Retinoic acid is required early during adult neurogenesis in the dentate gyrus

Abstract

Retinoic acid (RA) is commonly used in vitro to differentiate stem cell populations including adult neural stem cells into neurons; however, the in vivo function of RA during adult neurogenesis remains largely unexplored. We found that depletion of RA in adult mice leads to significantly decreased neuronal differentiation within the granular cell layer of the dentate gyrus. RA contribution to neurogenesis occurs early, for RA deficiency also results in a decrease in newborn cells expressing an immature neuronal marker. Furthermore, although proliferation is unaffected during RA absence, cell survival is significantly reduced. Finally, a screen for retinoid-induced genes identifies metabolic targets including the lipid transporters, CD-36 and ABCA-1, the lipogenic master regulator SREBP1c as well as components of the Wnt signaling pathway. Our results reveal RA as a crucial contributor to early stages of adult neurogenesis and survival in vivo.

Fig. 1.

Experimental design for the induction of retinoid deficiency in mice. Mothers were placed on RAD diets immediately after giving birth. Pups were maintained on this diet. Once retinoid depletion was attained, BrdUrd injections began for 6 days. On the 7th day, mice were killed, and the brains were used to analyze the effect of retinoid deficiency on proliferation in the DG. Three weeks later, additional mice were killed, and the brains were assessed for neuronal differentiation and survival. In parallel, additional mice were maintained on a RAS diet. For retinoid rescue, some RAD mice were placed on a RAS diet on day 103, at the start of BrdUrd treatment.

Fig. 2.

RA status does not affect proliferation in the adult DG. (A and B) Proliferating cells in the subgranular zone of the DG were counted based on expression of PCNA or BrdUrd labeling. (C) No difference in PCNA expression is observed between RAD and RAS mice.

Fig. 3.

Survival of new cells is decreased with retinoid deficiency. Fewer cells labeled with BrdUrd remain in the GCL 3 weeks later, when RA is not present (P = 0.015). Retinoid supplementation also does not rescue this phenotype (P = 0.031).

Fig. 4.

Neuronal differentiation is impaired in RAD and retinoid rescued mice. (A) BrdUrd-containing cells were analyzed for signs of differentiation by labeling with antibodies for NeuN (neuronal phenotype) or S100b (astrocytic phenotype). (B) Retinoid deficiency grossly impaired neuronal differentiation, as observed in mice 3 weeks after BrdUrd injection (P = 0.0001). Returning RA to the diet of similarly retinoid-deprived mice did not rescue neurogenesis (P = 0.0001).

Fig. 5.

RA is essential during early phases of neuronal differentiation. (A) Early dcx⁺ cells exhibit no processes or processes horizontal to the dentate blades. Late dcx⁺ cells exhibit long processes perpendicular to the blades. Dcx expression in newborn cells is reduced in the absence of RA, during early stages as well as during later stages (total P = 0.005, early P = 0.05, late P = 0.008). Retinoid supplementation does not rescue dcx expression in RAD mice (total P = 0.005, late P = 0.05). (B) Retinoid deficiency results in a significant decrease in dcx expression in RAD mice examined immediately after BrdUrd injections when neither proliferation nor survival were affected (P < 0.005). (C) Contribution by RA to neuronal differentiation is independent of survival requirements. Retinoid deficiency results in a significant decrease in dcx expression in retinoid deficient mice (P < 0.005) immediately after BrdUrd injections when neither proliferation nor survival was affected by retinoid depletion. (D) Rate of neurogenesis is affected by both rates of survival and neuronal differentiation. Total number of BrdUrd-labeled cells expressing dcx in RAD and retinoid-replenished mice is reduced to 29.1% and 28.5%, respectively, compared to RAS mice. Total number of BrdUrd and NeuN double-labeled cells are reduced to 18.2% and 16.2%, respectively, compared to RAS mice.