Physiological Role of the Zebrafish Retinol Receptor rbpr2 in Vision

 描述(申请人提供)：维生素A(视黄醇，ROL)缺乏和过量都会导致严重的视觉缺陷，包括失明和视网膜退行性疾病。Rol是11-顺式视黄醛(11-cisral)的前体，其功能是视觉生色团。因此，维持ROL的供应是全身和眼部产生维甲酸以支持视觉功能所必需的。哺乳动物不能从头合成维生素A，必须通过饮食获得维生素A前体。维生素A前体预计将跟随肠道中胆固醇/脂的命运；即促进吸收。长期以来，人们一直认为肠道对维生素A前体的摄取是通过被动扩散发生的；然而，现在提出了一种受体介导或促进饮食ROL的摄取，因为这不仅可以提供生理剂量的ROL，还可以防止已知与过量维生素A积累有关的全身和眼睛畸形。即使考虑到饮食中维生素A的持续供应对视觉功能的重要性，与饮食中维生素A的摄取和储存相关的信息仍然构成了一个知识空白；因此，研究促进ROL进入肠道细胞的受体对于眼部维甲酸的动态平衡可能会弥补这一点。在这项研究中，我们使用斑马鱼的幼虫，这是很好地建立了涉及眼部维甲酸稳态的基因的研究。这些基因的敲除重现了视觉表型，类似于在具有相应功能丧失突变的人类中观察到的表型。斑马鱼仔鱼的眼睛表现出典型的脊椎动物的形态，仔鱼在受精后5.5天就已经有了视觉表现。斑马鱼幼体还拥有许多与人类相同的胃肠道器官，以及参与吸收和处理脂质和维生素A前体的特殊细胞类型(肠道肠道细胞，Ents)。因此，在像斑马鱼这样的脊椎动物模型中，对受体的功能研究是可行的，建议在饮食中摄取和供应维生素A来维持眼部维甲酸的动态平衡。我们的初步结果，使用基于细胞的体外分析，显示了Rbpr2受体的质膜定位模式，这与参与促进ROL上调的受体的定位模式一致。为了确定rbpr2的组织分布模式，我们对斑马鱼进行了整装原位杂交(WISH)。我们发现Rbpr2分别在肠道肠道细胞和肝脏肝细胞中表达，这些组织和细胞被认为是调节饮食ROL摄取和储存的组织和细胞。使用吗啡基因敲除方法，我们观察到在受精后5.5天，rbpr2变体在视觉功能的视动反应(OKR)测试中显示出跳动频率和增益减少。这表明后期斑马鱼幼体中Rbpr2的表达降低，这些斑马鱼幼体依赖于外源饲料中的维生素A，其发色团浓度低于最适水平，表现为视觉功能下降。在这些初步数据的基础上，我们现在已经使用TALEN技术产生了F0 rbpr2突变体。Rbpr2-突变体是可行的，没有严重的发育或眼睛缺陷，因此功能分析是可行的。基于这些发现，我们将检验我们的假设，即rbpr2促进肠道对饮食中ROL的摄取，并且rbpr2的丢失将影响眼睛视黄醇水平，表现为视觉功能丧失。这一假说将在体外和体内模型中得到解决，实验的具体目标如下：特定目标1：确定Rbpr2摄取视黄醇的生化能力。具体目标2：确定rbpr2是否在支持光接收的情况下促进肠道ROL摄取以产生眼部发色团。这项拟议的研究将揭示斑马鱼中rbpr2的缺失如何减少维持视觉功能的视黄醇类物质的ROL供应。这项研究是否应该揭示rbpr2的作用 视黄醇稳态受体在视觉支持中的作用，如果这种受体的缺失影响发色团的产生和视觉功能，则可以通过调节参与饮食中视黄醇摄取和供应的受体来更好地探索解决人类因维生素A缺乏或过剩而导致的视觉缺陷的策略。

 DESCRIPTION (provided by applicant): Both vitamin A (retinol, ROL) deficiency and excess are known to cause severe visual defects including blindness and retinal degenerative diseases. ROL serves as the precursor for 11-cis-retinaldehyde (11-cis RAL), which functions as the visual chromophore. Therefore a sustained supply of ROL is required for systemic and ocular retinoid production in supporting visual function. Mammals, are unable to synthesize vitamin A de novo, and must obtain vitamin A precursors via the diet. Vitamin A precursors are predicted to follow the fate of cholesterol/lipids in the intestine; i.e. facilitated uptake. It was long assmed intestinal uptake of vitamin A precursors occurs by passive diffusion; however a receptor mediated or facilitated uptake of dietary ROL is now proposed as this would not only provide physiological doses of ROL but would also prevent systemic and eye malformations known to be associated with excessive vitamin A accumulation. Even given the importance of a continued supply of dietary vitamin A for visual function, information related to the uptake and storage of dietary vitamin A still constitutes a knowledge gap; therefore study of receptors facilitating ROL entry into intestinal cells for ocular retinoid homeostasis could remedy this. In this study, we ai to use the larva of the zebrafish, which is well established for studies of genes involved in ocula retinoid homeostasis. Knockdown of such genes have recapitulated visual phenotypes similar to those observed in humans with corresponding loss-of- function mutations. The zebrafish larval eye shows the typical vertebrate morphology and larvae have visual performance already at 5.5 days post fertilization (dpf). The larval zebrafish also possess many of the same gastrointestinal organs present in humans as well as specialized cell types involved in absorption and processing (intestinal enterocytes, ENTs) of lipids and vitamin A precursors. Therefore, functional studies of receptors, proposed in the dietary uptake and supply of vitamin A for ocular retinoid homeostasis in a vertebrate model such as the zebrafish are feasible. Our preliminary results, using in vitro cell based assays showed plasma membrane localization patterns for the Rbpr2 receptor, consistent with a localization pattern for a receptor involved in facilitated uptak of ROL. To determine tissue distribution patterns for rbpr2, we performed whole mount in-situ hybridization (WISH) in zebrafish. We found that Rbpr2 is expressed in intestinal enterocytes and liver hepatocytes, tissues and cells proposed to mediate dietary ROL uptake and storage respectively. Using a morpholino gene knockdown approach, we observed that at 5.5 days post fertilization rbpr2-morphants display reduced saccade frequency and gain in optokinetic response (OKR) tests for visual function. This indicated that reduced Rbpr2 expression in late stage zebrafish larvae, which are dependent on exogenous dietary sources for vitamin A, have suboptimal chromophore concentrations, manifesting in decreased visual function. Building on these preliminary data, we have now generated F0 rbpr2- mutants using the TALEN technology. Rbpr2-mutants are viable and show no severe developmental or eye defects, thus making functional analysis feasible. Based on these findings, we will test our hypothesis that rbpr2 facilitates dietary ROL uptake in the intestine and loss of rbpr2 will impact ocular retinoid level manifesting in loss of visual function. This hypothesis will be addressed in both in vitro and an i vivo model in the experiments of the following Specific Aims: Specific Aim 1: Determine the biochemical capabilities of Rbpr2 for retinol uptake. Specific Aim 2: Determine if rbpr2 facilitate intestinal ROL uptake for ocular chromophore production in the support of photoreception. The proposed studies will reveal how loss of rbpr2 in zebrafish decreases ROL supply for the production of retinoids in sustaining visual function. Should this study reveal a role of the rbpr2 receptor for retinol homeostasis in the support of vision and should an absence of such a receptor affects chromophore production and visual function, strategies aimed at addressing visual defects due to vitamin A deficiency or excess in humans could be better explored by modulating receptors involved in dietary retinol uptake and supply.