The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate-buffered saline (control) or two concentrations (2 × 10² and 2 × 10⁶ CFU mL⁻¹) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.

Summary The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.

Introductory paragraphThe pancreas is a central organ for human diseases that have a dramatic societal burden, such as pancreatic cancer and diabetes 1,2 . Non-coding cis-regulatory elements (CREs) of DNA control gene expression 3,4 , being required for proper pancreas function. Most disease-associated alleles 5,6 are noncoding, often overlapping with CREs 5 , suggesting that alterations in these regulatory sequences contribute to human pancreatic diseases by impairing gene expression. However, functional testing of CREs in vivo is not fully explored. Here we analysed histone modifications, transcription, chromatin accessibility and interactions, to identify zebrafish pancreas CREs and their human functional equivalents, uncovering disease-associated sequences across species. We found a human pancreatic enhancer whose deletion impairs the tumour suppressor gene ARID1A expression, conferring a potential tumour suppressor role to this non-coding sequence. Additionally, we identified a zebrafish ptf1a distal enhancer which deletion generates pancreatic agenesis, demonstrating the causality of this condition in humans 7 and the interspecies functional equivalency of enhancers. ResultsThe pancreas of zebrafish, a vertebrate model suitable for genetic manipulation 8 , shares many similarities with the human pancreas, including transcription factors (TFs) operating in similar genetic networks of pancreas development and function 9,10 . We observed that these similarities are extensible to organ structure ( Fig.1a) and cellular composition (SupplementaryFig.1), suggesting that shared genetic networks might operate through equivalent sets of CREs in both species.To identify CREs active in the zebrafish adult pancreas, we performed ChIP-seq for H3K27ac 11 , a key histone modification associated to active enhancers and ATAC-seq 12 , to identify regions of open chromatin. We have also performed HiChIP 13 against H3K4me3 14 to determine active promoters interacting with the uncovered enhancers ( Fig.1b). We found 14753 putative active enhancers, mostly in intergenic regions (57.8%), and 23298 putative active promoters corresponding to 9848 genes ( Fig.1c; SupplementaryTable1-3). To identify a subset of pancreas enhancers with higher tissue-specificity, we asked which of them are inactive in whole embryos (dome to 48hpf), finding that 7115 (48.2%) are active only in the differentiated adult pancreas (PsE; Fig.1d; SupplementaryTable4) while the remaining 7638 (51.8%) are also active during embryonic development (DevE). Interestingly, DevE presented 4 clusters (C1-4) with different activity dynamics during development (Fig.1d; SupplementaryFig.2; SupplementaryTable5).Pancreatic enhancers should activate the expression of genes in the pancreas. To test this, we identified the nearest genes to each putative pancreas enhancer 15 , observing that genes nearby PsE are enriched for exocrine pancreas expression (p<4.27e-9; SupplementaryFig.3a; SupplementaryTable6-7), detected by in situ hybridization. These results contrast with DevE, ...

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease.

Turbot, Scophthlalmus maximus, is a pleuronectiform fish that occurs in the northeast Atlantic along the European coast and in the Mediterranean Sea, and is produced in fish farms since the last quarter of the twentieth century. During a survey conducted in a turbot fish farm, nodular formations were occasionally observed in several organs, especially in the kidney and in the spleen. Microscopic observations showed that these nodules contained acid-fast bacilli. Molecular identification of the isolated bacteria revealed the Mycobacterium genus. Although no abnormal mortalities were evident morbidity was observed. The normal development and welfare of infected fish decrease and the condition factor, the haematocrit and haemoglobin concentration in blood decreases significantly with the increase of nodule abundance.

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