brca2-mutant zebrafish exhibit context- and tissue-dependent alterations in cell phenotypes and response to injury.

Cancer cells frequently co-opt molecular programs that are normally activated in specific contexts, such as embryonic development and the response to injury. Determining the impact of cancer-associated mutations on cellular phenotypes within these discrete contexts can provide new insight into how such mutations lead to dysregulated cell behaviors and subsequent cancer onset. Here we assess the impact of heritable BRCA2 mutation on embryonic development and the injury response using a zebrafish model (Danio rerio). Unlike most mouse models for BRCA2 mutation, brca2-mutant zebrafish are fully viable and thus provide a unique tool for assessing both embryonic and adult phenotypes. We find that maternally provided brca2 is critical for normal oocyte development and embryonic survival in zebrafish, suggesting that embryonic lethality associated with BRCA2 mutation is likely to reflect defects in both meiotic and embryonic developmental programs. On the other hand, we find that adult brca2-mutant zebrafish exhibit aberrant proliferation of several cell types under basal conditions and in response to injury in tissues at high risk for cancer development. These divergent effects exemplify the often-paradoxical outcomes that occur in embryos (embryonic lethality) versus adult animals (cancer predisposition) with mutations in cancer susceptibility genes such as BRCA2. The altered cell behaviors identified in brca2-mutant embryonic and adult tissues, particularly in adult tissues at high risk for cancer, indicate that the effects of BRCA2 mutation on cellular phenotypes are both context- and tissue-dependent.

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos.

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

A single codon insertion in PICALM is associated with development of familial subvalvular aortic stenosis in Newfoundland dogs.

Familial subvalvular aortic stenosis (SAS) is one of the most common congenital heart defects in dogs and is an inherited defect of Newfoundlands, golden retrievers and human children. Although SAS is known to be inherited, specific genes involved in Newfoundlands with SAS have not been defined. We hypothesized that SAS in Newfoundlands is inherited in an autosomal dominant pattern and caused by a single genetic variant. We studied 93 prospectively recruited Newfoundland dogs, and 180 control dogs of 30 breeds. By providing cardiac screening evaluations for Newfoundlands we conducted a pedigree evaluation, genome-wide association study and RNA sequence analysis to identify a proposed pattern of inheritance and genetic loci associated with the development of SAS. We identified a three-nucleotide exonic insertion in phosphatidylinositol-binding clathrin assembly protein (PICALM) that is associated with the development of SAS in Newfoundlands. Pedigree evaluation best supported an autosomal dominant pattern of inheritance and provided evidence that equivocally affected individuals may pass on SAS in their progeny. Immunohistochemistry demonstrated the presence of PICALM in the canine myocardium and area of the subvalvular ridge. Additionally, small molecule inhibition of clathrin-mediated endocytosis resulted in developmental abnormalities within the outflow tract (OFT) of Xenopus laevis embryos. The ability to test for presence of this PICALM insertion may impact dog-breeding decisions and facilitate reduction of SAS disease prevalence in Newfoundland dogs. Understanding the role of PICALM in OFT development may aid in future molecular and genetic investigations into other congenital heart defects of various species.