Biology subjects

Franco, S.

Publications and source records attributed to Franco, S..

7 recordsLinked to original sources

bioRxiv2026

Autonomous Dampening of Wnt Signaling Selects Zebrafish Spinal Cord Progenitors for an Oligodendrocyte Fate

Nervous system development relies on coordinated formation of neurons and glia from neural progenitor cells (NPCs). During spinal cord development, NPCs in the pMN domain of the ventral spinal cord produce motor neurons followed by oligodendrocyte precursor cells (OPCs), a subset of which differentiate as oligodendrocytes, the myelinating cell of the vertebrate central nervous system. Whereas prior research has outlined an extensive gene regulatory network driving oligodendrocyte lineage cell differentiation, less is known about the mechanisms that specify NPCs for oligodendrocyte fate. To elucidate these mechanisms, our lab recently generated a single-cell RNA sequencing and single-cell ATAC sequencing multi-omics dataset from the zebrafish spinal cord. The dataset identified a molecularly distinct subset of NPCs that appear fated to the oligodendrocyte lineage, which we refer to as pre-OPCs. Our data revealed that pre-OPCs are enriched for RNAs encoding factors that antagonize Wnt signaling, such as barhl2 and cachd1. The chromatin regions associated with these genes are highly accessible in pre-OPCs, suggesting that their expression is developmentally regulated. Using confocal light microscopy to visualize expression of a Wnt-responsive transgenic reporter, we found that Wnt signaling is downregulated in the spinal cord immediately prior to OPC specification. To explore how modulation of Wnt signaling influences OPC specification, we pharmacologically inhibited or activated Wnt signaling immediately prior to OPC specification. These experiments revealed that embryos treated with a Wnt inhibitor produced excess OPCs whereas those treated with a Wnt agonist had a deficit of OPCs. Additionally, CRISPR/Cas9 targeted mutations of barhl2 and cachd1 impaired OPC production. Finally, we identified wnt4b as a ventral source of Wnt and determined that loss of wnt4b enhances OPC specification. Together, our studies reveal pre-OPC modulation of ventral Wnt signaling as a mechanism of OPC specification in the spinal cord.

developmental biology↗

bioRxiv2026

Mechanically-mediated low-pressure cell membrane poration enables tunable intracellular delivery of traditionally impermeable cargoes in high throughput and clinical scale formats

Traditional intracellular delivery methods suffer from cargo inefficiencies, non-linear delivery kinetics, and cellular trauma. We designed a mechanically-mediated poration platform governed by deterministic, passive diffusion operating at low pressure which enables dose-dependent, cargo-agnostic intracellular delivery and preserves cellular homeostasis; key advantages include high-fidelity multiplexing, transient cell engineering, and direct-to-biology live-cell target engagement enabling development of novel intracellular delivery applications across drug discovery and cell therapy. We demonstrate examples including live-cell DEL discovery and MOA studies, complex and rapid cell therapy manufacturing, and assay development in sensitive primary cell types.

cell biology↗

bioRxiv2025

Gsx2 Regulates Oligodendrocyte Precursor Formation in the Zebrafish Spinal Cord

Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system, later in development. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.

developmental biology↗

bioRxiv2025

Aqueous two-phase bioinks for discrete packing and compartmentalisation of 3D bioprinted cells

The unparalleled ability of aqueous two-phase systems (ATPS) to reproduce microscale cellular and biomaterial compartmentalisation to selectively modulate cell behaviour and functionality is ideal for tissue engineering and regenerative medicine (TERM) purposes. Herein, we introduce new ATPS biomaterial inks for 3D bioprinting of water-in-water (W/W) emulsions, enabling precise cellular crowding for tissue regeneration in vitro and ex vivo. Gelatin methacryloyl (GelMA) was hierarchically dispersed in an alginic acid phase depending on sodium chloride (NaCl) concentration (0-36 g/L). Emulsion droplet size (12.8{+/-}2.6 {micro}m to 52.4{+/-}11.4 {micro}m) influenced degradation and spatial cell localisation (A549, C2C12, MG63). A microfluidic-assisted 3D bioprinting approach allowed fine-tuning of fibre structure adjusting ATPS deposition by modulating flow rates and printing speed. Rheological properties supported the findings of the two-phase partitioning, aiding the selection of the ATPS ink formulation for functional cell-laden construct fabrication. Encapsulation of C2C12 cells revealed enhanced cytoskeletal remodelling at higher salt concentrations. Increased GelMA phase promoted human bone marrow stromal cells (HBMSCs) crowding, mineral deposition and skeletal differentiation. In ovo studies demonstrated degradation control and vascular infiltration via salt modulation. Altogether, ATPS bioinks offer a versatile platform for the assembling of complex, hierarchical tissues with microscale precision, expanding biofabrication strategies for TERM applications.

bioengineering↗

bioRxiv2025

Novel antimicrobial activity of photoactive compounds originally synthesized for industrial applications

The emergence of antimicrobial resistance threatens advances achieved by medicine in the last century. This situation has been aggravated by the suboptimal outcome of screening campaigns to provide novel antibiotics. In this study, we took an alternative approach to discover new chemical scaffolds with antimicrobial activity. We screened a collection of photoactive compounds originally synthesized for industrial purposes and found that 4H-pyran-4-ylidenes are active against Gram-positive bacteria. Compounds belonging to this family displayed dose-dependent bactericidal activity against wild-type Staphylococcus aureus and Methicillin Resistant S. aureus (MRSA). Importantly, they were not cytotoxic to hepatic cell lines at the concentrations at which antimicrobial activity was observed. Resistance to 4H-pyran-4-ylidenes in S. aureus was achieved via mutations in the rny locus, likely via changes in mRNA levels of genes critical for their activity. Overall, we demonstrate that chemical libraries not originally intended for drug discovery can be a fruitful source of chemical diversity for the development of novel antimicrobials.

microbiology↗

bioRxiv2023

Developmental Characterization of Neuronal Migration Anomalies and Axon Proliferation in mTOR pathway-associated Malformations of Cortical Development

Drug-resistant epilepsy (DRE) is a prevalent problem in children that can lead to abnormal development and various psychiatric comorbidities. Malformations of cortical development (MCD) include focal cortical dysplasia, tuberous sclerosis complex and hemimegalencephaly, which are the most common pathologies among children who undergo surgical resection for treatment of DRE. These disorders share many histopathological features, including dyslamination of the cerebral cortex and enlarged neuronal somata. Recently, genetic mutations in the mammalian target of rapamycin (mTOR) signaling cascade have been shown to underpin most MCDs. Rodent models, including the RhebCA model, recapitulate histologic and physiologic aspects of human DRE. However, there have been few studies characterizing the developmental time point of the histological changes seen in MCDs. In this study, we use in utero electroporation to upregulate the Rheb protein (directly upstream of mTOR) in a focal area of the neocortex. We demonstrate that mTOR dysregulation leads to focal dyslamination and increased neuronal size that is histologically similar to MCD, which correlates to spontaneous recurrent seizures. We used immunohistochemistry to investigate neuronal lamination at several time points during development between E18 and P21 and show early differences in lamination that persisted through development. Furthermore, the increased axonal length associated with mTOR upregulation occurs early in development. Our study provides a time frame for the initial development of abnormal neuronal migration and cellular growth that occurs in MCDs, and our data supports that these anatomical changes may contribute to the formation of epileptic networks.

neuroscience↗

bioRxiv2018

HIV-1 Protease Evolvability is Affected by Synonymous Nucleotide Recoding

One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair re-engineered protease sequence including 38 (13%) synonymous mutations. WT and MAX viruses showed indistinguishable replication in MT-4 cells or PBMCs. Both viruses were subjected to serial passages in MT-4 cells with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (IC50 14.6 {+/-} 5.3 and 21.2 {+/-} 9 nM for ATV, and 5. 9 {+/-} 1.0 and 9.3 {+/-} 1.9 for DRV, respectively). Ultra-deep sequence clonal analysis revealed that both viruses harbored previously described resistance mutations to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in WT, and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in MAX. Remarkably, G48L and L89I are rarely found in vivo in PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P < 0.0001), suggesting higher selective pressure for change in this recoded virus. Our results indicate that HIV-1 protease position in sequence space delineates the evolution of its mutant spectra. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to the WT virus.\n\nIMPORTANCELarge-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a viruss position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease impacts the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and the virus with the recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotype was indistinguishable between the recoded virus and the WT virus, suggesting that the synonymously recoded protease HIV-1 and the WT protease virus were equally robust and evolvable.

evolutionary biology↗