CRISPR/Cas9 mediated genomic deletions protocol in zebrafish

Since its first application for site-directed mutagenesis in 2013, the CRISPR/Cas9 system revolutionized genome engineering. In this protocol, João Amorim, Chiara Perrod and collaborators, lead by José Bessa from i3S, present a validated workflow for the generation of targeted genomic deletions in zebrafish. Here, step-by-step details can be found from design, cloning and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotyping screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely-used approach in functional genetics.

The article entitled CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish was published in STAR protocols.

Genetic variants associated to type 2 diabetes can modulate endocrine enhancers in vivo

Several genome-wide association studies have shown a clear association between single nucleotide polymorphisms (SNPs) and type 2 diabetes, being the vast majority of these variants located in putative endocrine pancreatic enhancers. This suggests that these SNPs may modulate the enhancer activity and, consequently, gene expression. However, the correlation between SNPs and enhancer activity impairment and the in vivo validation are still poorly explored. Here, by using an in vivo mosaic transgenesis assays in zebrafish, Eufrásio and coworkers from Bessa’s lab at I3S, were able to identify 6 endocrine pancreatic enhancers. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. One of the latter, located in a SLC30A8 exon, results in an aminoacid substitution, being the canonical explanation for the risk of type 2 diabetes the decrease of SLC30A8 function. However, there are other studies that show other type 2 diabetes-associated SNPs that truncate SLC30A8 that can confer protection from this disease. The authors clarified this incongruence by showing that the SLC30A8 gain of function, mediated by the presence of one type 2 diabetes-associated SNP, is the explanation for the increased risk for the disease. To know how, read the full work in Diabetes, in the article entitled In vivo reporter assays uncover changes in enhancer activity caused by type 2 diabetes-associated single nucleotide polymorphisms”.

A conserved notochord enhancer controls pancreas development in Vertebrates

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. Here, João Amorim, Ana Gali-Macedo and collaborators from José Bessa’s team at I3S show that in zebrafish, the loss of function of Nog2, a Bmp antagonist expressed in the notochord, impairs beta-cell differentiation, compatible with the antagonistic role of Bmp in this process. In addition, the study shows 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 beta-cell differentiation. Tracing Nog2 diffusion, it was observed that Nog2 emanates from the notochord to the pancreatic progenitor domain. Finally, a notochord enhancer was found 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.

The article, entitled "A conserved notochord enhancer controls pancreas development in Vertebrates " was published in Cell Reports.

Axial progenitors and the trunk-to-tail transition

During the trunk-to-tail transition, axial progenitors relocate from the epiblast to the tail bud. Rita Aires and collaborators, lead by Moises Mallo from the IGC, show that this process entails a major regulatory switch, bringing tail bud progenitors under Gdf11 signaling control. After Gdf11-induced transition from trunk to tail development, Lin28 genes promote axial progenitor expansion until activation of Hox13 genes overrides this activity. Their work demonstrates that different gene networks regulate axial progenitors at the trunk versus tail levels. More information here.

The article entitled “Tail bud progenitor activity relies on a network comprising Gdf11, Lin28, and Hox13 genes” was published in Developmental Cell.

 

 

 

 

Fast screening platform for cancer therapy

 

Despite promising preclinical results, average response rates to anti-VEGF therapies, such as bevacizumab, are reduced for most cancers, while incurring in remarkable costs and side effects. Currently, there are no biomarkers available to select patients that can benefit from this therapy. Depending on the individual tumor, anti-VEGF therapies can either block or promote metastasis. In this context, an assay able to predict individual responses prior to treatment, including the impact on metastasis would prove of great value to guide treatment options. A team from the Champalimaud Foundation led by Rita Fior showed that zebrafish xenografts are able to reveal different responses to bevacizumab in just 4 days, evaluating not only individual tumor responses but also the impact on angiogenesis and micrometastasis. Importantly, the author perform proof-of-concept experiments where clinical responses in patients were compared with their matching zebrafish Patient-Derived Xenografts - zAvatars, opening the possibility of using the zebrafish model to screen bevacizumab therapy in a personalized manner.

The article entitled “Zebrafish xenografts as a fast screening platform for bevacizumab cancer therapy” was published in Communications Biology.