Prenatal stress increases IgA coating of offspring microbiota and exacerbates necrotizing enterocolitis-like injury in a sex-dependent manner.

Necrotizing enterocolitis (NEC) is an intestinal inflammatory disease with high morbidity and mortality that affects almost exclusively premature infants. Breast milk feeding is known to substantially lower NEC incidence, and specific components of breast milk, such as immunoglobulin (Ig) A, have been identified as mediating this protective effect. On the other hand, accumulating evidence suggests dysbiosis of the neonatal intestinal microbiome contributes to NEC pathogenesis. In mice, neonates can inherit a dysbiotic microbiome from dams that experience stress during pregnancy. Here we show that while prenatal stress lowers fecal IgA levels in pregnant mice, it does not result in lower levels of IgA in the breast milk. Nevertheless, coating of female, but not male, offspring microbiota by IgA is increased by prenatal stress. Accordingly, prenatal stress was found to alter the bacterial community composition in female neonates but not male neonates. Furthermore, female, but not male, offspring of prenatally stressed mothers exhibited more severe colonic tissue damage in a NEC-like injury model compared to offspring with non-stressed mothers. Our results point to prenatal stress as a possible novel risk factor for NEC and potentially reveal new avenues in NEC prevention and therapy.

Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene generated using CRISPR-Cas9 RNPs.

Nodal-related protein (ndr2) is amember of the transforming growth factor type ß superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.