Mark A. Krasnow

sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways.

Antagonists of several growth factor signaling pathways play important roles in developmental patterning by limiting the range of the cognate inducer. Here, we describe an antagonist of FGF signaling that patterns apical branching of the Drosophila airways. In wild-type embryos, the Branchless FGF induces secondary branching by activating the Breathless FGF receptor near the tips of growing primary branches. In sprouty mutants, the FGF pathway is overactive and ectopic branches are induced on the stalks of primary branches. We show that FGF signaling induces sprouty expression in the nearby tip cells, and sprouty acts nonautonomously and in a competitive fashion to block signaling to the more distant stalk cells. sprouty encodes a novel cysteine-rich protein that defines a new family of putative signaling molecules that may similarly function as FGF antagonists in vertebrate development.

branchless Encodes a Drosophila FGF Homolog That Controls Tracheal Cell Migration and the Pattern of Branching

The molecular basis for patterning of complex organ structures like the lung and insect tracheal system is unknown. Here, we describe the Drosophila gene branchless (bnl) and demonstrate that it is a key determinant of the tracheal branching pattern. bnl is required for tracheal branching and is expressed dynamically in clusters of cells surrounding the developing tracheal system at each position where a new branch will form and grow out. Localized misexpression of bnl can direct branch formation and outgrowth to new positions. Generalized misexpression activates later programs of tracheal gene expression and branching, resulting in massive networks of branches. bnl encodes a homolog of mammalian fibroblast growth factors (FGFs) and appears to function as a ligand for the breathless receptor tyrosine kinase, an FGF receptor homolog expressed on developing tracheal cells. The results suggest that this FGF pathway specifies the tracheal branching pattern by guiding tracheal cell migration during primary branch formation and then activating later programs of finer branching at the ends of growing primary branches.

Tube Morphogenesis: Making and Shaping Biological Tubes

Many organs are composed of epithelial tubes that transport vital fluids. Such tubular organs develop in many different ways and generate tubes of widely varying sizes and structures, but always with the apical epithelial surface lining the lumen. We describe recent progress in several diverse cell culture and genetic models of tube morphogenesis, which suggest apical membrane biogenesis, vesicle fusion, and secretion play central roles in tube formation and growth. We propose a unifying mechanism of tube morphogenesis that has been modified to create tube diversity and describe how defects in the tube size-sensing step can lead to polycystic kidney disease.

Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

The branching programme of mouse lung development

Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular programme is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs.

Coronary arteries form by developmental reprogramming of venous cells

Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus—the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.

Alveolar progenitor and stem cells in lung development, renewal and cancer

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

Cellular and Genetic Analysis of Wound Healing in Drosophila Larvae

To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution.

Developmental Control of Blood Cell Migration by the Drosophila VEGF Pathway

We show that a vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E or Vegf27Cb have no effect, but simultaneous inactivation of all three Vegf genes phenocopied the receptor mutant, and ectopic expression of Vegf27Cb redirected migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and we speculate that the ancestral function of VEGF pathways was to guide blood cell movement.

Transcriptional Activation and Repression by Ultrabithorax Proteins in Cultured Drosophila Cells

Homeotic genes of Drosophila melanogaster such as Ultrabithorax (Ubx) and Antennapedia (Antp) have long been thought to select metameric identity during development by controlling the expression of various target genes. Here we describe a cotransfection assay in cultured D. melanogaster cells that is used to demonstrate that Ubx proteins (UBX) can repress an Antp promoter fusion and activate a Ubx promoter fusion, activities predicted from genetic studies. We show (a) that UBX proteins regulated the level of accurately initiated Antp P1 and Ubx transcripts, (b) that activation of the Ubx promoter required a downstream cluster of UBX binding sites, and (c) that binding site sequences were sufficient to confer regulation on a heterologous promoter, regardless of their orientation or precise position. We conclude that UBX proteins are transcriptional repressors and activators, and that their actions are mediated by binding to promoter region sequences. Each member of the UBX protein family has similar regulatory abilities, but the properties of synthetic mutant forms suggest that UBX proteins may have a modular design similar to other transcriptional regulators.