Introduction and Background

• The embryo develops three main body axes (dorsal–ventral, anterior–posterior, and left–right). Correct left–right (LR) orientation is crucial for proper organ placement.
• This study explores how polarity proteins help establish the LR axis in a frog model (Xenopus).
• Two main ideas explain LR patterning:
  • Cilia-driven fluid flow: Tiny hair-like structures (cilia) create directional flows during later stages.
  • Cellular chirality and polarity: Intrinsic properties of individual cells (through apical–basal and planar cell polarity proteins) may set up early LR asymmetry.

Key Concepts and Terminology

• Cell Polarity: The spatial differences in the shape, structure, and function of cells.
  • Apical–Basal Polarity (ABP): The organization of a cell from its top (apical) to bottom (basal) side.
  • Planar Cell Polarity (PCP): The arrangement of cells within the plane of a tissue, similar to arranging tiles on a floor.
• Heterotaxia: A condition where organs are randomly positioned instead of following a normal left–right pattern (imagine the heart being on the wrong side).
• Serotonin (5HT): A signaling molecule that, in early development, helps create asymmetry.
• Tight Junctions (TJs): Structures that seal cells together, much like a gasket seals parts of a machine, ensuring proper cell communication.

Research Objectives and Questions

• Determine whether ABP and PCP proteins are required for the proper orientation of the LR axis.
• Examine if disrupting these proteins leads to misplacement of organs (heterotaxia) independent of cilia-driven mechanisms.
• Investigate the role of these proteins in early LR signaling (including localization of serotonin and the asymmetric expression of key genes like Xnr-1).
• Explore how early organizers communicate LR information to later organizers (the “big brother effect” in conjoined twins).

Experimental Methods and Step-by-Step Process

• Manipulation of Polarity Proteins:
  • Injected dominant negative (DN) constructs for proteins such as Par6 and aPKC to disrupt normal ABP.
  • Used morpholinos (antisense molecules) to knock down the PCP protein Vangl2 and others (e.g., diversin, disheveled, RSG1).
• Targeting Specific Cells:
  • Injections were made at early cleavage stages (e.g., at the one-cell or four-cell stage) to affect either cells contributing to the ciliated node (GRP) or cells that do not.
  • This allowed researchers to test if the effects on LR patterning are independent of cilia.
• Assays and Measurements:
  • Laterality Assay: Checking the positions of organs (heart, stomach, gall bladder) at tadpole stage.
  • In Situ Hybridization: Examining the expression pattern of the gene Xnr-1, which is normally expressed only on the left side.
  • Protein Localization: Using immunohistochemistry to monitor proteins like disheveled-2 (dsh2) and the distribution of serotonin (5HT).
  • Tight Junction Integrity: A biotin-labeling assay was performed to assess how well cells stay connected.
• Conjoined Twin Experiments (“Big Brother Effect”):
  • A secondary organizer was induced using the transcription factor XSiamois at the 16-cell stage.
  • Disruption of polarity proteins in either the primary or secondary organizer randomized the orientation of the LR axis in twins.

Results: What Did They Find?

• Disrupting ABP proteins (DNPar6, DNaPKC) leads to heterotaxia—organs are placed in random positions.
• Similarly, interference with PCP proteins (Vangl2, diversin, disheveled, RSG1) also randomizes LR orientation.
• The effects occur even when disruptions are made in cells that do not contribute to the ciliated node, showing that these pathways work independently of cilia.
• Alterations in cell polarity cause:
  • Mislocalization of serotonin (5HT), which normally becomes concentrated in one specific cell.
  • Disruption of tight junctions, meaning the “seals” between cells are compromised.
  • Abnormal expression of the left-side gene Xnr-1.
• In conjoined twin experiments, proper LR orientation of the secondary organizer depends on intact ABP and PCP signals from the primary organizer.

Conclusions and Implications

• Both ABP and PCP proteins are essential for correctly orienting the LR axis during early embryonic development.
• They operate through mechanisms that are independent of cilia-driven fluid flow.
• These proteins help establish early gradients and maintain cell–cell connections, which in turn instruct the proper positioning of organs.
• The study suggests that early cell polarity is a fundamental, conserved mechanism that ensures our organs develop in the right places.

Step-by-Step “Cooking Recipe” Summary

• Step 1: In the very early embryo, establish cell polarity using ABP and PCP proteins—imagine setting the table with clearly defined positions.
• Step 2: These proteins direct the placement of key ingredients (molecules like 5HT and genes such as Xnr-1) and maintain tight junctions (like sealing envelopes to keep messages intact).
• Step 3: When polarity proteins are disrupted, the “recipe” goes wrong—the signals become scrambled, and the ingredients are misplaced.
• Step 4: As a result, organs develop in random positions (heterotaxia), much like ingredients ending up in the wrong parts of a dish.
• Step 5: In twin experiments, if the early organizer’s signals are disrupted, even a later-induced organizer cannot correctly orient its LR axis.
• Step 6: Only when the polarity “chefs” work properly does the embryo achieve a well-organized body plan.

Key Takeaways

• Proper LR asymmetry is essential for health; misplacement can lead to serious defects.
• Cell polarity proteins (ABP and PCP) are like internal compasses that instruct cells on which way is left or right.
• These findings highlight an ancient, conserved mechanism that works independently of cilia.
• The study provides insights that could help understand and potentially correct laterality defects in humans.

观察与背景 (引言)

• 胚胎发育时会形成三个主要轴：背腹轴、前后轴和左右轴。正确的左右（LR）方向对于器官的正常定位至关重要。
• 本研究探讨了极性蛋白在青蛙（Xenopus）模型中如何建立左右轴。
• 关于左右模式的形成有两种主要观点：
  • 纤毛驱动的液体流动：在较晚阶段，细小的纤毛产生定向流动。
  • 细胞本身的手性和极性：通过顶-基（ABP）和面板极性（PCP）蛋白，细胞内部的固有特性在早期就可能建立左右不对称。

关键概念与术语

• 细胞极性：指细胞在形状、结构和功能上的空间差异。
  • 顶-基极性 (ABP)：细胞从顶部（顶）到基底（底）的组织方式。
  • 面板极性 (PCP)：细胞在组织平面内的排列方式，就像铺地砖一样。
• 异位症 (Heterotaxia)：器官位置随机分布，而不是遵循正常的左右模式（就像心脏长在错误的一边）。
• 血清素 (5HT)：一种在早期发育中帮助建立不对称性的信号分子。
• 紧密连接 (TJs)：细胞之间的密封结构，类似于机械中用来密封零件的垫圈，确保细胞之间的正确通讯。

研究目标与问题

• 确定顶-基极性和面板极性蛋白是否对正确定位左右轴至关重要。
• 检验破坏这些蛋白是否会导致器官位置混乱（异位症），且这种效应与纤毛机制无关。
• 研究这些蛋白在左右信号传递中的作用，包括血清素定位和关键基因（如Xnr-1）的不对称表达。
• 探讨早期组织器如何向后期组织器传递左右信息（即“哥哥效应”在连体双胞中的表现）。

实验方法与步骤

• 极性蛋白的操作：
  • 注射抑制性构建体（如DNPar6和DNaPKC）来破坏正常的顶-基极性。
  • 使用反义寡核苷酸（morpholinos）敲低面板极性蛋白Vangl2及其他蛋白（如diversin、disheveled、RSG1）。
• 特定细胞的靶向：
  • 在胚胎早期（如一细胞或四细胞阶段）注射，使影响限于参与纤毛结点（GRP）的细胞或不参与的细胞。
  • 从而测试这种效应是否与纤毛无关。
• 检测与测量：
  • 左右性检测：在蝌蚪阶段检查心脏、胃和胆囊的位置。
  • 原位杂交：观察通常仅在左侧表达的Xnr-1基因的表达模式。
  • 蛋白定位：通过免疫组化检测disheveled-2 (dsh2) 和血清素 (5HT) 的分布。
  • 紧密连接检测：利用生物素标记法检测细胞之间连接的完整性。
• 连体双胞实验（“哥哥效应”）：
  • 在16细胞阶段利用转录因子XSiamois诱导形成第二个组织器。
  • 在主要或次要组织器中破坏极性蛋白，观察左右轴定向是否被打乱。

结果：研究发现了什么？

• 破坏顶-基极性蛋白（DNPar6、DNaPKC）导致异位症——器官随机分布。
• 同样，干扰面板极性蛋白（Vangl2、diversin、disheveled、RSG1）也会随机化左右轴。
• 即使在不参与纤毛结点的细胞中破坏这些蛋白，也会影响左右不对称，说明这些途径独立于纤毛机制。
• 细胞极性改变导致：
  • 血清素（5HT）定位异常，本应集中在特定细胞的5HT信号变得混乱。
  • 紧密连接受损，类似于细胞之间的“密封条”失效。
  • Xnr-1基因的不对称表达异常。
• 在连体双胞实验中，如果主要组织器的信号受到干扰，则次要组织器也无法正确定向，验证了“哥哥效应”。

结论与意义

• 顶-基极性和面板极性蛋白对胚胎早期左右轴的正确定位至关重要。
• 它们通过独立于纤毛流动的机制发挥作用。
• 这些蛋白帮助建立早期信号梯度并维持细胞间连接，从而指导器官的正确定位。
• 这一机制可能是保守的，适用于其他脊椎动物，对理解和矫正左右不对称缺陷具有重要意义。

逐步“烹饪”式总结

• 第一步：在胚胎早期，通过顶-基极性和面板极性蛋白建立细胞极性，就像为餐桌安排座位。
• 第二步：这些蛋白指导关键成分（如5HT和Xnr-1基因）的定位，并维持紧密连接（类似于确保信息密封传递）。
• 第三步：若极性蛋白受到干扰，“菜谱”就会出错——信号混乱，成分放错位置。
• 第四步：结果，器官会随机分布（异位症），就像食材放错了位置。
• 第五步：在连体双胞实验中，若早期组织器信号受损，即使后期诱导的组织器也无法正确定向。
• 第六步：只有当极性“厨师”正常工作时，胚胎才能形成一个布局合理的身体计划。

关键要点总结

• 正确的左右不对称对于健康至关重要，错误的器官定位可能引发严重缺陷。
• 细胞极性蛋白就像内部指南针，指示细胞哪边是左、哪边是右。
• 研究表明，这一古老且保守的机制独立于纤毛机制发挥作用。
• 这些发现有助于理解并可能纠正人类左右不对称发育缺陷。