Biology subjects

Saunders, T. E.

Publications and source records attributed to Saunders, T. E..

9 recordsLinked to original sources

bioRxiv2018

MicroRNAs buffer genetic variation at specific temperatures during embryonic development

Successful embryogenesis requires the coordination of developmental events. Perturbations, such as environmental changes, must be buffered to ensure robust development. However, how such buffering occurs is currently unknown in most developmental systems. Here, we demonstrate that seven miRNAs are differentially expressed during Drosophila embryogenesis at varying temperatures within natural physiological ranges. Lack of miR-3-309, -31a, -310c, -980 or -984c causes developmental delays specifically at a given temperature. Detailed analysis on miR-310c and -984c shows that their targets are typically mis-expressed in mutant backgrounds, with phenotypes more pronounced at temperatures where miRNAs show highest expression in wild-type embryos. Our results show that phenotypes may arise at specific temperatures while remaining silent at others, even within typical temperature ranges. Our work uncovers that miRNAs mask genetic variation at specific temperatures to increase embryonic robustness, highlighting another layer of complexity in miRNA expression.

developmental biology↗

bioRxiv2018

The mirtron miR-1010 functions in concert with its host gene SKIP to maintain synaptic homeostasis

Mirtrons are non-canonical miRNAs arising by splicing and debranching from short introns. A plethora of introns have been inferred by computational analyses as potential mirtrons. Yet, few have been experimentally validated and their functions, particularly in relation to their host genes, remain poorly understood. Here, we found that larvae lacking the mirtron miR-1010 are unable to grow properly and pupariate. We show that miR-1010 downregulates nAcR{beta}2. Increase of cortical nAcR{beta}2 mediated by neural activity elevates the level of intracellular Ca2+, which in turn activates CaMKII and, further downstream, the transcription factor Adf-1. We reveal that Adf-1 initiates the expression of SKIP, the host gene of miR-1010. Preventing synaptic potentials from overshooting their optimal range requires both SKIP to temper synaptic potentials (incoherent feedforward loop) and miR-1010 to reduce nAcR{beta}2 mRNA levels (negative feedback loop). Our results demonstrate how a mirtron, in coordination with its host gene, contributes to maintaining homeostasis.

developmental biology↗

bioRxiv2018

Temporal development of Drosophila embryos is highly robust across a wide temperature range

Development is a precisely coordinated process in both space and time. Spatial precision has been quantified in a number of developmental systems, and, for example, such data has contributed significantly to our understanding of morphogen gradient interpretation. However, comparatively little quantitative analysis has been performed on timing and temporal coordination during development. Here, we use Drosophila to explore the temporal robustness of embryonic development within physiologically normal temperatures. We find that development is temporally very precise across a wide range of temperatures in all three Drosophila species investigated. However, we find temperature dependence in the heterochronicity. A simple model incorporating history-dependence can explain the developmental temporal trajectories. Interestingly, the history-dependence is temperature specific with either effective negative or positive feedback at different temperatures. We also find that embryos are surprisingly robust to shifting temperatures during embryogenesis. We further identify differences between tropical and temperate species that are suggestive of different potential mechanisms regulating temporal development depending on the local environment. Overall, our data shows that Drosophila embryonic development is temporally robust across a wide range of temperatures but there are species specific differences.

developmental biology↗

bioRxiv2018

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here we introduce morphogen-age measurements as a novel approach to retrieve key parameters in morphogen dynamics. Using a tandem fluorescent timer (tFT) as a protein-age sensor we find a gradient of increasing age of Bicoid (Bcd) along the anterior-posterior (AP) axis in the early Drosophila embryo. Quantitative analysis retrieves parameter that are most consistent with the synthesis-diffusion-degradation (SDD) model underlying Bcd-gradient formation, and rule out some other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bcd gradient formation, and demonstrate how morphogen age-information can complement knowledge about movement, abundance and distribution, which should be widely applicable for other systems.

developmental biology↗

bioRxiv2018

Pcdh18a-positive tip cells instruct notochord formation in zebrafish

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not known how these processes act together in a coordinated way during notochord formation. Analysing the tissue flow, we determined the displacement of the axial mesoderm and identified, relative to the ectoderm, an actively-migrating notochord tip cell population and a group of trailing notochordal plate cells. Molecularly, these tip cells express Protocadherin18a, a member of the cadherin superfamily. We show that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms these tip cells into a cohesive and fast migrating cell group. In turn, these tip cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework, and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation of the posterior, axial cells. Indeed, grafting experiments validated the predictions and induced ectopic notochord-like rods. Our findings indicate that the tip cells influence the trailing mesodermal cell sheet by inducing the formation of the notochord.

developmental biology↗

bioRxiv2017

Disrupting different Distal-less exons leads to ectopic and missing eyespots accurately modeled by reaction-diffusion mechanisms

Eyespots on the wings of nymphalid butterflies represent colorful examples of the process of pattern formation, yet the developmental origins and the mechanisms behind eyespot differentiation are still not fully understood. Here we re-examine the function of Distal-less (Dll) in eyespot development, which is still unclear. We show that CRISPR-Cas9 induced exon 2 mutations in Bicyclus anynana leads to exon skipping and ectopic eyespots on the wing. Exon 3 mutations, however, lead to null/missense transcripts, missing eyespots, lighter wing coloration, loss of scales, and a variety of other phenotypes implicating Dll in the process of eyespot differentiation. Reaction-diffusion modeling enabled exploration of the function of Dll in eyespot formation, and accurately replicated a wide-range of mutant phenotypes. These results confirm that Dll is a required activator of eyespot development, scale growth and melanization and point to a new mechanism of alternative splicing to achieve Dll over-expression phenotypes.

developmental biology↗

bioRxiv2017

Coupling optogenetics and light-sheet microscopy to study signal transduction in vivo

Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila {beta}-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of {beta}-catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

developmental biology↗

bioRxiv2017

Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo

Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of morphogen decoding.

developmental biology↗

bioRxiv2016

The architectural balance of the Ventral Nerve Cord depends on the level of JNK signaling activity

Central Nervous System (CNS) organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyzed the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered, in a subset of pioneer neurons. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating, during early embryogenesis, JNK signaling activity in pioneer neurons, directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization.

developmental biology↗