Biology subjects

Melchionda, M.

Publications and source records attributed to Melchionda, M..

6 recordsLinked to original sources

bioRxiv2026

Minimal essential requirements for neural tube self-organisation

The reliable generation of diverse cell types in precise proportions is essential for the formation of functional tissues during embryonic development. Three-dimensional organoid models derived from pluripotent stem cells (PSCs) provide powerful systems for identifying principles governing tissue self-organisation. Neural tube organoids (NTOs), initiated from single PSCs with a pulse of retinoic acid (RA), self-organise into structures containing floorplate cells that secrete SHH morphogen to pattern adjacent neural tissue. Yet, how the initial cellular diversity arises and how appropriate cell-type proportions are allocated has remained unclear. Here, using time-resolved single-cell transcriptomics, quantitative immunofluorescence, and dynamical systems modelling we show that RA triggers a transient co-expression state of the transcription factors PAX6 and FOXA2 from which cells asynchronously resolve into two opposing fates: PAX6 neural precursors and FOXA2 floorplate precursors. PAX6 and FOXA2 are both necessary and sufficient to reconstitute self-organisation, establishing these transcription factors as key determinants of emergent tissue pattern. Rather than operating as a simple feed-forward system in which cells are guided solely by RA, feedback between the alternative cell fates, mediated by BMP signalling from floorplate precursors, determines cell type proportions and ensures reproducible cell-type diversity in each NTO. This dual expression state was also identified in mouse embryos, demonstrating how in vitro models inform in vivo biology. These findings establish a general design strategy - symmetry breaking through opposing fate determinants coupled to proportioning via signal feedback control - that may operate broadly across developmental contexts to generate tissues with predictable cellular compositions.

developmental biology↗

bioRxiv2025

Predictable Engineering of Signal-DependentCis-Regulatory Elements

Cis-regulatory elements (CREs) control how genes respond to external signals, but the principles governing their structure and function remain poorly understood. While differential transcription factor binding is known to regulate gene expression, how CREs integrate the amount and combination of inputs to secure precise spatiotemporal profiles of gene expression remains unclear. Here, we developed a high-throughput combinatorial screening strategy, that we term NeMECiS, to investigate signal- dependent synthetic CREs (synCREs) in differentiating mammalian stem cells. By concatenating fragments of functional CREs from genes that respond to Sonic Hedgehog in the developing vertebrate neural tube, we found that CRE activity follows hierarchical design rules. While individual 200-base-pair fragments showed minimal activity, their combinations generated thousands of functional signal-responsive synCREs, many exceeding the activity of natural sequences. Statistical modelling revealed CRE function can be decomposed into specific quantitative contributions in which sequence fragments combine through a multiplicative rule, tuned by their relative positioning and spacing. These findings provide a predictive framework for CRE redesign, which we used to engineer synthetic CREs that alter the pattern of motor neuron differentiation in neural tissue. These findings establish quantitative principles for engineering synthetic regulatory elements with programmable signal responses to rewire genetic circuits and control stem cell differentiation, providing a basis for understanding developmental gene regulation and designing therapeutic gene expression systems.

developmental biology↗

bioRxiv2023

Clonal neural tube organoids self-organise floorplate through BMP-mediated cluster competition

The neural tube (NT) has been a hallmark example of embryonic induction and patterning whereby the notochord induces an organiser, the floorplate, that secretes Sonic Hedgehog (SHH) to pattern the surrounding field of neural progenitors. On the other hand, NT organoids (NTOs) formed from embryonic stem cells (ESCs) undergo spontaneous floorplate formation and patterning in the absence of their normal embryonic inducers. Understanding how stem cells undergo regulative organiser formation is a central challenge in biology. Here, we investigated the self-organisation of a SHH-expressing floorplate organiser using clonal NTOs. Expression of FOXA2, a floorplate transcription factor, was initially spatially scattered before resolving into multiple clusters. These FOXA2+ clusters underwent competition and physical sorting, resulting in a stable "winning" floorplate. We identified BMP signalling as a key governor of long-range cluster competition. FOXA2+ clusters expressed BMP4 ligand suppressing FOXA2 in receiving cells, while simultaneously expressing the BMP-inhibitor NOGGIN to secure FOXA2+ cluster survival. Genetic mutation of Noggin perturbed the floorplate not only in NTOs but also in vivo at the mid-hindbrain region of the mouse NT. These results demonstrate how the floorplate can form autonomously without its well-known inducer, the notochord, suggesting redundant mechanisms ensuring robustness. Defining molecular pathways that govern organiser self-organisation is critical in harnessing the developmental plasticity of stem cells toward directed tissue engineering.

developmental biology↗

bioRxiv2020

Temporal patterning of the central nervous system by a shared transcription factor code

The molecular mechanisms that ensure the reproducible generation of neuronal diversity in the vertebrate nervous system are incompletely understood. Here we provide evidence of a temporal patterning program consisting of cohorts of transcription factors expressed in neurons generated at successive developmental timepoints. This program acts in parallel to spatial patterning, diversifying neurons throughout the nervous system and in neurons differentiated in-vitro from stem cells. We demonstrate the TGF{beta} signalling pathway controls the pace of the temporal program. Furthermore, targeted perturbation of components of the temporal program, Nfia and Nfib, reveals their requirement for the generation of late-born neuronal subtypes. Together, our results provide evidence for the existence of a previously unappreciated global temporal program of neuronal subtype identity and suggest that the integration of spatial and temporal patterning programs diversifies and organises neuronal subtypes in the vertebrate nervous system.

developmental biology↗

bioRxiv2019

Species-specific developmental timing is associated with global differences in protein stability in mouse and human

What determines the pace of embryonic development? Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which these operate can vary substantially between species. For example, the same genetic programme, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme runs twice as fast in mouse as in human. We provide evidence that this is neither due to differences in signalling, nor the genomic sequence of genes or their regulatory elements. Instead, we find an approximately two-fold increase in protein stability and cell cycle duration in human cells compared to mouse. This can account for the slower pace of human development, indicating that global differences in key kinetic parameters play a major role in interspecies differences in developmental tempo.

developmental biology↗

bioRxiv2018

Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord

The coordinated spatial and temporal regulation of gene expression in the vertebrate neural tube determines the identity of neural progenitors and the function and physiology of the neurons they generate. Progress has been made deciphering the gene regulatory programmes responsible for this process, however, the complexity of the tissue has hampered the systematic analysis of the network and the underlying mechanisms. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions of the developing mouse neural tube between embryonic days (e)9.5-e13.5. We confirmed the data accurately recapitulates neural tube development, allowing us to identify new markers for specific progenitor and neuronal populations. In addition, the analysis highlighted a previously underappreciated temporal component to the mechanisms generating neuronal diversity and revealed common features in the sequence of transcriptional events that lead to the differentiation of specific neuronal subtypes. Together the data provide a compendium of gene expression for classifying spinal cord cell types that will support future studies of neural tube development, function, and disease.

developmental biology↗