Introduction and Background

• This study explores a novel role for serotonin transport in setting up left-right (LR) body asymmetry during early embryonic development in both frog (Xenopus) and chick embryos.
• Serotonin (5-hydroxytryptamine or 5-HT) is a chemical messenger known for regulating mood and other neural functions; however, it also plays a key role in early developmental processes before the nervous system forms.
• The research focuses on two main transporters:
  • SERT (serotonin transporter): Removes serotonin from the cell surface using sodium gradients.
  • VMAT (vesicular monoamine transporter): Packages serotonin into vesicles for storage and later release, using proton gradients.
• The paper investigates how inhibiting these transporters affects the normal LR patterning of organs such as the heart, gut, and gallbladder.

Research Goals

• To determine whether SERT and VMAT are required for establishing consistent left-right asymmetry during early embryonic development.
• To understand the timing and spatial aspects of serotonin transport in relation to the LR patterning cascade.
• To explore if interfering with serotonin transport can randomize the normal position (laterality) of internal organs.

Key Terms and Definitions

• Serotonin (5-HT): A neurotransmitter involved in mood regulation and embryonic signaling. Think of it as a “messenger” that helps cells talk to each other.
• SERT (Serotonin Transporter): A protein that moves serotonin from the space outside the cell into the cell. It acts like a vacuum cleaner that cleans up extra serotonin.
• VMAT (Vesicular Monoamine Transporter): A protein that helps store serotonin inside small bubbles (vesicles) within the cell, much like packing supplies for later use.
• Left-Right Asymmetry: The normal, consistent difference in the position and shape of organs on the left and right sides of the body.
• Heterotaxia: A condition where organs do not follow their usual left-right pattern, resulting in a mix-up of positions.
• In Situ Hybridization: A laboratory method used to detect specific RNA sequences in tissues, helping locate where certain genes are active.

Materials and Methods

• Cloning and Probe Preparation:
  • Chick SERT and VMAT genes were cloned using RNA extracted from stage 23 chicken embryos.
  • Fragments of these genes were amplified via PCR and then inserted into vectors for in situ hybridization.
• Xenopus (Frog) Drug Exposure:
  • Frog embryos were treated with various pharmacological inhibitors (e.g., fluoxetine, imipramine, citalopram, alaproclate for SERT; reserpine and TBZOH for VMAT) during early cleavage stages.
  • After treatment until stage 16, the embryos were washed and allowed to develop until stage 45.
• Chick Embryo Drug Exposure:
  • Chick embryos were cultured in ovo with small openings in the eggshell to allow the introduction of inhibitors.
  • They were exposed to SERT and VMAT blockers (fluoxetine and reserpine) early in development and then fixed for later analysis.
• Scoring and Analysis:
  • The position (situs) of organs such as the heart, gut, and gallbladder was examined under a microscope.
  • Embryos showing reversal or randomization of organ positions were counted as having heterotaxia.
• Molecular Loss of Function:
  • A dominant negative mutant of SERT (D98G) was microinjected into specific blastomeres at the 4-cell stage to interfere with normal SERT function.
  • This approach helped pinpoint which cells are most sensitive to serotonin transport disruption.

Step-by-Step Experimental Procedures

• Preparation:
  • Extract RNA from embryos and perform reverse transcription to obtain cDNA of SERT and VMAT.
  • Use PCR with degenerate primers to amplify the target gene segments.
  • Clone the amplified fragments into expression vectors for probe creation.
• Drug Exposure in Xenopus:
  • Dejelly the embryos and divide them into control and treatment groups.
  • Add specific concentrations of SERT or VMAT inhibitors to the treatment groups from fertilization until stage 16.
  • After drug treatment, wash embryos thoroughly and allow them to develop until stage 45.
  • Score the embryos under a microscope to determine the rate of organ laterality defects.
• Drug Exposure in Chick Embryos:
  • Create a small opening in the eggshell and remove some albumin to reduce pressure.
  • Inject a mixture of inhibitors in albumin into the egg.
  • Seal the egg and incubate at 37.5 °C until the desired developmental stage is reached.
  • Fix the embryos and perform in situ hybridization to examine the expression of left-side markers like Shh and Nodal.
• Microinjection of Mutant SERT:
  • Synthesize capped mRNA for the nonfunctional SERT mutant (D98G).
  • Inject the mRNA into specific blastomeres (right ventral, right dorsal, left dorsal, or left ventral) at the 4-cell stage.
  • Allow embryos to develop and then score the organ laterality to determine which cell lineage is most affected.

Results in Xenopus (Frog) Embryos

• Exposure to SERT inhibitors (e.g., fluoxetine, imipramine) and VMAT inhibitors (e.g., reserpine, TBZOH) led to a significant percentage of embryos showing heterotaxia.
• Timing was crucial: embryos exposed from fertilization to early cleavage (up to stage 7) were most affected.
• The maximum effect observed was around 20–27% heterotaxia, meaning many embryos had one or more organs on the wrong side.
• Control treatments (using vehicle or a norepinephrine uptake blocker like nisoxetine) did not show these defects.

Results in Chick Embryos

• Both SERT and VMAT are expressed in the primitive streak and Hensen’s node – key organizers in chick development.
• In situ hybridization showed that SERT expression appears as a punctate pattern in the ectoderm and mesoderm, while VMAT is more uniformly expressed in the mesoderm.
• Exposure to fluoxetine and reserpine randomized the expression of early left-side markers such as Shh and Nodal, with 36–38% of embryos displaying bilateral expression instead of the normal left-sided pattern.
• This indicates that proper serotonin transport is necessary for maintaining the normal asymmetrical patterning of the embryo.

Molecular and Mechanistic Insights

• Microinjection of the dominant negative SERT mutant (D98G) demonstrated that interference with normal SERT function leads to laterality defects.
• Embryos injected in the right ventral blastomere exhibited the highest rates of heterotaxia, suggesting that this cell lineage is particularly dependent on serotonin transport for proper LR patterning.
• The study suggests that SERT and VMAT function upstream of known asymmetric gene cascades (e.g., XNR-1 in frogs and Shh in chicks).

Discussion and Implications

• The results provide strong evidence that serotonin transport is an early and essential step in establishing LR asymmetry.
• This new role for SERT and VMAT suggests that the movement of serotonin across cell membranes is crucial for setting up the directional signals during embryogenesis.
• Metaphorically, imagine the embryo as a kitchen where ingredients must be distributed correctly for the recipe (normal organ placement) to turn out well; serotonin transport acts like a delivery service ensuring ingredients reach the right side of the kitchen.
• The findings may have broader implications for understanding birth defects related to laterality and caution in the use of serotonin reuptake inhibitors during pregnancy.

Key Conclusions

• SERT and VMAT are vital for establishing left-right asymmetry in both frog and chick embryos.
• Interference with serotonin transport leads to randomization of organ positioning, highlighting its upstream role in the LR patterning cascade.
• The right ventral blastomere in frog embryos is particularly sensitive to disruption of SERT function.
• This research expands our understanding of how early embryonic signaling guides the formation of body plans and may inform future studies on developmental disorders.

观察与背景

• 本研究探索了血清素转运在两栖动物（非洲爪蟾）和鸡胚发育早期左右不对称（LR）形成中的新作用。
• 血清素（5-羟色胺或5-HT）不仅在调节情绪和神经功能中起作用，还在神经系统形成前的早期发育过程中发挥关键作用。
• 研究主要关注两种转运蛋白：
  • SERT（血清素转运蛋白）：利用钠离子梯度将血清素从细胞外运入细胞内，可比作“吸尘器”。
  • VMAT（囊泡单胺转运蛋白）：将血清素装入囊泡中储存以备释放，类似于将食材打包储存备用。
• 论文探讨了抑制这些转运蛋白如何影响器官（如心脏、肠道和胆囊）的正常左右定位。

研究目标

• 确定SERT和VMAT是否对胚胎早期左右不对称的建立至关重要。
• 阐明血清素转运在左右模式形成级联反应中的时空作用。
• 探讨干扰血清素转运是否会导致内脏器官位置随机化（异位现象）。

关键术语与定义

• 血清素 (5-HT)：一种神经递质，既参与情绪调节，也参与胚胎早期信号传递，可视为细胞之间传递信息的“信使”。
• SERT（血清素转运蛋白）：负责将血清素从细胞外转入细胞内，类似于将多余的信使收集起来。
• VMAT（囊泡单胺转运蛋白）：将血清素储存在囊泡中，类似于为未来使用打包储存。
• 左右不对称：指体内器官在左右两侧的正常、固定的差异。
• 异位现象：器官未按通常左右分布排列，导致位置混乱的状况。
• 原位杂交：一种实验方法，用于检测组织中特定RNA序列，帮助确定基因的活跃区域。

材料与方法

• 克隆与探针制备：
  • 从23期鸡胚中提取RNA，并逆转录得到SERT和VMAT的cDNA。
  • 使用PCR扩增目标基因片段，并克隆入表达载体，以便后续原位杂交使用。
• 非洲爪蟾胚胎药物处理：
  • 在去除透明带后，将胚胎分组，对照组与实验组分别处理。
  • 向实验组中加入SERT抑制剂（如氟西汀、伊米普明等）或VMAT抑制剂（如利血平、TBZOH），从受精到16期均持续处理。
  • 药物处理结束后，彻底清洗胚胎，并允许其发育至45期，再进行左右定位的评分。
• 鸡胚药物处理：
  • 在蛋壳上打一个小孔，去除部分蛋清以减小内部压力。
  • 将含有SERT和VMAT抑制剂（氟西汀和利血平）的混合溶液注入蛋内。
  • 密封蛋壳并在37.5 °C条件下孵育，直至达到预定发育阶段，然后固定胚胎进行原位杂交分析。
• 评分与分析：
  • 使用显微镜观察胚胎中诸如心脏、肠道和胆囊的位置。
  • 若器官出现位置反转或随机化，则判定为存在异位现象。
• 分子功能丧失实验：
  • 制备SERT的显性负突变体（D98G）的合成mRNA，并注射到4细胞期的特定细胞中以干扰正常SERT功能。
  • 通过这种方法，可以确定哪些细胞对血清素转运干扰最为敏感。

实验步骤详解

• 准备阶段：
  • 从胚胎中提取RNA，进行逆转录以获得SERT和VMAT的cDNA。
  • 使用含有退火引物的PCR扩增目标片段，并将其克隆进表达载体中以备探针制备。
• 爪蟾胚胎药物处理：
  • 将胚胎分成对照组和实验组，对照组仅加入溶剂。
  • 向实验组中加入特定浓度的SERT或VMAT抑制剂，从受精开始至16期持续处理。
  • 处理结束后彻底清洗胚胎，并继续孵育至45期，再进行器官定位的观察和记录。
• 鸡胚药物处理：
  • 在蛋壳上打孔并移除少量蛋清。
  • 注入含药物的混合溶液后，密封蛋壳并孵育至预定发育阶段。
  • 固定胚胎并通过原位杂交检测早期左侧标记基因（如Shh和Nodal）的表达。
• 微注射实验：
  • 合成SERT D98G突变体的mRNA，并在4细胞期将其注射到特定细胞中（如右腹侧细胞）。
  • 发育至适当阶段后，通过观察器官位置来评估注射对左右定位的影响。

非洲爪蟾胚胎结果

• 使用SERT和VMAT抑制剂处理的胚胎中，大量出现器官左右位置随机化现象（异位现象）。
• 从受精到早期裂解期（至7期）的药物处理效果最显著。
• 异位发生率约为20%–27%，即部分胚胎中一个或多个器官位置出现反转。
• 对照组（仅使用溶剂或使用选择性去甲肾上腺素抑制剂的组）未观察到明显缺陷。

鸡胚结果

• SERT和VMAT在原始条和Hensen结节中表达，这些区域是鸡胚体内的主要组织组织中心。
• 原位杂交显示，SERT在外胚层和中胚层中呈现点状表达，而VMAT主要在中胚层中均匀表达。
• 使用氟西汀和利血平处理后，早期左侧标记基因Shh和Nodal的表达出现双侧分布，约36%–38%的胚胎表现出左右表达混乱。
• 这一结果表明，正常的血清素转运对于维持胚胎左右不对称的正确模式至关重要。

分子机制与探讨

• 显性负突变体SERT (D98G)的微注射实验显示，干扰正常SERT功能会导致器官左右定位异常。
• 右腹侧细胞注射后出现的异位率最高，提示该细胞谱系在左右模式形成中对血清素转运特别敏感。
• 数据表明SERT和VMAT在已知的不对称基因级联（例如爪蟾中的XNR-1、鸡胚中的Shh）之前发挥作用。

讨论与意义

• 研究证明血清素转运在胚胎左右不对称形成中的早期且关键作用。
• 这种新发现提示，通过调控细胞膜上血清素的分布，可以指导胚胎细胞在左右方向上的命运，就像厨房中调料被正确配送以确保食谱完美一样。
• 结果对理解与左右不对称相关的先天缺陷具有潜在意义，并对孕期使用选择性血清素再摄取抑制剂提出了警示。
• 该研究为进一步探究胚胎发育中信号分子如何控制体轴提供了新的分子机制视角。

关键结论

• SERT和VMAT在非洲爪蟾和鸡胚中均对左右不对称的建立起关键作用。
• 干扰血清素转运导致器官位置随机化，证明其在不对称基因级联前的作用。
• 右腹侧细胞对SERT功能的干扰尤为敏感，提示其在左右模式形成中占有重要地位。
• 研究扩展了我们对胚胎体轴形成机制的认识，并可能为未来预防左右定位相关的先天缺陷提供依据。