Biology subjects

Fernandes, V. M.

Publications and source records attributed to Fernandes, V. M..

8 recordsLinked to original sources

bioRxiv2026

Glia generate distinct visual processing centres by locally inhibiting ERK activity in an optic lobe neuroepithelium

Epithelial patterning is fundamental to organ development. Extensive work has focused on how neuroepithelia are patterned to generate diverse progenitors, yet how a single neuroepithelium is partitioned to produce distinct processing centres is poorly understood. Here, we focus on the Drosophila outer proliferation centre neuroepithelium, which generates the medulla and lamina visual processing centres. Medulla neuroblasts are produced by a proneural wave that initiates at the medial margin of the neuroepithelium and moves laterally propagated by EGFR-ERK signalling, whereas lamina precursors arise at the lateral neuroepithelial margin and have been proposed to require photoreceptor-derived Hedgehog. Here, we show that Hedgehog signalling is dispensable for lamina precursor specification but instead promotes their survival. In contrast, suppressing ERK and apoptosis together is sufficient to drive ectopic lamina precursor development. We find that cortex glia secrete the EGF antagonist Argos, which accumulates at the lateral neuroepithelium, thus repressing ERK activity locally. Together, our findings reveal a glia-mediated, extrinsic patterning mechanism that suppresses EGFR-ERK signalling in the lateral neuroepithelium, protecting these cells from the proneural wave and instructing lamina over medulla fate. Summary statementGlial cells locally control ERK signalling to partition a developing tissue, enabling distinct structures to arise from a common neuroepithelium.

developmental biology↗

bioRxiv2025

Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

Inherited retinal diseases are a class of genetically heterogenous disorders characterized by mutations in genes required for retinal function, resulting in progressive loss of vision in human patients. One such deletion mutation in the translation termination factor, HBS1L, results in a suite of developmental anomalies in human patients, including progressive vision loss defects. HBS1L, and its interaction partner Pelota (Pelo), are required for recycling stalled ribosomes on mRNAs, but the specific mRNA target causative of vision defects seen with HBS1L deletion remains unknown. Further, the specific cell types in the visual system that require HBS1L for proper development and function are also unknown. Here, we discover that loss of the Drosophila HBS1L homolog, Hbs1, results in reduced expression of the stress responsive Activating Transcription Factor 4 (ATF4) which is encoded by an mRNA containing multiple upstream open reading frames (uORFs) in its 5 leader. We corroborate these results in cultured human cells, where we find that HBS1L and Pelo promote translation reinitiation at the ATF4 ORF by facilitating proper translation termination at the preceding uORFs. Like human HBS1L deficiency patients, loss of function Drosophila mutants for Hbs1, pelo, and ATF4 show vision defects as measured by electroretinograms (ERG). Depleting Hbs1 in lamina neurons replicated the ERG defects seen in Hbs1 mutants, suggesting that Hbs1-Pelo is required for proper lamina neuron function. Further confocal analysis of Hbs1 mutants revealed vacuolization defects in the lamina layer, which are indicative of defective synapse formation between lamina neurons and photoreceptors. Strikingly, restoring ATF4 expression in the lamina partially rescued ERG defects in Hbs1 mutants, indicating that ATF4 is likely a relevant mRNA target regulated by Hbs1-Pelo in these cells. Together, our data support a model wherein Hbs1-Pelo mediated translation regulation of ATF4 in lamina neurons underlies the inherited retinal disease caused by HBS1L deletion.

genetics↗

bioRxiv2025

Morphogen and juxtacrine signalling dynamically integrate to specify cell fates with single-cell resolution

Morphogen gradients guide tissue patterning but do not act in isolation. How they integrate with other signalling modalities, like juxtacrine signalling, and how these integrations influence pattern resolution and robustness remain unclear. We address this in the Drosophila lamina, where columns of precursors are patterned with single-cell resolution into motion-processing neurons, dependent on a photoreceptor-derived Hedgehog gradient and glial-orchestrated differentiation. Combining experiments and theory, we show that glial-induced ERK activity drives Delta expression in lamina precursors, generating a graded Notch activity pattern. Notch restricts Hedgehog morphogen relay and enhances positional information. Glia act as timekeepers, scheduling ERK-driven differentiation after Hedgehog and Notch patterns are established. Thus, Notch and ERK dynamically integrate with Hedgehog to encode positional information, enabling reproducible cell fate patterning with single-cell resolution.

developmental biology↗

bioRxiv2022

A Drosophila glial cell atlas reveals that transcriptionally defined cell types can be morphologically diverse

Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system, where glia are categorized into five main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single cell RNA sequencing data of Drosophila glia in two well-characterized tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the ventral nerve cord. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila, and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.

neuroscience↗

bioRxiv2022

Photoreceptors generate neuronal diversity in their target field through a Hedgehog morphogen gradient in Drosophila

Defining the origin of neuronal diversity is a major challenge in developmental neurobiology. The Drosophila visual system is an excellent paradigm to study how cellular diversity is generated. Photoreceptors from the eye disc grow their axons into the optic lobe and secrete Hedgehog (Hh) to induce the lamina, such that for every unit eye there is a corresponding lamina unit made up of post-mitotic precursors stacked into columns. Each differentiated column contains five lamina neuron types (L1-L5), making it the simplest neuropil in the optic lobe, yet how this diversity is generated was unknown. Here, we found that Hh pathway activity is graded along the distal-proximal axis of lamina columns and further determined that this gradient in pathway activity arises from a gradient of Hh ligand. We manipulated Hh pathway activity cell-autonomously in lamina precursors and non-cell autonomously by inactivating the Hh ligand, and by knocking it down in photoreceptors. These manipulations showed that different thresholds of activity specify unique cell identities, with more proximal cell types specified in response to progressively lower Hh levels. Thus, our data establish that Hh acts as a morphogen to pattern the lamina. Although, this is the first such report during Drosophila nervous system development, our work uncovers a remarkable similarity with the vertebrate neural tube, which is patterned by Sonic Hedgehog. Altogether, we show that differentiating neurons can regulate the neuronal diversity of their distant target fields through morphogen gradients.

developmental biology↗

bioRxiv2022

A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila

Extracellular Signal-Regulated Kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active Epidermal Growth Factor Receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, heterogeneity in ERK activity in retinal basal glia correlated with the direction of migration of individual cells. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases. Summary StatementWe describe a reporter to study the dynamics of ERK signalling in Drosophila, use it to measure signalling in individual cells over time, and monitor development.

developmental biology↗

bioRxiv2021

Differentiation signals from glia are fine-tuned to set neuronal numbers during development

Neural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. We used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains [~]6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the extra precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. Here, we showed that a glial population called the outer chiasm giant glia (xgO), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to EGF from photoreceptors. By forcing neuronal differentiation in the lamina, we uncovered that though fated to die, the extra precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. We found that the row of precursors nearest to xgO differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the extra precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron.

developmental biology↗

bioRxiv2020

An immobilization technique for long-term time-lapse imaging of explanted Drosophila tissues

Time-lapse imaging is an essential tool to study dynamic biological processes that cannot be discerned from fixed samples alone. However, imaging cell- and tissue-level processes in intact animals poses numerous challenges if the organism is opaque and/or motile. Explant cultures of intact tissues circumvent some of these challenges, but sample drift remains a considerable obstacle. We employed a simple yet effective technique to immobilize tissues in medium-bathed agarose. We applied this technique to study multiple Drosophila tissues from first-instar larvae to adult stages in various orientations and with no evidence of anisotropic pressure or stress damage. Using this method, we were able to image fine features for up to 18 hours and make novel observations. Specifically, we report that fibers characteristic of quiescent neuroblasts are inherited by their basal daughters during reactivation; that the lamina in the developing visual system is assembled roughly 2-3 columns at a time; that lamina glia positions are dynamic during development; and that the nuclear envelopes of adult testis cyst stem cells do not break down completely during mitosis. In all, we demonstrate that our protocol is well-suited for tissue immobilization and long-term live imaging, enabling new insights into tissue and cell dynamics in Drosophila.

developmental biology↗