Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila.

A BMP gradient is essential for patterning the dorsal-ventral axis of invertebrate and vertebrate embryos. The extracellular BMP binding protein Short Gastrulation (Sog) in Drosophila plays a key role in BMP gradient formation. In this study, we combine genome editing, structural and developmental approaches to study Sog function in Drosophila. We generate a sog knockout fly stock, which allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, we test the requirement for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, we show that the sogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo. Additionally, we use experimental negative stain EM imaging and hydrodynamic data to validate the AlphaFold structure prediction for Sog. The model suggests a more compact shape than the vertebrate ortholog Chordin and conformational flexibility between the C-terminal von Willebrand C domains. We discuss how this altered compactness may contribute to mechanistic differences in Sog and Chordin function during BMP gradient formation. This article has an associated First Person interview with the first author of the paper.

The developing wing crossvein of Drosophila melanogaster: a fascinating model for signaling and morphogenesis.

The Drosophila wing has been used as a model for studying tissue growth, morphogenesis and pattern formation. The wing veins of Drosophila are composed of two distinct structures, longitudinal veins and crossveins. Although positional information of longitudinal veins is largely defined in the wing imaginal disc during the larval stage, crossvein primordial cells appear to be naive until the early pupal stage. Here, we first review how wing crossveins have been investigated in the past. Then, the developmental mechanisms underlying crossvein formation are summarized. This review focuses on how a conserved trafficking mechanism of BMP ligands is utilized for crossvein formation, and how various co-factors play roles in sustaining BMP signalling. Recent findings further reveal that crossvein development serves as an excellent model to address how BMP signal and dynamic cellular processes are coupled. This comprehensive review illustrates the uniqueness, scientific value and future perspectives of wing crossvein development as a model.

BMP antagonists in tissue development and disease.

Bone morphogenic proteins (BMPs) are important growth regulators in embryogenesis and postnatal homeostasis. Their tight regulation is crucial for successful embryonic development as well as tissue homeostasis in the adult organism. BMP inhibition by natural extracellular biologic antagonists represents the most intensively studied mechanistic concept of BMP growth factor regulation. It was shown to be critical for numerous developmental programs, including germ layer specification and spatiotemporal gradients required for the establishment of the dorsal-ventral axis and organ formation. The importance of BMP antagonists for extracellular matrix homeostasis is illustrated by the numerous human connective tissue disorders caused by their mutational inactivation. Here, we will focus on the known functional interactions targeting BMP antagonists to the ECM and discuss how these interactions influence BMP antagonist activity. Moreover, we will provide an overview about the current concepts and investigated molecular mechanisms modulating BMP inhibitor function in the context of development and disease.

Distinct Roles of Broadly Expressed Repressors Support Dynamic Enhancer Action and Change in Time.

How broadly expressed repressors regulate gene expression is incompletely understood. To gain insight, we investigated how Suppressor of Hairless-Su(H)-and Runt regulate expression of bone morphogenetic protein (BMP) antagonist short-gastrulation via the sog_Distal enhancer. A live imaging protocol was optimized to capture this enhancer's spatiotemporal output throughout the early Drosophila embryo, finding in this context that Runt regulates transcription initiation, Su(H) regulates transcription rate, and both factors control spatial expression. Furthermore, whereas Su(H) functions as a dedicated repressor, Runt temporally switches from repressor to activator. Our results demonstrate that broad repressors play temporally distinct roles and contribute to dynamic gene expression. Both Run and Su(H)'s ability to influence the spatiotemporal domains of gene expression may serve to counterbalance activators and function in this manner as important regulators of the maternal-to-zygotic transition in early embryos.

A Developmental Program Truncates Long Transcripts to Temporally Regulate Cell Signaling.

Rapid mitotic divisions and a fixed transcription rate limit the maximal length of transcripts in early Drosophila embryos. Previous studies suggested that transcription of long genes is initiated but aborted, as early nuclear divisions have short interphases. Here, we identify long genes that are expressed during short nuclear cycles as truncated transcripts. The RNA binding protein Sex-lethal physically associates with transcripts for these genes and is required to support early termination to specify shorter transcript isoforms in early embryos of both sexes. In addition, one truncated transcript for the gene short-gastrulation encodes a product in embryos that functionally relates to a previously characterized dominant-negative form, which maintains TGF-ß signaling in the off-state. In summary, our results reveal a developmental program of short transcripts functioning to help temporally regulate Drosophila embryonic development, keeping cell signaling at early stages to a minimum in order to support its proper initiation at cellularization.

N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs.

Disorders of N-linked glycosylation are increasingly reported in the literature. However, the targets that are responsible for the associated developmental and physiological defects are largely unknown. Bone morphogenetic proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. Here, we investigate the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). We identify conserved N-glycosylated sites and describe the effect of mutating these residues on BMP pathway activity in Drosophila Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, we provide evidence that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.

zen and the art of phenotypic maintenance: canalization of embryonic dorsal-ventral patterning in Drosophila.

We recently uncovered a novel genetic mechanism that generates the phenotypic uniformity, or canalization, of BMP signaling and cell fate specification during patterning of the dorsal-ventral (D/V) axis in D. melanogaster embryos. We went on to show that other wild-type Drosophila species lack this canalizing genetic circuitry and, consequently, have non-robust D/V patterning. In this review, we propose molecular mechanisms that may give rise to stereotyped BMP signaling, and we identify an additional species that could have decanalized D/V patterning. Extension of these analyses could in turn help explain why canalization is not a universal necessity for species survival.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-ß superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.

Image analysis and empirical modeling of gene and protein expression.

Protein gradients and gene expression patterns are major determinants in the differentiation and fate map of the developing embryo. Here we discuss computational methods to quantitatively measure the positions of gene expression domains and the gradients of protein expression along the dorsal-ventral axis in the Drosophila embryo. Our methodology involves three layers of data. The first layer, or the primary data, consists of z-stack confocal images of embryos processed by in situ hybridization and/or antibody stainings. The secondary data are relationships between location, usually an x-axis coordinate, and fluorescent intensity of gene or protein detection. Tertiary data comprise the optimal parameters that arise from fits of the secondary data to empirical models. The tertiary data are useful to distill large datasets of imaged embryos down to a tractable number of conceptually useful parameters. This analysis allows us to detect subtle phenotypes and is adaptable to any set of genes or proteins with a canonical pattern. For example, we show how insights into the Dorsal transcription factor protein gradient and its target gene ventral-neuroblasts defective (vnd) were obtained using such quantitative approaches.