the epithelium that: - alter the way that the epithelium responds to mesodermal signals - signal back to the mesoderm to pattern the mesoderm (in mammals, not in flies!) 18
Drosophila as a model system for branching morphogenesis - much of our knowledge about the cellular mechanisms involved in forming branched organs stems from research on Drosophila trachea development - remains by far the best characterized branching process genetic tractability - more than 50 genes required for normal branching morphogenesis have been identified in forward genetic screens; minimal functional redundancy relative simplicity - the whole tracheal system is composed of only ~1600 cells ease of visualization - e.g. fluorescence markers, time lapse video microscopy *many of the mechanisms, both molecular and cellular, are conserved in mammals* The Drosophila life cycle ADULT PUPAL stage 5 days L1 EMBRYONIC stages 1 day G L3 R O L2 W T H LARVAL stages 4 days 19
The Drosophila larval tracheal system - air-filled network of interconnected epithelial tubes containing some 10,000 branches - transports oxygen and other gases from outside to essentially every cell of each internal tissue - bilaterally symmetrical, segmentally repeated pattern (lateral view of Drosophila embryo) 25 µm adapted from Ghabrial et al. 2003 Timelapse imaging of tracheal development Time-lapse video of developing embryo with tracheal cells expressing GFP http://flymove.uni-muenster.de/ 20
General scheme for branching morphogenesis from Affolter et al., 2003 The basic process of branching: - specification of a competence zone/primordium - epithelium invagination/budding into mesenchyme - branch initiation - branch outgrowth Specifying the tracheal primordium ~ 5 h after egg laying (AEL): - the trachea arise from 20 placodes on the embryonic surface ectoderm Trh - each placode consists of ~ 20 cells - first morphological sign of the placodes is expression of the transcription factor Trachealess (Trh) 21
Signalling - Specifying tracheal primordia - the Trachealess (Trh) bhlh-pas domain transcription factor is the earliest expressed trachea-specific gene; expressed throughout tracheal development - functions as a heterodimer together with Tango, a more broadly expressed bhlh-pas domain transcription factor - shortly after Trh, Ventral veinless (Vvl), a POU domain transcription factor, is also expressed in tracheal placode cells and throughout subsequent stages - the combinatorial activity of Trh/Tango and Vvl triggers early tracheal gene expression Trh + Tango Vvl tdf peb btl dof rho tkv tracheal development ~ 5-7 h after egg laying (AEL): Tracheal pit formation - cells in the placodes invaginate (apical constriction) to form the tracheal pits - as they invaginate, each cell undergoes two rounds of cell division, giving rise to a total of 80 cells per pit * these are the last cell divisions during formation of the trachea; after this point, there is no change in tracheal cell number * antibody staining against trachea lumen protein from Ghabrial et al. 2003 22
Primary branching ~ 7 h AEL: - primary branches begin to bud out from tracheal pits - form at 6 stereotypical positions (per hemisegment) - branches are formed by between 4 and 20 cells Dorsal branch Dorsal trunks Visceral branch Lateral trunk Ganglionic branch from Ghabrial et al. 2003 7-10 h AEL: Primary branching - primary branches lengthen along defined paths - involves extensive cell rearrangements (cell intercalation) and cell shape changes from Ghabrial et al. 2003 from Affolter et al., 2003 23
FGF s Fibroblast Growth Factor signalling drives primary branching - a large family of secreted polypeptide growth factors (22 in humans, 3 in Drosophila) FGF receptors (FGFR s) - receptor tyrosine kinases that are activated upon FGF binding (4 in humans, 2 in Drosophila) Implicated in diverse developmental processes including patterning, differentiation, cell proliferation, cell survival *FGF s are potent chemoattractants, stimulate cell migration* Fibroblast Growth Factor Signalling IN DROSOPHILA TRACHEA: FGF = Branchless (Bnl) FGFR = Breathless (Btl) adapter = Dof Transcription factor = Pointed (Pnt) and Yan from Thisse and Thisse, 2005 24
Tracheal placode cells are competent to respond to FGF A critical function of the early tracheal transcription factors is to activate expression of Btl/FGFR and Dof (adapter) in placode cells Trh + Tango Vvl tdf peb btl dof rho tkv tracheal development Klambt et al., 1992 Vincent et al., 1998 Btl Dof results in the establishment of FGF competence zones FGF signaling is required for tracheal branching wild-type ligand mutant receptor mutant adapter mutant Ectopic FGF is sufficient to induce tracheal cell migration ectopic Bnl ligand ectopic tracheal branch from Sutherland et al., 1996 Vincent et al., 1998 25
FGF induces tracheal cell migration Bnl/FGF induces migration of two tip cells in each primary branch toward the source of the ligand - FGF induces changes in gene expression and cytoskeletal organization in the tip cells, which are closest to the source of FGF - Btl/FGFR is a transcriptional target of Bnl/FGF signalling - autoregulation Remaining cells in each primary branch are pulled by tip cells from Affolter et al., 2003 Dynamic FGF expression drives branching Bnl/FGF is first expressed in a stereotyped pattern in 6 cell clusters surrounding each tracheal placode - presumably under control of global A/P, D/V, segment patterning systems from Sutherland et al., 1996 26
Secondary branching - ~ 10 h AEL ~ 24 unicellular secondary branches/hemisegment begin to sprout out from primary branches, at stereotypical locations - from ~10-12 h AEL, fusion cells will fuse with their partners in neighbouring segments or on the opposite side of the same segment to form a continuous network from Uv et al., 2003 Tertiary branching - terminal cells undergo extensive unicellular (tertiary) branching during larval life - make intimate contact with most cells of the internal organs high magnification view of terminal trachea branch cell contacting cells in the mature larval gut (blue nuclei) from Ghabrial et al. 2003 27
Dynamic FGF expression drives secondary branching stage 12 stage 13 stage 14 from Sutherland et al., 1996 - when migrating tracheal cells reach the first bnl/fgf sources, they stop - Bnl/FGF expression is downregulated, then re-initiated in more distant cell clusters, again in a stereotyped pattern (mechanism unknown ) - exposure to this second wave of Bnl/FGF signalling induces secondary branching (e.g. specification of terminal cells) Tertiary branch formation also requires Bnl/FGF - at later stages, FGF is expressed in response to hypoxia rather than global patterning cues - recruits tertiary branches from terminal tracheal cells to cells needing oxygen from Ghabrial et al. 2003 28
How does reiterative Bnl/FGF signalling produce different responses each time? - each exposure to Bnl/FGF changes subsequent response First exposure Second exposure Third exposure Trh+Tango Vvl Bnl/FGF Bnl/FGF Bnl/FGF placode specification Btl/FGFR Dof Btl/FGFR Dof Btl/FGFR Dof Btl/FGFR Dof MAPK MAPK MAPK Pnt Blistered 1 branching program Pnt 2 branching program Blistered (= Serum response Factor) 3 branching program Sprouty (Spry) controls branch point choice MESODERM EPITHELIUM - loss of Sprouty causes excessive 2 and terminal branch formation Bnl/FGF Btl/FGFR Dof MAPK Pnt 2 branching program Sprouty - sprouty is a Bnl/FGF target gene in branching epithelia (conserved) - Sprouty functions a general intracellular inhibitor of receptor tyrosine kinase signalling - negative feedback inhibition - in its absence, FGF signalling in tip cells is too strong, causing them to branch at the wrong time or in the wrong place 29
Is it as simple as follow the FGF? No - many other factors feed into branching morphogenesis Several signalling pathways are involved in defining branch identity (BMP) (Wnt) ligand expression pattern tracheal branching pattern if tracheal cells unable to respond to ligand adapted from Kerman et al., 2006 Other pathfinding cues - although Bnl/FGF acts as a chemoattractant, migrating tracheal cells are dependent on other cues in their physical environment for finding the correct path: - different branches depend on different adhesion molecules (e.g. integrins) for proper pathfinding - some branches follow physical cues - e.g. grooves between muscles, surfaces of other cells - to reach their destination from Wolf and Schuh, 2000 blue = mesodermal bridge cell wild-type mutant lacking bridge cell 30
Branching morphogenesis in mammals Two major difference between branching morphogenesis in mammals and flies: 1. Epithelia-to-mesenchyme signalling is essential in mammalian organs - Drosophila trachea are simple monolayered epithelial tubes - in mammals, the epithelia of branched organs are intimately associated with mesenchyme-derived tissues (e.g. blood vessels) - these supporting tissues have to be recruited and organized in a coordinated manner by the branching epithelial cells 2. Growth of the branching network is driven by cell proliferation; no direct evidence for cell migration (e.g. filopodia) during the process in mammals Kidney structure and function branching network: ureter-pelvis-major calyx-minor calyx-collecting ducts-nephron (1) (1) (3-5) (10-20) (thousands?) (~10 6 ) main functions: filter blood, remove metabolic waste products into urine, homeostasis (regulate ph, ionic concentration, blood volume, blood pressure) 31
Visualizing mouse kidney branching - studies of branching greatly aided by imaging of developing kidneys from HoxB7-GFP transgenic mice time-lapse imaging of explant grown in culture grown in vivo from Watanabe and Costantini 2004 The kidney derives from the ureteric bud from Costantini 2006 from Dressler 2006 - kidney derived from the caudal end of the nephric duct - E10.5 - ureteric bud invades metanephric mesenchyme - E11.5 - ureteric bud branches for the first time - T-shape 32
Branching in the developing kidney - ~8 rapid rounds of branching between E11 and E15.5 - followed by period of elongation of the branches formed in rounds 6-8; forms the long unbranched collecting ducts - branching resumes ~ 3 more rounds of branching in the cortex - ~3/4 of branching events are bifurcations (primary branching - at bud tips) from Costantini 2006 - ~6% branches originate from trunks (secondary branching) The Gdnf/Ret signalling pathway Glial-derived neurotrophic factor (Gdnf) is a peptide growth factor GFRα1 is glycosphingolipid-anchored co-receptor that works with Ret Ret is a receptor tyrosine kinase that signals through the typical RTK effector pathways (MAPK, PLC-γ, etc.) 33
Gdnf/Ret signalling in the kidney Ret is expressed in the ureteric bud Gfrα1 is expressed in the ureteric bud and metanephric mesenchyme Gdnf is expressed in the metanephric mesenchyme Sainio et al., 1997 Gdnf promotes sprouting of the ureteric bud Gdnf signalling is necessary for ureteric budding wild-type Gdnf -/- Gdnf is sufficient to induce ureteric budding wild-type misexpressing Gdnf throughout the nephric duct 34
Dynamic expression of Gdnf also drives later branching in the kidney Ret is a transcriptional target of Gdnf/Ret signalling Ret becomes restricted to branching tips from Costantini and Shakya, 2006 Gdnf becomes restricted to undifferentiated mesenchyme at kidney periphery Ret signalling is required in bud tips from Shakya et al., 2005 Is Gdnf a chemoattractant in the developing kidney? 35
Gdnf doesn t seem to guide branching from Shakya et al., 2005 Why is Gdnf/Ret signalling required in tip cells? Ret is a proto-oncogene could it be promoting cell proliferation? BrdU - bud tip swells by cell proliferation to form ampulla prior to branching - lack of Ret -/- cells in bud tips could reflect their failure to proliferate in the ureteric bud, prior to budding from Michael and Davies, 2004 Costantini and Shakya, 2006 36
Signaling within the epithelium - Sprouty function is conserved in kidney Sprouty1 is a target of Gdnf signalling in the ureteric bud Sprouty1 mutants show ectopic buds early and excessive branching later from Basson et al., 2005 and 2006 Signaling from epithelium to mesenchyme Bud tips induce mesenchyme to form nephrons - ureteric bud gives rise to segments up to collecting ducts 37
Importance of physical cues for branching - altered branching frequencies/patterns observed upon manipulation of: extracellular matrix molecules matrix receptors matrix remodelling proteins - extracellular matrix can play several roles: facilitating/resisting branching - e.g. fibronectin in salivary gland) Sakai et al., 2003 regulating signalling - e.g. ligand co-receptors, sequestering ligands Good references for those interested GENERAL: Wang, S., Sekiguchi, R., Daley, W.P., and Yamada, K.M. (2017). Patterned cell and matrix dynamics in branching morphogenesis. Journal of Cell Biology 216: 559-70. Quintin, S., Gally, C., and Labouesse, M. (2008). Epithelial morphogenesis in embryos: asymmetries, motors, and brakes. Trends in Genetics 24:221-230. Lu, P., Sternlicht, M.D., and Werb, Z. (2006). Comparative Mechanisms of Branching Morphogenesis in Diverse Systems. J. Mammary Gland Biol. Neoplasia 11:213-28. TRACHEA: Ghabrial, A., Luschnig, S., Metzstein, M.M., and Krasnow, M.A. (2003). Branching Morphogenesis of the Drosophila Tracheal System. Annu. Rev. Cell Dev. Bio. 19:623-47. KIDNEY: Short, K.M. and Smyth, I.M. (2016). The contribution of branching morphogenesis to kidney development and disease. Nature Reviews Nephrology 12: 754-67. Costantini, F. (2006). Renal branching morphogenesis: concepts, questions, and recent advances. Differentiation 74:402-421. TUBULOGENESIS: Lubarsky, B. and Krasnow, M. (2003). Tube Morphogenesis: Making and Shaping Biological Tubes. Cell 112:19-28. 38