Josep Rotllant

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.

Melanocortins regulate pigmentation via melanocortin receptors (MCRs), which are highly conserved across vertebrates. Unlike other MCRs, the melanocortin 2 receptor (MC2R) is exclusively activated by ACTH; however, its role in pigmentation remains unclear. Using CRISPR/Cas9-generated mc2r knockout zebrafish, we demonstrated that the loss of mc2r in zebrafish results in impaired interrenal steroidogenesis and pronounced hyperpigmentation characterized by an increased number of melanophores and xanthophores while preserving normal patterning. Transcriptomic analyses revealed the upregulation of genes involved in melanosome formation, melanin synthesis, lipid metabolism, and carotenoid accumulation. These findings demonstrate that, in addition to controlling steroidogenesis, mc2r plays a key role in pigment cell development and metabolic regulation.

Flatfish metamorphosis involves dramatic tissue remodeling, including the migration of one eye to the opposite side of the body, enabling the transition from pelagic to benthic life. While this process requires precise transcriptional regulation, the role of epigenetic mechanisms remains poorly understood. Here, we investigate DNA methylation dynamics during turbot metamorphosis using reduced-representation bisulfite sequencing (RRBS) across three key stages: pre-metamorphosis, climax, and post-metamorphosis. We identified stage-specific methylation patterns, with more than 31% of hypermethylated regions emerging during the climax phase—coinciding with upregulated dnmt3a (de novo methyltransferase) and altered expression of photoreceptor adaptation genes. Critically, the migrating and non-migrating eyes exhibited divergent methylation and expression of retinal ganglion cell (RGC) regulators (eomesa, tbr1b), linking epigenetic changes to asymmetric ocular development. Our results suggest that DNA methylation may play a role in visual system remodeling, particularly in processes associated with RGC-mediated eye migration and light-sensing adaptation, providing new understanding of the epigenetic regulation of vertebrate metamorphosis.

Parathyroid hormone 4 (Pth4) is an evolutionarily conserved member of the PTH family, expressed in hypothalamic neurons and lost in eutherian mammals. In order to elucidate its role in mineral homeostasis and skeletal development, a pth4 knockout ( pth4 ^KO ) zebrafish line was generated using CRISPR/Cas9 and transcriptomic profiling was conducted across six key tissues: brain, kidney, intestine, gills, scales, and bone. The results obtained demonstrated that the loss of Pth4 led to pronounced disturbances in calcium and phosphate homeostasis, skeletal deformities, and widespread tissue-specific transcriptional alterations. Notably, dysregulation of mineral regulatory genes— such as fgf23, phex , and slc34a1a was particularly evident in the kidney, suggesting disruption of the FGF23-Klotho axis. In parallel, differential expression of extracellular matrix genes ( col1a1a, col10a1a, col11a1 ) and matrix remodeling enzymes ( mmp9, mmp13a, mmp2 ) in bone and scales indicated impaired skeletal remodeling. Together, these findings highlight a pivotal role for Pth4 in the endocrine and local regulation of mineral metabolism and skeletal integrity, expanding our understanding of PTH family functions in vertebrate physiology.

The ALMS1 gene plays a crucial role in maintaining cellular homeostasis through its involvement in primary cilium assembly, cytoskeletal regulation, and signalling pathways such as NOTCH and TGF-β. Pathogenic variants in ALMS1 are associated with Alström Syndrome (ALMS), a multi-systemic ciliopathy characterised by neurosensory deficits, metabolic disorders, and multi-organ fibrosis. To better understand the tissue-dependent role of ALMS1 , we utilised CRISPR/Cas9 technology to develop a zebrafish model with alms1 depletion. Multi-tissue transcriptomic profiling revealed that alms1 depletion has pleiotropic effects on gene expression, with the brain and eyes displaying the most pronounced transcriptomic alterations, including disrupted ciliary function and immune dysregulation. Inflammatory and innate immune pathways along with glutamatergic synapse-related processes were significantly affected in the brain and eyes but with different gene expression signatures. The analysis further highlights tissue-specific processes, primarily associated with organ dysfunction. Additionally, our findings underscore the role of alms1 in regulating age-associated gene expression profiles in the brain, suggesting a link between ciliary dysfunction and accelerated brain ageing. Comparative analyses with Bardet-Biedl Syndrome iPSC models revealed shared pathways, reinforcing the potential of ciliopathies as models for ageing-related disorders. This study provides novel insights into the tissue-specific functions of alms1 and the molecular mechanisms underlying ALMS, paving the way for the development of targeted therapeutic strategies.

Flatfish metamorphosis is characterized by extensive tissue remodeling, associated with a transition from pelagic to benthic lifestyle, being the migration of one eye the most dramatic change. Epigenetic mechanisms exert a pivotal role in developmental programs. This study investigates the DNA methylation profiles of migrating and non-migrating eyes using reduced-representation bisulfite sequencing (RRBS) during three developmental stages of turbot: pre-metamorphosis, climax and post-metamorphosis. Over 31% of all identified regions were hypermethylated during climax stage in both eyes, coinciding with elevated expression of the dnmt3a gene, responsible for de novo methylation. Additionally, transcription factors crucial for retinal ganglion cells (RGCs) development, including the eomesa and tbr1b , exhibited differential methylation and expression between the migrating and non-migrating eye during the climax phase. These findings underscore the significance of DNA methylation in the intricate remodeling of the visual system during turbot metamorphosis, particularly regarding RGC-mediated ocular migration and the transmission of visual signals.

The regulation of gene expression plays a pivotal role in the complex metamorphic process of flatfishes, which oversees the dynamic alterations that occur during their transition from a pelagic to a benthic way of life. Flatfish metamorphosis is characterized by substantial modifications in both form and function, which are initiated by thyroid hormones. Epigenetic mechanisms play a vital role in the regulation of gene expression during this transformative process. This study examines the molecular mechanisms underlying flatfish metamorphosis by integrating multi-omics data that provide information on chromatin status, DNA methylation profile, and gene expression at three key stages (pre-metamorphosis, climax and post-metamorphosis) in the turbot ( Scophthalmus maximus ) brain, a complex organ that plays a critical role in the regulation of this intricate process. The analysis of DNA methylation patterns revealed major epigenetic dynamic changes during the different metamorphosis stages. Specifically, the methylation levels exhibited a typical bimodal distribution in the pre-metamorphic stage, transitioned to intermediate levels of methylation (around 50%) during the climax stage, and reverted to a bimodal distribution in the post-metamorphic stage. Notably, DMRs were identified in regions of open chromatin that colocalized with CpG islands. Moreover, our results indicate an inverse relationship between DNA methylation and transcriptional activity in regions near the transcription start sites (TSSs), where high levels of methylation correspond to low expression and low levels of methylation corresponds to high gene transcription. This study thereby elucidates the regulatory impact of methylation on gene expression during the metamorphosis process in the flatfish brain.

This study demonstrates that common octopuses ( Octopus vulgaris Cuvier 1797) do not produce cortisol, cortisone, or corticosterone in response to stress. Using liquid chromatography tandem mass spectrometry, it was also shown that octopuses have a low absorption rate of cortisol from water but a high absorption rate of 17β-estradiol (E2). The findings support the evolutionary argument that octopuses are unlikely to exhibit a stress response mediated by vertebrate-like corticosteroids.

Over the past decade, there has been a growing interest in common octopus aquaculture, prompted by several factors such as the increase in market demand, the decline in overall fisheries and the search for more sustainable food resources. Nevertheless, this interest has raised concerns about the potential impact of large-scale production and intensified farming practices in the future. This study aims to establish a baseline understanding of the natural microbial communities that inhabit skin mucus of the common octopus, describe its core microbiota, and assess the impact of captive rearing on these communities and animal welfare. The skin microbiome of wild-captured octopuses was compared with that of reared in aquaculture using 16S ribosomal RNA metabarcoding. The findings show that the core microbiota of octopus mucosal skin is dominated by the phyla Bacteroidota and Pseudomonadota, with Aurantivirga, Pseudofulvibacter, and Rubritalea being the most abundant genera. Despite differences in abundance, the microbiota composition and diversity between wild and aquaculture octopuses were similar. Wild samples had an enrichment of Gammaproteobacteria, including potentially pathogenic species such as Vibrio spp., Photobacterium swingsii, and Lactococcus garvieae, which were less prevalent or absent in aquaculture samples. KEGG functional pathways predictions indicated a higher enrichment in functional pathways related to xenobiotic remediation in wild samples, reflecting their need to adapt to a more variable and potentially contaminated environment. This is the first study to characterize the mucosal skin microbiome of the common octopus and to compare wild and aquaculture specimens. The results suggest that current aquaculture practices support animal welfare through the use of controlled hatchery environments and high-quality water conditions. These insights are valuable for the development of sustainable and responsible aquaculture practices, with the octopus microbiome serving as a potential biomarker for health status and animal welfare.