Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain

In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects

Klf15 Is Critical for the Development and Differentiation of Drosophila Nephrocytes

Insect nephrocytes are highly endocytic scavenger cells that represent the only invertebrate model for the study of human kidney podocytes. Despite their importance, nephrocyte development is largely uncharacterised. This work tested whether the insect ortholog of mammalian Kidney Krüppel-Like Factor (Klf15), a transcription factor required for mammalian podocyte differentiation, was required for insect nephrocyte development. It was found that expression of Drosophila Klf15 (dKlf15, previously known as Bteb2) was restricted to the only two nephrocyte populations in Drosophila, the garland cells and pericardial nephrocytes. Loss of dKlf15 function led to attrition of both nephrocyte populations and sensitised larvae to the xenotoxin silver nitrate. Although pericardial nephrocytes in dKlf15 loss of function mutants were specified during embryogenesis, they failed to express the slit diaphragm gene sticks and stones and did not form slit diaphragms. Conditional silencing of dKlf15 in adults led to reduced surface expression of the endocytic receptor Amnionless and loss of in vivo scavenger function. Over-expression of dKlf15 increased nephrocyte numbers and rescued age-dependent decline in nephrocyte function. The data place dKlf15 upstream of sns and Amnionless in a nephrocyte-restricted differentiation pathway and suggest dKlf15 expression is both necessary and sufficient to sustain nephrocyte differentiation. These findings explain the physiological relevance of dKlf15 in Drosophila and imply that the role of KLF15 in human podocytes is evolutionarily conserve

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns

Protonation-Gated Dual Photochromism of a Chromene–Styryl Dye Hybrid

A novel hybrid bisphotochromic system involving a chromene residue and a styryl dye fragment is described. Initially, the compound shows almost no photochromism due to intramolecular energy and electron transfer between the chromophores. Protonation of the hybrid system blocks the transfer processes and unlocks a dual photochromic activity that includes ring opening of the chromene fragment and E Z isomerization of the styryl dye residue. Deprotonation of any photoinduced form resets the system to the initial photoinactive form

Grecocyclines: New angucyclines from streptomyces sp. Acta 1362

Two novel angucyclines were isolated from the streptomycete Acta 1362. The strain was of particular interest regarding the production of characteristic metabolites that were detected by HPLC-diode array profiling of the extracts. Grecocycline A and B were isolated and their structures were determined, Grecocycline A shows cytotoxic activity and grecocycline B inhibits protein tyrosine phosphatase IB. Moreover, shunt product grecocyline C was isolated and its structure was determined. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA

Defective Decapentaplegic Signaling Results in Heart Overgrowth and Reduced Cardiac Output in Drosophila

During germ-band extension, Decapentaplegic (Dpp) signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. We showed previously that a second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Here we report that, via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. Our data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. Our study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects