Dietary vitamin A during pregnancy is required for the formation of secondary lymphoid organs of the developing embryo and affects the offspring’s immune competence in adulthood. This comparison of pregnant mice on diets completely lacking vitamin A, and with low and high vitamin A content, reveals a role for maternal vitamin A metabolites — notably retinoic acid — in establishing innate immunity in the developing fetus. The vitamin is required for the formation of secondary lymphoid organs during embryonic development and affects the offspring's immune competence during adulthood. The impact of nutritional status during fetal life on the overall health of adults has been recognized1; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed2,3. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells2,3,4,5. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Embryonic lymph node formation requires lymphoid tissue–inducer cells. Mebius and colleagues show that neurons adjacent to lymph anlagen synthesize retinoic acid, which triggers expression of the chemokine CXCL13 needed for the initial attraction of lymphoid tissue–inducer cells. The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue–inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5–14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons.

Loss of Crumbs homologue 1 (CRB1) function causes either the eye disease Leber congenital amaurosis or progressive retinitis pigmentosa, depending on the amount of residual CRB1 activity and the genetic background. Crb1 localizes specifically to the sub-apical region adjacent to the adherens junction complex at the outer limiting membrane in the retina. We show that it is associated here with multiple PDZ protein 1 (Mupp1), protein associated with Lin-7 (Pals1 or Mpp5) and Mpp4. We have produced Crb1^-/- mice completely lacking any functional Crb1. Although the retinas are initially normal, by 3-9 months the Crb1^-/- retinas develop localized lesions where the integrity of the outer limiting membrane is lost and giant half rosettes are formed. After delamination of the photoreceptor layer, neuronal cell death occurs in the inner and outer nuclear layers of the retina. On moderate exposure to light for 3 days at 3 months of age, the number of severe focal retinal lesions significantly increases in the Crb1^-/- retina. Crb2, Crb3 and Crb1 interacting proteins remain localized to the sub-apical region and therefore are not sufficient to maintain cell adhesion during light exposure in Crb1^-/- retinas. Thus we propose that during light exposure Crb1 is essential to maintain, but not assemble, adherens junctions between photoreceptors and Müller glia cells and prevents retinal disorganization and dystrophy. Hence, light may be an influential factor in the development of the corresponding human diseases.

Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration. We achieved to adapt a commercial 3^rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified. We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.