Functional analysis of genes and cis-regulatory elements involved in the specification of optic cup territories identified by scRNAseq and scATACseq.

ABSTRACTMotivation: In all metazoans, sight depends on the intimate relation and combined function between photoreceptors and pigmented cells, which derive from common precursors through the bifurcation of a single gene regulatory network (GRNs) into two mutually exclusive developmental programs; the neural retina (NR) and retinal-pigmented epithelium (RPE). Studying how GRNs guide the differentiation of these tissues is an essential step to better understand the molecular bases of retinal degenerative diseases and the dynamics of embryonic development. Here we use the development of the optic cup in zebrafish (Danio rerio) as a vertebrate model organism, while continuing a well-established line of work that previously identified active cis-regulatory elements in each NR/RPE domain and captured the transition states of genetic networks using ATAC-seq and scRNA-seq epigenomics techniques respectively from different pools of sorted cells derived out from distinct domains of the optic cup at several stages of development. In this study we plan to functionally analyze a battery of candidate genes that are relevant transcription factor/targets in multiome analysis from ATAC-seq and slightly to strongly overexpressed in pseudobulk differential expression analysis from scRNAseq at 18-somite stage. Among the genes chosen for functional analysis, there are 5 from the retina, 6 from the RPE, 2 from the optic stem, 2 from the diencephalon and another 2 from the telencephalon, together with characterized genes whose expression are well-known to serve as experimental controls: vsx2, bhlhe40, vax1, gbx2 and emx1 respectively for each type of tissue.Methods: To carry out the functional analysis of the previously identified genes we are following a fluorescent in situ hybridization (FISH) protocol performe on wildtype zebrafish embryos fixed at 18h post-fertilization. This is a powerful and commonly used technique in whole-mounted embryos to assess phenotype, allowing us via super-resolution microscopy to examine temporal and spatial patterns of gene expression by visualizing its mRNA with a fluorescent riboprobe able to specifically recognize a DNA sequence. The first step was the identification of species-specific mRNA sequences and the design of primers for each candidate gene to reduce the risk of non-specific binding of the riboprobes or background signal. The synthesis of riboproteins was carried out after amplifying and purifying the cDNA sequences ordered

A Yap-dependent mechanoregulatory program sustains cell migration for embryo axis assembly

The assembly of the embryo’s primary axis is a fundamental landmark for the establishment of the vertebrate body plan. Although the morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are well-known transcriptional mechanotransducers, yet their role in gastrulation remains elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure, due to reduced displacement and migratory persistence in mutant cells. Accordingly, we identified genes involved in cytoskeletal organization and cell-ECM adhesion as potentially direct Yap targets. Dynamic analysis of live sensors and downstream targets reveal that Yap is acting in migratory cells, promoting cortical actin and focal adhesions recruitment. Our results indicate that Yap coordinates a mechanoregulatory program to sustain intracellular tension and maintain the directed cell migration for embryo axis development

Mutation of vsx genes in zebrafish highlights the robustness of the retinal specification network

Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors' specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR/Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted toward photoreceptor or Müller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species

Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.This work is supported by the following grants: (I) To J.-R.M.-M.: From the Spanish Ministry of Science, Innovation, and Universities (MICINN): BFU2017-86339P with FEDER funds, MDM-2016-0687 and PY20_00006/Junta de Andalucía. (II) To O.B. Australian Research Council (ARC) Discovery Project (DP190103852). (III) To F.-J.D.-C.: Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018). (IV) To P.B.: BFU2016-75412-R with FEDER funds; PCIN-2015-176-C02-01/ERA-Net Neuron ImprovVision, and a CBMSO Institutional grant from the Fundación Ramón Areces. (V) To both J.-R.M.-M. and P.B.: BFU2016-81887-REDT, as well as Fundación Ramón Areces-2016 (Supporting L.B.)

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes

Impact of CoQ deficiency on embryonic development in zebrafish

Trabajo presentado en EMBO Workshop. Developmental metabolism: flows of energy, matter, and information, celebrado en Heidelberg (Alemania) del 12 al 15 de septiembre de 2023.Coenzyme Q (CoQ) is a redox-active lipid with a prominent role in the mitochondrial respiratory chain. CoQ is also involved in other redox processes being the electron acceptor for specific mitochondrial dehydrogenases. Primary CoQ deficiencies are rare mitochondrial conditions, biochemically characterised by a reduction in CoQ, caused by biallelic mutations in any of the -at least- 11 COQ genes participating in its biosynthesis. Remarkably, patients show a broad spectrum of clinical manifestations, severity, and age of onset, but a clear genotype-phenotype correlation is still lacking. We hypothesise that the disease unfolding due to defects in specific COQ genes could be different during development and would determine severity, the age of onset and the affected tissues. Modelling rare diseases is a promising approach to overcoming the lack of epidemiological studies. Danio rerio (zebrafish) is a convenient vertebrate model to study embryogenesis due to its straightforward genetic manipulation, the highly efficient, external and easy-to-control oocyte fertilisation and their transparent embryos that make them easy to monitor. We have generated a collection of CRISPR-Cas9 F0 knockout zebrafish models carrying a high rate of biallelic mutations in all known genes involved in CoQ biosynthesis. These somatic F0 mutants enable a high throughput evaluation of loss-of-function phenotypes during early development. Moreover, we have generated a stable coq6 zebrafish knockout line which will allow us to monitor the unfolding of the disease at later embryonic stages. Our work will contribute to close the gap between the knowledge of the regulation of CoQ biosynthesis during development and its coordination with mitochondrial biogenesis. The functional and physiological characterisation of our animal models will help to better understand CoQ deficiencies in humans.Peer reviewe

Systems biology approaches to understand gene specification networks in the zebrafish retina

Trabajo presentado en 27th European Association for Vision and Eye Research (EVER) Congress, celebrado en Valencia (España) del 03 al 05 de noviembre de 2023.The ontogeny of the vertebrate retina has been a topic of interest to developmental biologists and human geneticists for many decades. Understanding the unfolding of the genetic program that transforms a field of progenitors' cells into a functionally complex and multi-layered sensory organ is a formidable challenge. Although classical genetic studies succeeded in identifying the key regulators of retina specification, we are far from understanding the architecture of their gene regulatory networks (GRNs) and thus from predicting their behaviour. A goal in our group during the last years has been to analyse the bifurcation of the GRNs specifying the neural retina and RPE domains, as cells in these two compartments acquire their distinct shape; within a few hours window in zebrafish. We have characterized transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish to identify active cis-regulatory modules and enriched TF motives within them (Buono et al Nat com 2021). We have shown extensive network redundancy and context-dependent activity for the core TFs within each domain. In line with this finding, we have shown that the simultaneous mutation of the key specifiers vsx1 and vsx2 by CRISPR/Cas9, which results in severe visual impairment and bipolar cells depletion, does not interfere with neural retina specification. We concluded that although important cis-regulatory remodelling (ATAC-seq) occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level (RNA-seq). Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network (Letelier & Buono et al Biorxiv 2022). We are currently complementing previous efforts by applying single-cell methods to the analysis of neural retina and RPE networks. We have generated transcriptome and epigenome (chromium 10x multiome scRNAseq¿+¿scATACseq) datasets in zebrafish to obtain predictive information for the identification of novel genes and cis-regulatory elements involved in the specification of retinal domains. These datasets are a valuable resource to reconstruct the final architecture of the retinal GRNs.Peer reviewe

Modeling Planarian Regeneration: A Primer for Reverse-Engineering the Worm

A mechanistic understanding of robust self-assembly and repair capabilities of complex systems would have enormous implications for basic evolutionary developmental biology as well as for transformative applications in regenerative biomedicine and the engineering of highly fault-tolerant cybernetic systems. Molecular biologists are working to identify the pathways underlying the remarkable regenerative abilities of model species that perfectly regenerate limbs, brains, and other complex body parts. However, a profound disconnect remains between the deluge of high-resolution genetic and protein data on pathways required for regeneration, and the desired spatial, algorithmic models that show how self-monitoring and growth control arise from the synthesis of cellular activities. This barrier to progress in the understanding of morphogenetic controls may be breached by powerful techniques from the computational sciences—using non-traditional modeling approaches to reverse-engineer systems such as planaria: flatworms with a complex bodyplan and nervous system that are able to regenerate any body part after traumatic injury. Currently, the involvement of experts from outside of molecular genetics is hampered by the specialist literature of molecular developmental biology: impactful collaborations across such different fields require that review literature be available that presents the key functional capabilities of important biological model systems while abstracting away from the often irrelevant and confusing details of specific genes and proteins. To facilitate modeling efforts by computer scientists, physicists, engineers, and mathematicians, we present a different kind of review of planarian regeneration. Focusing on the main patterning properties of this system, we review what is known about the signal exchanges that occur during regenerative repair in planaria and the cellular mechanisms that are thought to underlie them. By establishing an engineering-like style for reviews of the molecular developmental biology of biomedically important model systems, significant fresh insights and quantitative computational models will be developed by new collaborations between biology and the information sciences

Planarian cell number depends on Blitzschnell, a novel gene family that balances cell proliferation and cell death

Control of cell number is crucial to define body size during animal development and to restrict tumoral transformation. The cell number is determined by the balance between cell proliferation and cell death. Although many genes are known to regulate those processes, the molecular mechanisms underlying the relationship between cell number and body size remain poorly understood. This relationship can be better understood by studying planarians, flatworms that continuously change their body size according to nutrient availability. We identified a novel gene family, blitzschnell (bls), which consists of de novo and taxonomically restricted genes that control cell proliferation:cell death ratio. Their silencing promotes faster regeneration and increases cell number during homeostasis. Importantly, this increase in cell number only leads to an increase in body size in a nutrient-rich environment; in starved planarians silencing results in a decrease in cell size and cell accumulation that ultimately produces overgrowths. bls expression is down-regulated after feeding and related with the Insulin/Akt/mTOR network activity, suggesting that the bls family evolved in planarians as an additional mechanism by which to restrict cell number in nutrient-fluctuating environments