Tubulogenesis: Lipid-lining the path to sparkling gas filling.

Clearance of liquid and gas filling of airways is vital for animal respiration. New research shows that a surfactant film of exosomal-derived lipids is built at the air-liquid interface of Drosophila airways before gas filling. Coordinated lysosomal and vesicular pathways synergize to assemble this lipid layer, which is essential for respiration and survival.

Time-Lapse Imaging and Morphometric Analysis of Tracheal Development in Drosophila.

Detailed and quantitative analyses of the cellular events underlying the formation of specific organs or tissues is essential to understand the general mechanisms of morphogenesis and pattern formation. Observation of live tissues or whole-mount fixed specimens has emerged as the method of choice for identifying and quantifying specific cellular and tissular structures within the organism. In both cases, cell and subcellular structure identification and good quality image acquisition for these analyses are essential. Many markers for live imaging and fixed tissue are now available for detecting cell membranes, subcellular structures, and extracellular structures like the extracellular matrix (ECM). Combination of live imaging and analysis of fixed tissue is ideal to obtain a general and detailed picture of the events underlying embryonic development. By applying morphometric methods to both approaches, we can, in addition, obtain a quantitative evaluation of the specific parameters under investigation in morphogenetic and cell biological studies. In this chapter, we focus on the development of the tracheal system of Drosophila melanogaster, which provides an ideal paradigm to understand the formation of branched tubular organs. We describe the most used methods of imaging and morphometric analysis in tubulogenesis using mainly (but not exclusively) examples from embryonic development. We cover embryo preparation for fixed and live analysis of tubulogenesis, together with methods to visualize larval tracheal terminal cell branching and lumen formation. Finally, we describe morphometric analysis and quantification methods using fluorescent images of tracheal cells.

Cytoskeletal players in single-cell branching morphogenesis.

Branching networks are a very common feature of multicellular animals and underlie the formation and function of numerous organs including the nervous system, the respiratory system, the vasculature and many internal glands. These networks range from subcellular structures such as dendritic trees to large multicellular tissues such as the lungs. The production of branched structures by single cells, so called subcellular branching, which has been better described in neurons and in cells of the respiratory and vascular systems, involves complex cytoskeletal remodelling events. In Drosophila, tracheal system terminal cells (TCs) and nervous system dendritic arborisation (da) neurons are good model systems for these subcellular branching processes. During development, the generation of subcellular branches by single-cells is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. In this review, we describe the current knowledge on cytoskeletal regulation of subcellular branching, based on the terminal cells of the Drosophila tracheal system, but drawing parallels with dendritic branching and vertebrate vascular subcellular branching.

Virtual meeting, real and sound science: report of the 17th Meeting of the Spanish Society for Developmental Biology (SEBD-2020).

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.

Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching.

Subcellular lumen formation by single-cells involves complex cytoskeletal remodelling. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation in Drosophila melanogaster, but not much is known on the what leads to the growth of these subcellular luminal branches or makes them progress through a particular trajectory within the cytoplasm. Here, we have identified that the spectraplakin Short-stop (Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the tracheal terminal cell (TC) cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal luminal branching points in the embryonic and larval tracheal TC leading to cells with extra-subcellular lumina. These data provide the first evidence for a role for spectraplakins in single-cell lumen formation and branching.

Nucleotide excision repair genes shaping embryonic development.

Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA lesions. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human disorders caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated ageing. All three syndromes include developmental abnormalities, indicating an important role for optimal transcription and for NER in protecting against spontaneous DNA damage during embryonic development. Here, we review the current knowledge on genes that function in NER that also affect embryonic development, in particular the development of a fully functional nervous system.

Centrosomes in Branching Morphogenesis.

The centrosome, a major microtubule organizer, has important functions in regulating the cytoskeleton as well as the position of cellular structures and orientation of cells within tissues. The centrosome serves as the main cytoskeleton-organizing centre in the cell and is the classical site of microtubule nucleation and anchoring. For these reasons, centrosomes play a very important role in morphogenesis, not just in the early stages of cell divisions but also in the later stages of organogenesis. Many organs such as lung, kidney and blood vessels develop from epithelial tubes that branch into complex networks. Cells in the nervous system also form highly branched structures in order to build complex neuronal networks. During branching morphogenesis, cells have to rearrange within tissues though multicellular branching or through subcellular branching, also known as single-cell branching. For highly branched structures to be formed during embryonic development, the cytoskeleton needs to be extensively remodelled. The centrosome has been shown to play an important role during these events.

Blimp-1 Mediates Tracheal Lumen Maturation in Drosophila melanogaster.

The specification of tissue identity during embryonic development requires precise spatio-temporal coordination of gene expression. Many transcription factors required for the development of organs have been identified and their expression patterns are known; however, the mechanisms through which they coordinate gene expression in time remain poorly understood. Here, we show that hormone-induced transcription factor Blimp-1 participates in the temporal coordination of tubulogenesis in Drosophila melanogaster by regulating the expression of many genes involved in tube maturation. In particular, we demonstrate that Blimp-1 regulates the expression of genes involved in chitin deposition and F-actin organization. We show that Blimp-1 is involved in the temporal control of lumen maturation by regulating the beginning of chitin deposition. We also report that Blimp-1 represses a variety of genes involved in tracheal maturation. Finally, we reveal that the kinase Btk29A serves as a link between Blimp-1 transcriptional repression and apical extracellular matrix organization.

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse.

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms.

SNARE complex in axonal guidance and neuroregeneration.

Through complex mechanisms that guide axons to the appropriate routes towards their targets, axonal growth and guidance lead to neuronal system formation. These mechanisms establish the synaptic circuitry necessary for the optimal performance of the nervous system in all organisms. Damage to these networks can be repaired by neuroregenerative processes which in turn can re-establish synapses between injured axons and postsynaptic terminals. Both axonal growth and guidance and the neuroregenerative response rely on correct axonal growth and growth cone responses to guidance cues as well as correct synapses with appropriate targets. With this in mind, parallels can be drawn between axonal regeneration and processes occurring during embryonic nervous system development. However, when studying parallels between axonal development and regeneration many questions still arise; mainly, how do axons grow and synapse with their targets and how do they repair their membranes, grow and orchestrate regenerative responses after injury. Major players in the cellular and molecular processes that lead to growth cone development and movement during embryonic development are the Soluble N-ethylamaleimide Sensitive Factor (NSF) Attachment Protein Receptor (SNARE) proteins, which have been shown to be involved in axonal growth and guidance. Their involvement in axonal growth, guidance and neuroregeneration is of foremost importance, due to their roles in vesicle and membrane trafficking events. Here, we review the recent literature on the involvement of SNARE proteins in axonal growth and guidance during embryonic development and neuroregeneration.

SNARE proteins play a role in motor axon guidance in vertebrates and invertebrates.

Axonal growth and guidance rely on correct growth cone responses to guidance cues, both in the central nervous system (CNS) and in the periphery. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the cross-talk mechanisms between guidance and membrane dynamics and turnover in the axon. Our studies have shown that Netrin-1/deleted in colorectal cancer signaling triggers exocytosis through the SNARE Syntaxin-1 (STX-1) during the formation of commissural pathways. However, limited in vivo evidence is available about the role of SNARE proteins in motor axonal guidance. Here we show that loss-of-function of SNARE complex members results in motor axon guidance defects in fly and chick embryos. Knock-down of Syntaxin-1, VAMP-2, and SNAP-25 leads to abnormalities in the motor axon routes out of the CNS. Our data point to an evolutionarily conserved role of the SNARE complex proteins in motor axon guidance, thereby pinpointing an important function of SNARE proteins in axonal navigation in vivo. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 963-974, 2017.

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects.

A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea.

The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation.

Drosophila chitinous aECM and its cellular interactions during tracheal development.

The morphology of organs, and hence their proper physiology, relies to a considerable extent on the extracellular matrix (ECM) secreted by their cells. The ECM is a structure contributed to and commonly shared by many cells in an organism that plays an active role in morphogenesis. Increasing evidence indicates that the ECM not only provides a passive contribution to organ shape but also impinges on cell behaviour and genetic programmes. The ECM is emerging as a direct modulator of many aspects of cell biology, rather than as a mere physical network that supports cells. Here, we review how the apical chitinous ECM is generated in Drosophila trachea and how cells participate in the formation of this supracellular structure. We discuss recent findings on the molecular and cellular events that lead to the formation of this apical ECM (aECM) and how it is influenced and affects tracheal cell biology.

The Hedgehog Signalling Pathway in Cell Migration and Guidance: What We Have Learned from Drosophila melanogaster.

Cell migration and guidance are complex processes required for morphogenesis, the formation of tumor metastases, and the progression of human cancer. During migration, guidance molecules induce cell directionality and movement through complex intracellular mechanisms. Expression of these molecules has to be tightly regulated and their signals properly interpreted by the receiving cells so as to ensure correct navigation. This molecular control is fundamental for both normal morphogenesis and human disease. The Hedgehog (Hh) signaling pathway is evolutionarily conserved and known to be crucial for normal cellular growth and differentiation throughout the animal kingdom. The relevance of Hh signaling for human disease is emphasized by its activation in many cancers. Here, I review the current knowledge regarding the involvement of the Hh pathway in cell migration and guidance during Drosophila development and discuss its implications for human cancer origin and progression.

Drosophila melanogaster Hedgehog cooperates with Frazzled to guide axons through a non-canonical signalling pathway.

We report that the morphogen Hedgehog (Hh) is an axonal chemoattractant in the midline of Drosophila melanogaster embryos. Hh is present in the ventral nerve cord during axonal guidance and overexpression of hh in the midline causes ectopic midline crossing of FasII-positive axonal tracts. In addition, we show that Hh influences axonal guidance via a non-canonical signalling pathway dependent on Ptc. Our results reveal that the Hh pathway cooperates with the Netrin/Frazzled pathway to guide axons through the midline in invertebrates.