Slit is secreted by cells at the midline of the central nervous system, where it binds to Roundabout (Robo) receptors and functions as a potent repellent. We found that migrating mesodermal cells in vivo respond to Slit as both an attractant and a repellent and that Robo receptors are required for both functions. Mesoderm cells expressing Robo receptors initially migrate away from Slit at the midline. A few hours after migration, these same cells change their behavior and require Robo to extend toward Slit-expressing muscle attachment sites. Thus, Slit functions as a chemoattractant to provide specificity for muscle patterning.
During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad–mediated cell adhesion.
Heart morphogenesis requires the coordinated regulation of cell movements and cell-cell interactions between distinct populations of cardiac precursor cells. Little is known about the mechanisms that organize cardiac cells into this complex structure. In this study, we analyzed the role of Slit, an extracellular matrix protein and its transmembrane receptors Roundabout (Robo) and Roundabout2 (Robo2) during morphogenesis of the Drosophila heart tube, a process analogous to early heart formation in vertebrates. During heart assembly, two types of progenitor cells align into rows and coordinately migrate to the dorsal midline of the embryo, where they merge to assemble a linear heart tube. Here we show that cardiac-specific expression of Slit is required to maintain adhesion between cells within each row during dorsal migration. Moreover, differential Robo expression determines the relative distance each row is positioned from the dorsal midline. The innermost CBs express only Robo, whereas the flanking pericardial cells express both receptors. Removal of robo2 causes pericardial cells to shift toward the midline, whereas ectopic robo2 in CBs drives them laterally, resulting in an unfused heart tube. We propose a model in which Slit has a dual role during assembly of the linear heart tube, functioning to regulate both cell positioning and adhesive interactions between migrating cardiac precursor cells.cell adhesion ͉ dorsal vessel ͉ heart morphogenesis
The formation of a multinucleated muscle fiber from individual myoblasts is a complex morphological event that requires dramatic cytoskeletal rearrangements. This multistep process includes myoblast fusion, myotube migration and elongation, myotube target recognition, and finally attachment to form a stable adhesion complex. Many of the studies directed towards understanding the developmental process of muscle morphogenesis at the cellular level have relied on forward genetic screens in model systems such as Drosophila melanogaster for mutations affecting individual stages in myogenesis. Through the analyses of these gene products, proteins that regulate the actin or microtubule cytoskeleton have emerged as important players in each of these steps. We recently demonstrated that RacGAP50C, an essential protein that functions as a cytoskeletal regulator during cell division, also plays an important role in organizing the polarized microtubule network in the elongating myotube. Here we review the current literature regarding Drosophila myogenesis and illustrate several steps of muscle development with respect to the diverse roles that the cytoskeleton plays during this process. Furthermore, we discuss the significance of cytoskeletal coordination during these multiple steps.
The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. We have identified a novel postmitotic function for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. We show that the localization of the MT nucleator γ-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and that this change in localization depends on RacGAP50C. RacGAP50C and γ-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants γ-tubulin remains dispersed throughout the cytoplasm. Furthermore, we show that the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute γ-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process.
Drosophila embryonic dorsal vessel (DV) morphogenesis is a highly stereotyped process that involves the migration and morphogenesis of 52 pairs of cardioblasts (CBs) in order to form a linear tube. This process requires spatiotemporally-regulated localization of signaling and adhesive proteins in order to coordinate the formation of a central lumen while maintaining simultaneous adhesion between CBs. Previous studies have shown that the Slit/Roundabout and Netrin/Unc5 repulsive signaling pathways facilitate site-specific loss of adhesion between contralateral CBs in order to form a luminal space. However, the concomitant mechanism by which attraction initiates CB outgrowth and discrete localization of adhesive proteins remains poorly understood. Here we provide genetic evidence that Netrin signals through DCC (Deleted in Colorectal Carcinoma)/UNC-40/Frazzled (Fra) to mediate CB outgrowth and attachment and that this function occurs prior to and independently of Netrin/UNC-5 signaling. fra mRNA is expressed in the CBs prior to and during DV morphogenesis. Loss-of-fra-function results in significant defects in cell shape and alignment between contralateral CB rows. In addition, CB outgrowth and attachment is impaired in both fra loss- and gain-of-function mutants. Deletion of both Netrin genes (NetA and NetB) results in CB attachment phenotypes similar to fra mutants. Similar defects are also seen when both fra and unc5 are deleted. Finally we show that Fra accumulates at dorsal and ventral leading edges of paired CBs, and this localization is dependent upon Netrin. We propose that while repulsive guidance mechanisms contribute to lumen formation by preventing luminal domains from coming together, site-specific Netrin/Frazzled signaling mediates CB attachment.
The embryonic heart tube is formed by the migration and subsequent midline convergence of two bilateral heart fields. In Drosophila the heart fields are organized into two rows of cardioblasts (CBs). While morphogenesis of the dorsal ectoderm, which lies directly above the Drosophila dorsal vessel (DV), has been extensively characterized, the migration and concomitant fundamental factors facilitating DV formation remain poorly understood. Here we provide evidence that DV closure occurs at multiple independent points along the A-P axis of the embryo in a “buttoning” pattern, divergent from the zippering mechanism observed in the overlying epidermis during dorsal closure. Moreover, we demonstrate that a genetically distinct subset of CBs is programmed to make initial contact with the opposing row. To elucidate the cellular mechanisms underlying this process, we examined the role of Rho GTPases during cardiac migration using inhibitory and overexpression approaches. We found that Cdc42 shows striking cell-type specificity during DV formation. Disruption of Cdc42 function specifically prevents CBs that express the homeobox gene tinman from completing their dorsal migration, resulting in a failure to make connections with their partnering CBs. Conversely, neighboring CBs that express the orphan nuclear receptor, seven-up, are not sensitive to Cdc42 inhibition. Furthermore, this phenotype was specific to Cdc42 and was not observed upon perturbation of Rac or Rho function. Together with the observation that DV closure occurs through the initial contralateral pairing of tinman-expressing CBs, our studies suggest that the distinct buttoning mechanism we propose for DV closure is elaborated through signaling pathways regulating Cdc42 activity in this cell type.
The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.
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