Protein Purification and Western Blot Detection from Single Zebrafish Embryo.

Characterization of a protein of interest during development is essential for functional studies. A general strategy for understanding the function of a particular protein involves the generation of null mutations, or treatment with drugs, that interfere with its activity. To demonstrate that the synthesis, stability, or activity of a protein has been affected, accurate and efficient detection of low amounts of protein is essential. This can be achieved by immunohistochemistry or by western blot. Here we describe a method for the detection of proteins from single de-yolked zebrafish embryos. This procedure includes a fixation step and the concomitant elimination of lipids from the yolk cell. We show that this approach allows the rapid analysis of proteins in embryos without having to manually remove the yolk. This method provides a convenient alternative for genotyping of mutant embryos as early as the 128 cell stage. In addition, in drug- or morpholino-treated embryos, the correlation between the penetrance of a phenotype and the concentration of a protein present may be established.

smarce1 mutants have a defective endocardium and an increased expression of cardiac transcription factors in zebrafish.

SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes and facilitate tissue-specific gene regulation during development. BAF57/SMARCE1 is a BAF complex subunit encoded in animals by a single gene and is a component of all mammalian BAF complexes. In vivo, the loss of SMARCE1 would lead to the formation of deficient combinations of the complex which might present limited remodeling activities. To address the specific contribution of SMARCE1 to the function of the BAF complex, we generated CRISPR/Cas9 mutations of smarce1 in zebrafish. Smarce1 mutants showed visible defects at 72 hpf, including smaller eyes, abnormal body curvature and heart abnormalities. Gene expression analysis revealed that the mutant embryos displayed defects in endocardial development since early stages, which led to the formation of a misshapen heart tube. The severe morphological and functional cardiac problems observed at 4 dpf were correlated with the substantially increased expression of different cardiac transcription factors. Additionally, we showed that Smarce1 binds to cis-regulatory regions of the gata5 gene and is necessary for the recruitment of the BAF complex to these regions.

The developmental and pathogenic roles of BAF57, a special subunit of the BAF chromatin-remodeling complex.

Mammalian SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes. BAF57 is a subunit of the BAF complexes; it is encoded only in higher eukaryotes and is present in all mammalian assemblies. Its main structural feature is a high-mobility group domain, the DNA-binding properties of which suggest that BAF57 may play topological roles as the BAF complex enters or exits the nucleosome. BAF57 displays specific interactions with a number of proteins outside the BAF complex. Through these interactions, it can accomplish specific functions. In the embryo, BAF57 is responsible for the silencing of the CD4 gene during T-cell differentiation, and during the repression of neuronal genes in non-neuronal cells, BAF57 interacts with the transcriptional corepressor, Co-REST, and facilitates repression. Extensive work has demonstrated a specific role of BAF57 in regulating the interactions between BAF and nuclear hormone receptors. Despite its involvement in oncogenic pathways, new generation sequencing studies do not support a prominent role for BAF57 in the initiation of cancer. On the other hand, evidence has emerged to support a role for BAF57 as a metastasis factor, a prognosis marker and a therapeutic target. In humans, BAF57 is associated with disease, as mutations in this gene predispose to important congenital disorders, including menigioma disease or the Coffin-Siris syndrome. In this article, we present an exhaustive analysis of the BAF57 molecular and biochemical properties, cellular functions, loss-of-function phenotypes in living organisms and pathological manifestations in cases of human mutations.