Adequate supply of choline, an essential nutrient, is necessary to support proper brain development. Whether prenatal choline availability plays a role in development of the visual system is currently unknown. In this study, we addressed the role of in utero choline supply for the development and later function of the retina in a mouse model. We lowered choline availability in the maternal diet during pregnancy and assessed proliferative and differentiation properties of retinal progenitor cells (RPCs) in the developing prenatal retina, as well as visual function in adult offspring. We report that low choline availability during retinogenesis leads to persistent retinal cytoarchitectural defects, ranging from focal lesions with displacement of retinal neurons into subretinal space to severe hypocellularity and ultrastructural defects in photoreceptor organization. We further show that low choline availability impairs timely differentiation of retinal neuronal cells, such that the densities of early‐born retinal ganglion cells, amacrine and horizontal cells, as well as cone photoreceptor precursors, are reduced in low choline embryonic d 17.5 retinas. Maintenance of higher proportions of RPCs that fail to exit the cell cycle underlies aberrant neuronal differentiation in low choline embryos. Increased RPC cell cycle length, and associated reduction in neurofibromin 2/Merlin protein, an upstream regulator of the Hippo signaling pathway, at least in part, explain aberrant neurogenesis in low choline retinas. Furthermore, we find that animals exposed to low choline diet in utero exhibit a significant degree of intraindividual variation in vision, characterized by marked functional discrepancy between the 2 eyes in individual animals. Together, our findings demonstrate, for the first time, that choline availability plays an essential role in the regulation of temporal progression of retinogenesis and provide evidence for the importance of adequate supply of choline for proper development of the visual system.—Trujillo‐Gonzalez, I., Friday, W. B., Munson, C. A., Bachleda, A., Weiss, E. R., Alam, N. M., Sha, W., Zeisel, S. H., Surzenko, N. Low availability of choline in utero disrupts development and function of the retina. FASEB J. 33, 9194–9209 (2019). http://www.fasebj.org

Development of the cerebral cortex may be influenced by the composition of the maternal gut microbiota. To test this possibility, we administered probiotic Lactococcus lactis in drinking water to mouse dams from day 10.5 of gestation until pups reached postnatal day 1 (P1). Pups were assessed in a battery of behavioral tests starting at 10 weeks old. We found that females, but not males, exposed to probiotic during prenatal development spent more time in the center of the open field and displayed decreased freezing time in cue associated learning, compared to controls. Furthermore, we found that probiotic exposure changed the density of cortical neurons and increased the density of blood vessels in the cortical plate of P1 pups. Sex-specific differences were observed in the number of mitotic neural progenitor cells, which were increased in probiotic exposed female pups. In addition, we found that probiotic treatment in the latter half of pregnancy significantly increased plasma oxytocin levels in mouse dams, but not in the offspring. These results suggest that exposure of naïve, unstressed dams to probiotic may exert sex-specific long-term effects on cortical development and anxiety related behavior in the offspring.

24 Development of the cerebral cortex may be influenced by the composition of the maternal gut 25 microbiota. To test this possibility, we administered probiotic Lactococcus lactis in the drinking 26 water to mouse dams from day 10.5 of gestation and until pups reached postnatal day 1 (P1).27 Pups were assessed in a battery of behavioral tests starting at 10 weeks old. We found that 28 females, but not males, exposed to probiotic during prenatal development spent more time in the 29 center of the open field and also displayed decreased freezing time in cue associated learning, 30 compared to controls. Furthermore, we found that probiotic exposure changes the densities of 31 cortical neurons and increases the density of blood vessels in the cortical plate of P1 pups. Sex-32 specific differences were observed in the numbers of mitotic neural progenitor cells, which were 33 increased in probiotic exposed female pups. In addition, we found that probiotics treatment 34 throughout pregnancy significantly increased plasma oxytocin levels in mouse dams, but not in 35 the offspring. These results suggest that exposure of naïve, unstressed dams to probiotic may 36 exert sex-specific long-term effects on cortical development and anxiety related behavior in the 37 offspring. 38 39 40 41 Introduction 42 Neocortical development is influenced by a variety of environmental factors, including maternal 43 nutrition, stress and exposure to pathogens. In terms of cortical patterning and growth, the impact 3 44 of essential micronutrients, such as choline, has been well-characterized; yet less is known on 45 how food-derived microbiota, e.g. probiotics, may be altering cortical development at different 46 stages [1] . While the fetal gut is sterile before birth, it is increasingly clear that factors from the 47 maternal microbiota can induce specific patterns of gene expression in the fetal gut, as well as in 48 the brain [1][2][3] . After birth, microbial colonization of the infant gastrointestinal tract has been 49 shown to have widespread influence on brain development and behavior [4] . For example, mice 50 whose gut microbiota were depleted via antibiotics or who were raised in a germ-free facility 51 demonstrate exaggerated responses to stress and social stimuli, and display neurochemical and 52 brain structural abnormalities [5, 6] . Recently, neonatal exposure to probiotics, such as Bifidus 53 longum and Lactobacillus rhamnosous, have been shown to reverse maladaptive learning 54 behaviors in innately anxious mice [7] . Probiotics have also alleviated behavioral effects in other 55 models of anxiety disorders, such as those induced by maternal separation, social defeat or other 56 types of early life stress [8][9][10] . Mounting data on brain-gut axis has revealed several pathways 57 where probiotic treatment can affect microbiota populations, brain signaling, and subsequently 58 anxiety in mouse models of disease. However, there is less consensus on the behavioral effects 59 of probiotics in naïve, wild type mice which h...