The endocycle is a modified cell cycle that lacks M phase. Endocycles are well known for enabling continued growth of post-mitotic tissues. By contrast, we discovered pre-mitotic endocycles in precursors of Drosophila rectal papillae ( papillar cells). Unlike all known proliferative Drosophila adult precursors, papillar cells endocycle before dividing. Furthermore, unlike diploid mitotic divisions, these polyploid papillar divisions are frequently error prone, suggesting papillar structures may accumulate long-term aneuploidy. Here, we demonstrate an indispensable requirement for pre-mitotic endocycles during papillar development and also demonstrate that such cycles seed papillar aneuploidy. We find blocking pre-mitotic endocycles disrupts papillar morphogenesis and causes organismal lethality under high-salt dietary stress. We further show that pre-mitotic endocycles differ from post-mitotic endocycles, as we find only the M-phase-capable polyploid cells of the papillae and female germline can retain centrioles. In papillae, this centriole retention contributes to aneuploidy, as centrioles amplify during papillar endocycles, causing multipolar anaphase. Such aneuploidy is well tolerated in papillae, as it does not significantly impair cell viability, organ formation or organ function. Together, our results demonstrate that pre-mitotic endocycles can enable specific organ construction and are a mechanism that promotes highly tolerated aneuploidy.

No abstract

The collective polarization of cellular structures and behaviors across a tissue plane is a near universal feature of epithelia known as planar cell polarity (PCP). This property is controlled by the core PCP pathway, which is comprised of highly conserved membrane-associated protein complexes that localize asymmetrically at cell junctions. Here we introduce three new mouse models for investigating the localization and dynamics of transmembrane PCP proteins Celsr1, Fz6, and Vangl2. Using the skin epidermis as a model, we characterize and verify the expression, localization and function of endogenously-tagged Celsr1-3xGFP, Fz6-3xGFP and tdTomato-Vangl2 fusion proteins. Live imaging of Fz6-3xGFP in basal epidermal progenitors reveals that the polarity of the tissue is not fixed through time. Rather asymmetry dynamically shifts during cell rearrangements and divisions, while global, average polarity of the tissue is preserved. We show using super-resolution STED imaging that Fz6-3xGFP and tdTomato-Vangl2 can be resolved, enabling us to observe their complex localization along junctions. We further explore PCP fusion protein localization in the trachea and neural tube, and discover new patterns of PCP expression and localization throughout the mouse embryo.

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.

A hallmark of mammalian lungs is the fractal nature of the bronchial tree. In the adult, each successive generation of airways is a fraction of the size of the parental branch. This fractal structure is physiologically beneficial, as it minimizes the energy needed for breathing. Achieving this pattern likely requires precise control of airway length and diameter, as the branches of the embryonic airways initially lack the fractal scaling observed in those of the adult lung. In epithelial monolayers and tubes, directional growth can be regulated by the planar cell polarity (PCP) complex. Here, we comprehensively characterized the roles of PCP-complex components in airway initiation, elongation, and widening during branching morphogenesis of the murine lung. Using tissue-specific knockout mice, we surprisingly found that branching morphogenesis proceeds independently of PCP-component expression in the developing airway epithelium. Instead, we found a novel, Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme. Specifically, mesenchymal loss of Vangl1/2 leads to defects in branch initiation, elongation, and widening. At the cellular level, we observe changes in the shape of smooth muscle cells that indicate a potential defect in collective mesenchymal rearrangements, which we hypothesize are necessary for lung morphogenesis. Our data thus reveal an explicit function for Vangl that is independent of the core PCP complex, suggesting a functional diversification of PCP components in vertebrate development. These data also reveal an essential role for the embryonic mesenchyme in generating the fractal structure of airways of the mature lung.

The planar cell polarity (PCP) complex orients cytoskeletal and multicellular organization throughout vertebrate development. PCP is speculated to function in formation of the murine lung, where branching morphogenesis generates a complex tree of tubular epithelia whose distal tips expand dramatically during sacculation in preparation for gas exchange after birth. Here, using tissue-specific knockouts, we show that the PCP complex is dispensable in the airway epithelium for sacculation. Rather, we find a novel, Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme: loss of Vangl1/2 inhibits mesenchymal thinning and expansion of the saccular epithelium. Further, loss of mesenchymal Wnt5a mimics the sacculation defects observed in Vangl2-mutant lungs, implicating mesenchymal Wnt5a/Vangl signaling as a key regulator of late lung morphogenesis. By mathematically modeling sacculation, we predict that the process of sacculation requires a fluid mesenchymal compartment. Finally, lineage-tracing and cell-shape analyses are consistent with the pulmonary mesenchyme acting as a fluid tissue, and suggest that loss of Vangl1/2 likely impacts the ability of mesenchymal cells to exchange neighbors. Our data thus uncover an explicit function for Vangl and the pulmonary mesenchyme during late lung morphogenesis to actively shape the saccular epithelium.

Nature has evolved a variety of mechanisms to build epithelial trees of diverse architectures within different organs and across species. Epithelial trees are elaborated through branch initiation and extension, and their morphogenesis ends with branch termination. Each of these steps of the branching process can be driven by the actions of epithelial cells themselves (epithelial-intrinsic mechanisms) or by the cells of their surrounding tissues (epithelial-extrinsic mechanisms). Here, we describe examples of how these mechanisms drive each stage of branching morphogenesis, drawing primarily on studies of the lung, kidney, salivary gland, mammary gland, and pancreas, all of which contain epithelial trees that form through collective cell behaviors. Much of our understanding of epithelial branching morphogenesis comes from experiments using mice, but we also include examples here from avian and reptilian models. Throughout, we highlight how distinct mechanisms are employed in different organs and species to build epithelial trees. We also highlight how similar morphogenetic motifs are used to carry out conserved developmental programs or repurposed to support novel ones. Understanding the unique strategies used by nature to build branched epithelia from across the tree of life can help to inspire creative solutions to problems in tissue engineering and regenerative medicine.

Accumulating evidence suggests the evolutionarily conserved Ras/mitogen activated protein kinase (MAPK) signaling pathway directs distinct biological consequences that depend on distinct intensity states. Given this intensity-dependence, we hypothesized that different levels of Ras/MAPK signaling may be subjected to regulation by different sets of proteins. To identify such 'differential' signaling modifiers, we turned to the Drosophila eye as a model. First, we created flies whereby mutant active Ras V12 expressed in the eyes was enriched with either rare or commonly occurring codons. We show that this codon manipulation can generate either low or high levels of Ras protein and MAPK signaling, and correspondingly a mild or severe rough-eye phenotype. Using this novel platform to control Ras/MAPK signaling, we performed a wholegenome haploinsufficiency deficiency screen, which yielded 15 deficiencies that modify the rougheye phenotype in either high or low MAPK signaling states, but not both. We then mapped the underlying gene from one deficiency to the gene RpS21. Disrupting RpS21 expression increases MAPK signaling and enhances the rough-eye phenotype specifically when Ras V12 is encoded by rare codons (low signaling), and RpS21 negatively regulates Ras protein and MAPK signaling in several contexts. We also provide evidence that the MAPK pathway promotes expression of RpS21, providing potential negative feedback. Taken together, these data reveal intensityspecific regulation of Ras/MAPK signaling, with the first candidate of this class being RpS21.