Surface tension determines tissue shape and growth kinetics.

The collective self-organization of cells into three-dimensional structures can give rise to emergent physical properties such as fluid behavior. Here, we demonstrate that tissues growing on curved surfaces develop shapes with outer boundaries of constant mean curvature, similar to the energy minimizing forms of liquids wetting a surface. The amount of tissue formed depends on the shape of the substrate, with more tissue being deposited on highly concave surfaces, indicating a mechano-biological feedback mechanism. Inhibiting cell-contractility further revealed that active cellular forces are essential for generating sufficient surface stresses for the liquid-like behavior and growth of the tissue. This suggests that the mechanical signaling between cells and their physical environment, along with the continuous reorganization of cells and matrix is a key principle for the emergence of tissue shape.

A Behavioral Assay to Study Effects of Retinoid Pharmacology on Nervous System Development in a Marine Annelid.

Autonomous animal locomotion, such as swimming, is modulated by neuronal networks acting on cilia or muscles. Understanding how these networks are formed and coordinated is a complex scientific problem, which requires various technical approaches. Among others, behavioral studies of developing animals treated with exogenous substances have proven to be a successful approach for studying the functions of neuronal networks. One such substance crucial for the proper development of the nervous system is the vitamin A-derived morphogen retinoic acid (RA). In the larva of the marine annelid Platynereis dumerilii , for example, RA is involved in the specification and differentiation of individual neurons and responsible for orchestrating the swimming behavior of the developing larva. Here, we report a workflow to analyze the effects of RA on the locomotion of the P. dumerilii larva. We provide a protocol for both the treatment with RA and the recording of larval swimming behavior. Additionally, we present a pipeline for the analysis of the obtained data in terms of swimming speed and movement trajectory. This chapter thus summarizes the methodology for analyzing the effects of a specific drug treatment on larval swimming behavior. We expect this approach to be readily adaptable to a wide variety of pharmacological compounds and aquatic species.

Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila.

Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.

Drosophila brain development: closing the gap between a macroarchitectural and microarchitectural approach.

Neurobiologists address neural structure, development, and function at the level of "macrocircuits" (how different brain compartments are interconnected; what overall pattern of activity they produce) and at the level of "microcircuits" (how connectivity and physiology of individual neurons and their processes within a compartment determine the functional output of this compartment). Work in our lab aims at reconstructing the developing Drosophila brain at both levels. Macrocircuits can be approached conveniently by reconstructing the pattern of brain lineages, which form groups of neurons whose projections form cohesive fascicles interconnecting the compartments of the larval and adult brain. The reconstruction of microcircuits requires serial section electron microscopy, due to the small size of terminal neuronal processes and their synaptic contacts. Because of the amount of labor that traditionally comes with this approach, very little is known about microcircuitry in brains across the animal kingdom. Many of the problems of serial electron microscopy reconstruction are now solvable with digital image recording and specialized software for both image acquisition and postprocessing. In this chapter, we introduce our efforts to reconstruct the small Drosophila larval brain and discuss our results in light of the published data on neuropile ultrastructure in other animal taxa.

Iron-regulatory protein-1 (IRP-1) is highly conserved in two invertebrate species--characterization of IRP-1 homologues in Drosophila melanogaster and Caenorhabditis elegans.

Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, we now identify the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.

Oocyte polarity depends on regulation of gurken by Vasa.

Vasa, a DEAD box mRNA helicase similar to eIF4A, is involved in pole plasm assembly in the Drosophila oocyte and appears to regulate translation of oskar and nanos mRNAs. However, several vasa alleles exhibit a wide range of early oogenesis phenotypes. Here we report a detailed analysis of Vasa function during early oogenesis using novel as well as previously identified hypomorphic vasa alleles. We find that vasa is required for the establishment of both anterior-posterior and dorsal-ventral polarity of the oocyte. The polarity defects of vasa mutants appear to be caused by a reduction in the amount of Gurken protein at stages of oogenesis critical for the establishment of polarity. Vasa is required for translation of gurken mRNA during early oogenesis and for achieving wild-type levels of gurken mRNA expression later in oogenesis. A variety of early oogenesis phenotypes observed in vasa ovaries, which cannot be attributed to the defect in gurken expression, suggest that vasa also affects expression of other mRNAs.