Biology subjects

Racioppi, C.

Publications and source records attributed to Racioppi, C..

5 recordsLinked to original sources

bioRxiv2026

Evolution of CTCF underlies the establishment of loop extrusion as a predominant chromatin folding mechanism in vertebrates

CTCF is a DNA-binding protein that emerged in the last bilaterian ancestor and that plays a central role in 3D genome organization. Yet, despite its relevance, genomic studies have revealed notable differences in CTCF function across animal clades. In vertebrates, CTCF is essential for TAD organization through cohesin-mediated loop extrusion, whereas its contribution to chromatin organization outside this clade appears to be more limited. Here we investigate the evolution of CTCF as an architectural factor through a cross-species functional replacement approach, in which orthologs from divergent bilaterian lineages are expressed in either mouse embryonic stem cells or Drosophila embryos. Chromosome conformation analyses demonstrated that the ability of CTCF to mediate loop extrusion-dependent TAD formation is encoded within the vertebrate protein. Yet, this property alone is insufficient to establish vertebrate-like genome architecture in an invertebrate background, suggesting the existence of additional lineage-specific factors. ChIP-seq analyses further revealed that CTCF DNA-binding specificity has been progressively refined during bilaterian evolution, with the vertebrate lineage exhibiting a preferential shift towards distal genomic regions that is consistent with an expansion of regulatory domains. Notably, despite failing to form TADs, the ortholog from a non-vertebrate chordate partially rescued the lethality associated with the loss of the vertebrate CTCF protein, indicating that its architectural and regulatory functions are evolutionary independent. Further Hi-C analyses in tunicate and cyclostome species suggest that the ability of CTCF to act as a loop extrusion barrier was already present in the last common ancestor of all vertebrates. Altogether, our findings establish the diversification of CTCF regulation and function as key drivers for the specialization of bilaterian 3D genome organization.

genomics↗

bioRxiv2020

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate

GATA4/5/6 transcription factors play essential, conserved roles in heart development. How these factors mediate the transition from multipotent mesoderm progenitors to a committed cardiac fate is unclear. To understand how GATA4/5/6 modulate cell fate decisions we labelled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at pre-cardiac time points spanning gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was dramatically altered. In addition to the expected absence of cardiac mesoderm, we observed a concomitant expansion of cranial-pharyngeal mesoderm. Functional genetic analyses in zebrafish and the invertebrate chordate Ciona, which possess a single GATA4/5/6 homolog, revealed an essential and cell-autonomous role for GATA4/5/6 in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, the maintenance and repression of GATA4/5/6 activity plays a critical, evolutionarily conserved role in early development.

developmental biology↗

bioRxiv2019

A cis-regulatory change underlying the motor neuron-specific loss of terminal selector gene expression in immotile tunicate larvae

The evolutionary history of animal body plans cannot be fully reconstructed without considering the roles of both novelties and losses. Some of the more remarkable examples of massively parallel evolutionary losses in animals comes from many species in the tunicate genus Molgula that have independently lost the swimming larva and instead develop as tail-less, immotile larvae that bypass the period of swimming and dispersal observed in other tunicates, marine invertebrate chordates that alternate between motile larval and sessile adult life cycle stages. The larvae of Molgula occulta and other tail-less species do not fully develop structures that are essential for swimming behavior, including notochord, tail muscles, and otolith, and loss-of-function mutations have been identified in various genes required for the differentiation of these tissues. However, little is known about the extent of development of the larval nervous system in M. occulta. While differentiated neurons might in principle be entirely dispensable to the non-swimming larva, the adult has a fully functional nervous system like any other tunicate. To further investigate this conundrum, we studied the specification and patterning of the M. occulta Motor Ganglion, which is the key central nervous system compartment that drives the motor movements of swimming tunicate larvae. We found that the expression patterns of important regulators of MG neuron subtype specification are highly conserved during the development of the non-swimming larvae of M. occulta, suggesting that the gene networks regulating their expression are largely intact in this species, despite the loss of swimming ability. However, we identified a M. occulta-specific reduction in expression of the important motor neuron terminal selector gene Ebf (Collier/Olf/EBF or COE) in the Motor Ganglion. Although M. occulta Ebf is predicted to encode a fully functional protein, its expression was reduced in developing motor neurons when compared to species with swimming larvae, which was corroborated by measuring allele-specific expression of Ebf in interspecific hybrid embryos produced by crossing M. occulta with the closely related swimming species M. oculata. Comparative reporter construct experiments also revealed a specific cis-regulatory sequence change that underlies the reduced expression of M. occulta Ebf in motor neurons, but not in other tissues and cell types. This points to a potential mechanism for arresting larval motor neuron differentiation in the non-swimming larvae of this species.

evolutionary biology↗

bioRxiv2019

Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices

In embryos, lineage-specific profiles of chromatin accessibility control gene expression by modulating transcription, and thus impact multipotent progenitor states and subsequent fate choices. Subsets of cardiac and pharyngeal/head muscles share a common origin in the cardiopharyngeal mesoderm, but the chromatin landscapes that govern multipotent progenitors competence and early fate choices remain largely elusive. Here, we leveraged the simplicity of the chordate model Ciona to profile chromatin accessibility through stereotyped transitions from naive Mesp+ mesoderm to distinct fate-restricted heart and pharyngeal muscle precursors. An FGF-Foxf pathway acts in multipotent progenitors to establish cardiopharyngeal-specific patterns of accessibility, which govern later heart vs. pharyngeal muscle-specific expression profiles, demonstrating extensive spatiotemporal decoupling between early cardiopharyngeal enhancer accessibility and late cell-type-specific activity. Combinations of cis-regulatory elements with distinct chromatin accessibility profiles are required to activate of Ebf and Tbx1/10, two key determinants of cardiopharyngeal fate choices. We propose that this higher order combinatorial logic increases the repertoire of regulatory inputs that control gene expression, through either accessibility and/or activity, thus fostering spatially and temporally accurate fate choices.

developmental biology↗

bioRxiv2018

A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolated a pan-LPM enhancer in the zebrafish draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captured the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncovered specific drl reporter activity in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

developmental biology↗