Brain bilateral asymmetry - insights from nematodes, zebrafish, and Drosophila.

Chirality is a fundamental trait of living organisms, encompassing the homochirality of biological molecules and the left-right (LR) asymmetry of visceral organs and the brain. The nervous system in bilaterian organisms displays a lateralized organization characterized by the presence of asymmetrical neuronal circuits and brain functions that are predominantly localized within one hemisphere. Although body asymmetry is relatively well understood, and exhibits robust phenotypic expression and regulation via conserved molecular mechanisms across phyla, current findings indicate that the asymmetry of the nervous system displays greater phenotypic, genetic, and evolutionary variability. In this review we explore the use of nematode, zebrafish, and Drosophila genetic models to investigate neuronal circuit asymmetry. We discuss recent discoveries in the context of body-brain concordance and highlight the distinct characteristics of nervous system asymmetry and its cognitive correlates.

Biological effects of the loss of homochirality in a multicellular organism.

Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-L-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism's health, we use chiral-selective in vivo assays to detect protein-bound non-L-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. We find that altering the in vivo chiral balance creates a 'heterochirality syndrome' with impaired caspase activity, increased tumour formation, and premature death. Our work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.

A dynamic and mosaic basement membrane controls cell intercalation in Drosophila ovaries.

Basement membranes (BM) are extracellular matrices assembled into complex and highly organized networks essential for organ morphogenesis and function. However, little is known about the tissue origin of BM components and their dynamics in vivo Here, we unravel the assembly and role of the BM main component, Collagen type IV (ColIV), in Drosophila ovarian stalk morphogenesis. Stalks are short strings of cells assembled through cell intercalation that link adjacent follicles and maintain ovarian integrity. We show that stalk ColIV has multiple origins and is assembled following a regulated pattern leading to a unique BM organisation. Absence of ColIV leads to follicle fusion, as observed upon ablation of stalk cells. ColIV and integrins are both required to trigger cell intercalation and maintain mechanically strong cell-cell attachment within the stalk. These results show how the dynamic assembly of a mosaic BM controls complex tissue morphogenesis and integrity.

The Drosophila actin nucleator DAAM is essential for left-right asymmetry.

Left-Right (LR) asymmetry is essential for organ positioning, shape and function. Myosin 1D (Myo1D) has emerged as an evolutionary conserved chirality determinant in both Drosophila and vertebrates. However, the molecular interplay between Myo1D and the actin cytoskeleton underlying symmetry breaking remains poorly understood. To address this question, we performed a dual genetic screen to identify new cytoskeletal factors involved in LR asymmetry. We identified the conserved actin nucleator DAAM as an essential factor required for both dextral and sinistral development. In the absence of DAAM, organs lose their LR asymmetry, while its overexpression enhances Myo1D-induced de novo LR asymmetry. These results show that DAAM is a limiting, LR-specific actin nucleator connecting up Myo1D with a dedicated F-actin network important for symmetry breaking.

A standardized nomenclature and atlas of the male terminalia of Drosophila melanogaster.

Animal terminalia represent some of the most diverse and rapidly evolving structures in the animal kingdom, and for this reason have been a mainstay in the taxonomic description of species. The terminalia of Drosophila melanogaster, with its wide range of experimental tools, have recently become the focus of increased interest in the fields of development, evolution, and behavior. However, studies from different disciplines have often used discrepant terminologies for the same anatomical structures. Consequently, the terminology of genital parts has become a barrier to integrating results from different fields, rendering it difficult to determine what parts are being referenced. We formed a consortium of researchers studying the genitalia of D. melanogaster to help establish a set of naming conventions. Here, we present a detailed visual anatomy of male genital parts, including a list of synonymous terms, and suggest practices to avoid confusion when referring to anatomical parts in future studies. The goal of this effort is to facilitate interdisciplinary communication and help newcomers orient themselves within the exciting field of Drosophila genitalia.

The Drosophila insulin pathway controls Profilin expression and dynamic actin-rich protrusions during collective cell migration.

Understanding how different cell types acquire their motile behaviour is central to many normal and pathological processes. Drosophila border cells represent a powerful model for addressing this issue and to specifically decipher the mechanisms controlling collective cell migration. Here, we identify the Drosophila Insulin/Insulin-like growth factor signalling (IIS) pathway as a key regulator in controlling actin dynamics in border cells, independently of its function in growth control. Loss of IIS activity blocks the formation of actin-rich long cellular extensions that are important for the delamination and the migration of the invasive cluster. We show that IIS specifically activates the expression of the actin regulator chickadee, the Drosophila homolog of Profilin, which is essential for promoting the formation of actin extensions and migration through the egg chamber. In this process, the transcription factor FoxO acts as a repressor of chickadee expression. Altogether, these results show that local activation of IIS controls collective cell migration through regulation of actin homeostasis and protrusion dynamics.

Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry.

The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. Here, we show that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2), to shape a productive LRO flow. Our findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry.

A Conserved Role of the Unconventional Myosin 1d in Laterality Determination.

Anatomical and functional asymmetries are widespread in the animal kingdom [1, 2]. In vertebrates, many visceral organs are asymmetrically placed [3]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [1]. Nodal signaling is widely used [4], and many, but not all, vertebrates use cilia for symmetry breaking [5]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs [6, 7]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [8], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.

Polycomb and Hox Genes Control JNK-Induced Remodeling of the Segment Boundary during Drosophila Morphogenesis.

In segmented tissues, anterior and posterior compartments represent independent morphogenetic domains, which are made of distinct lineages separated by boundaries. During dorsal closure of the Drosophila embryo, specific "mixer cells" (MCs) are reprogrammed in a JNK-dependent manner to express the posterior determinant engrailed (en) and cross the segment boundary. Here, we show that JNK signaling induces de novo expression of en in the MCs through repression of Polycomb (Pc) and release of the en locus from the silencing PcG bodies. Whereas reprogramming occurs in MCs from all thoracic and abdominal segments, cell mixing is restricted to the central abdominal region. We demonstrate that this spatial control of MC remodeling depends on the antagonist activity of the Hox genes abdominal-A and Abdominal-B. Together, these results reveal an essential JNK/en/Pc/Hox gene regulatory network important in controlling both the plasticity of segment boundaries and developmental reprogramming.

Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo.

Tissue morphogenesis relies on proper differentiation of morphogenetic domains, adopting specific cell behaviours. Yet, how signalling pathways interact to determine and coordinate these domains remains poorly understood. Dorsal closure (DC) of the Drosophila embryo represents a powerful model to study epithelial cell sheet sealing. In this process, JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converges and interacts (anterior/posterior -AP- determination; segmentation genes, such as Wnt/Wingless; TGFß/Decapentaplegic). To better characterize properties of the LE morphogenetic domain, we sought out new JNK target genes through a genomic approach: 25 were identified of which 8 are specifically expressed in the LE, similarly to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domain. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, our results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing.

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

The Atypical Cadherin Dachsous Controls Left-Right Asymmetry in Drosophila.

Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. Here, we propose a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. We further show that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry.

Diversity and convergence in the mechanisms establishing L/R asymmetry in metazoa.

Differentiating left and right hand sides during embryogenesis represents a major event in body patterning. Left-Right (L/R) asymmetry in bilateria is essential for handed positioning, morphogenesis and ultimately the function of organs (including the brain), with defective L/R asymmetry leading to severe pathologies in human. How and when symmetry is initially broken during embryogenesis remains debated and is a major focus in the field. Work done over the past 20 years, in both vertebrate and invertebrate models, has revealed a number of distinct pathways and mechanisms important for establishing L/R asymmetry and for spreading it to tissues and organs. In this review, we summarize our current knowledge and discuss the diversity of L/R patterning from cells to organs during evolution.

Starvation induces FoxO-dependent mitotic-to-endocycle switch pausing during Drosophila oogenesis.

When exposed to nutrient challenge, organisms have to adapt their physiology in order to balance reproduction with adult fitness. In mammals, ovarian follicles enter a massive growth phase during which they become highly dependent on gonadotrophic factors and nutrients. Somatic tissues play a crucial role in integrating these signals, controlling ovarian follicle atresia and eventually leading to the selection of a single follicle for ovulation. We used Drosophila follicles as a model to study the effect of starvation on follicle maturation. Upon starvation, Drosophila vitellogenic follicles adopt an 'atresia-like' behavior, in which some slow down their development whereas others enter degeneration. The mitotic-to-endocycle (M/E) transition is a critical step during Drosophila oogenesis, allowing the entry of egg chambers into vitellogenesis. Here, we describe a specific and transient phase during M/E switching that is paused upon starvation. The Insulin pathway induces the pausing of the M/E switch, blocking the entry of egg chambers into vitellogenesis. Pausing of the M/E switch involves a previously unknown crosstalk between FoxO, Cut and Notch that ensures full reversion of the process and rapid resumption of oogenesis upon refeeding. Our work reveals a novel genetic mechanism controlling the extent of the M/E switch upon starvation, thus integrating metabolic cues with development, growth and reproduction.

The myosin ID pathway and left-right asymmetry in Drosophila.

Drosophila is a classical model to study body patterning, however left-right (L/R) asymmetry had remained unexplored, until recently. The discovery of the conserved myosin ID gene as a major determinant of L/R asymmetry has revealed a novel L/R pathway involving the actin cytoskeleton and the adherens junction. In this process, the HOX gene Abdominal-B plays a major role through the control of myosin ID expression and therefore symmetry breaking. In this review, we present organs and markers showing L/R asymmetry in Drosophila and discuss our current understanding of the underlying molecular genetic mechanisms. Drosophila represents a valuable model system revealing novel strategies to establish L/R asymmetry in invertebrates and providing an evolutionary perspective to the problem of laterality in bilateria.

In vivo characterization of dynein-driven nanovectors using Drosophila oocytes.

Molecular motors transport various cargoes including vesicles, proteins and mRNAs, to distinct intracellular compartments. A significant challenge in the field of nanotechnology is to improve drug nuclear delivery by engineering nanocarriers transported by cytoskeletal motors. However, suitable in vivo models to assay transport and delivery efficiency remain very limited. Here, we develop a fast and genetically tractable assay to test the efficiency and dynamics of fluospheres (FS) using microinjection into Drosophila oocytes coupled with time-lapse microscopy. We designed dynein motor driven FS using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and characterized in real time the efficiency of the FS movement according to its linker's sequence. Results show that the conserved LC8 binding motif allows fast perinuclear nanoparticle's accumulation in a microtubule and dynein dependent mechanism. These data reveal the Drosophila oocyte as a new valuable tool for the design of motor driven nanovectors.

Drosophila left/right asymmetry establishment is controlled by the Hox gene abdominal-B.

In Drosophila, left/right (LR) asymmetry is apparent in the directional looping of the gut and male genitalia. The dextral orientation of the organs depends on the activity of a single gene, MyosinID (myoID), whose mutation leads to a fully inverted LR axis, thus revealing the activity of a recessive sinistral pathway. Here, we present the identification of the Hox gene Abdominal-B (Abd-B) as an upstream regulator of LR determination. This role appears distinct from its function in anteroposterior patterning. We show that the Abd-Bm isoform binds to regulatory sequences of myoID and controls MyoID expression in the organ LR organizer. Abd-Bm is also required for the sinistral pathway. Thus, when Abd-B activity is missing, no symmetry breaking occurs and flies develop symmetrically. These findings identify the Hox gene Abd-B as directing the earliest events of LR asymmetry establishment in Drosophila.

DE-Cadherin regulates unconventional Myosin ID and Myosin IC in Drosophila left-right asymmetry establishment.

In bilateria, positioning and looping of visceral organs requires proper left-right (L/R) asymmetry establishment. Recent work in Drosophila has identified a novel situs inversus gene encoding the unconventional type ID myosin (MyoID). In myoID mutant flies, the L/R axis is inverted, causing reversed looping of organs, such as the gut, spermiduct and genitalia. We have previously shown that MyoID interacts physically with ß-Catenin, suggesting a role of the adherens junction in Drosophila L/R asymmetry. Here, we show that DE-Cadherin co-immunoprecipitates with MyoID and is required for MyoID L/R activity. We further demonstrate that MyoIC, a closely related unconventional type I myosin, can antagonize MyoID L/R activity by preventing its binding to adherens junction components, both in vitro and in vivo. Interestingly, DE-Cadherin inhibits MyoIC, providing a protective mechanism to MyoID function. Conditional genetic experiments indicate that DE-Cadherin, MyoIC and MyoID show temporal synchronicity for their function in L/R asymmetry. These data suggest that following MyoID recruitment by ß-Catenin at the adherens junction, DE-Cadherin has a twofold effect on Drosophila L/R asymmetry by promoting MyoID activity and repressing that of MyoIC. Interestingly, the product of the vertebrate situs inversus gene inversin also physically interacts with ß-Catenin, suggesting that the adherens junction might serve as a conserved platform for determinants to establish L/R asymmetry both in vertebrates and invertebrates.

Regulation and activity of JNK signaling in the wing disc peripodial membrane during adult morphogenesis in Drosophila.

Thorax closure in Drosophila is a process during adult morphogenesis in which the anterior ends of the presumptive notum of the two wing imaginal discs fuse to make a seamless thorax. Similar to dorsal closure during embryogenesis, this process is regulated by plegic and JNK signaling pathways. Despite the fact that Peripodial Membrane (PM) cells do not contribute to the formation of any adult structure, they are known to facilitate the process of thorax closure. Here we show that JNK signaling is activated only in a subset of PM cells, known as medial edge cells. While the mechanism that activates JNK signaling specifically in the medial edge cells of the PM is still not understood, the results presented here show that the pair rule gene odd skipped is required to ensure that JNK signaling is not activated anywhere else in the wing disc. Medial edge cells of the PM are elongated in shape, while the remaining PM cells are hexagonal. Down regulation of JNK signaling in the medial edge cells results in defective thorax closure in adult flies. It also causes the transformation of the morphology of medial edge cells into hexagonal shape. Conversely, activation of JNK signaling in hexagonal cells of the PM causes transformation of their morphology to elongated shape. Thus, similar to dorsal closure during embryogenesis, JNK-mediated elongation of medial edge cells is functionally correlated to the process of thorax closure.