SummaryIntracellular targeting of mRNAs has long been recognized as a means to produce proteins locally, but has only recently emerged as a prevalent mechanism used by a wide variety of polarized cell types. Localization of mRNA molecules within the cytoplasm provides a basis for cell polarization, thus underlying developmental processes such as asymmetric cell division, cell migration, neuronal maturation and embryonic patterning. In this review, we describe and discuss recent advances in our understanding of both the regulation and functions of RNA localization during animal development.Key words: RNA localization, RNA transport, Local translation, Cell polarity, Post-transcriptional gene regulation Introduction Establishment of cell polarity is crucial for the execution of developmental programmes governing key processes, including specification of cell fates, individual or collective cell movements and specialization of somatic cell types. Cell polarization depends on the asymmetric segregation of organelles and various molecules within the cell. Polarized accumulation of RNA molecules was first visualized nearly 30 years ago, when -actin mRNA was found to be asymmetrically localized within ascidian eggs and embryos (Jeffery et al., 1983). Following this, the discovery of the first localized maternal mRNAs in Xenopus (Rebagliati et al., 1985) and Drosophila oocytes (Frigerio et al., 1986;Berleth et al., 1988) provided evidence for the earlier proposal that localized RNA determinants could be responsible for early embryonic patterning (Kandler-Singer and Kalthoff, 1976). mRNAs were soon found to be asymmetrically distributed within differentiated somatic cells, such as fibroblasts (Lawrence and Singer, 1986), oligodendrocytes (Trapp et al., 1987) and neurons (Garner et al., 1988), and to colocalize with their encoded proteins, establishing intracellular transport of mRNAs as a potential mechanism used to target the production of selected proteins to discrete sites.Significant improvements in RNA detection methods led to the identification of a growing number of localized mRNAs. Still, in the early 2000s, the set of described targeted mRNAs was limited to ~100 (reviewed by Bashirullah et al., 1998;Palacios and St Johnston, 2001) and the process of mRNA localization was thought to be restricted to specific cell types. However, recent genome-wide analyses (see Table 1) have changed this view dramatically, and strongly suggest that subcellular targeting of mRNAs is a prevalent mechanism used by polarized cells to establish functionally distinct compartments (Fig. 1). Particularly striking was the discovery that >70% of the 2314 expressed transcripts analysed in a highresolution in situ hybridization screen were subcellularly localized in Drosophila embryos (Lécuyer et al., 2007). Moreover, hundreds to thousands of mRNAs have been detected in cellular compartments as diverse as the mitotic apparatus (Blower et al., 2007;Sharp et al., 2011), pseudopodia (Mili et al., 2008), dendrites (Moccia et al., 2003;Poon et a...
In mammals, the JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is activated in response to cytokines and growth factors to control blood cell development, proliferation and cell determination. In Drosophila, a conserved JAK/STAT signaling pathway controls segmentation in embryos, as well as blood cell development and other processes in larvae and adults. During embryogenesis, transduction of the Unpaired [Upd; also known as Outstretched (Os)] ligand through the JAK/STAT pathway requires Domeless, a putative membrane protein with distant homology to vertebrate type I cytokine receptors. We have isolated domeless(dome) in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, we show that dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process,dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, the Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. We also show that domefunctions downstream of upd and that both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether,our data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis.
Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis.
Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues [1-4]. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches [5]. Here, we identify the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting [6-12]. We show that whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, we demonstrate that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. Our study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation.
The basement membrane (BM) represents a barrier to cell migration, which has to be degraded to promote invasion. However, the role and behaviour of the BM during the development of pre-invasive cells is only poorly understood. Drosophila border cells (BCs) provide an attractive genetic model in which to study the cellular mechanisms underlying the migration of mixed cohorts of epithelial cells. BCs are made of two different epithelial cell types appearing sequentially during oogenesis: the polar cells and the outer BCs. Here, we show that the pre-invasive polar cells undergo an unusual and asymmetrical apical capping with major basement membrane proteins, including the two Drosophila Collagen IV α chains, Laminin A and Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical transcytosis mechanism that involves the small GTPase Drab5. Apical capping is transient and is followed by rapid shedding prior to the initiation of BC migration, suggesting that the apical cap blocks migration. Consistently,non-migratory polar cells remain capped. We further show that JAK/STAT signalling and recruitment of outer BCs are required for correct shedding and migration. The dynamics of the BM represents a marker of migratory BC,revealing a novel developmentally regulated behaviour of BM coupled to epithelial cell invasiveness.
Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart. Developmental Dynamics 239:3303-3311,
Axonal transport is essential for the initial growth, maintenance and synaptic plasticity of axons, and altered axonal transport has been observed in different models of neurodegenerative pathologies. Dissecting the mechanisms underlying axonal transport in developing or degenerating brains requires dynamic imaging of axonal cargo movement in living samples. Whereas methods exist to image axonal transport in Drosophila larval neurons, they are not suitable to follow this process during metamorphosis, when brains undergo extensive remodeling. Here we present a simple method that enables confocal imaging of both fast and slow axonal transport in Drosophila pupal brain explants. We describe how to prepare chambers adapted for live imaging, how to maintain brain explants under physiological conditions and how to monitor and quantitatively analyze the movement of fluorescently labeled cargoes. This protocol requires minimal equipment and is ideally suited for experiments that combine genetics, optogenetics and pharmacological approaches. The brains can be prepared for image acquisition in 1.5 h, and the protocol can be performed easily in any fly laboratory.
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