What Was Observed? (Introduction)

• The study investigated how cell polarity proteins control left–right (LR) orientation during early embryonic development in Xenopus (frog embryos).
• It compared two main models: one where cilia‐driven fluid flow determines LR asymmetry and another where intrinsic cellular chirality (cell polarity) plays a key role.
• The findings support that both apical–basal (ABP) and planar cell polarity (PCP) proteins are crucial for establishing consistent LR asymmetry, independent of ciliary functions.

Key Terms and Concepts

• Left–Right (LR) Asymmetry: The organized placement of internal organs (such as the heart, liver, and stomach) on either side of the body.
• Apical–Basal Polarity (ABP): The orientation of cells from their top (apical) to bottom (basal) surfaces; key proteins include Par6 and aPKC.
• Planar Cell Polarity (PCP): The coordinated alignment of cells within the plane of a tissue; involves proteins like Vangl2, diversin, disheveled, and RSG1.
• Heterotaxia: A condition where organ placement is randomized or misoriented.
• GRP (Gastrocoel Roof Plate): A ciliated structure in Xenopus embryos that normally contributes to LR asymmetry but is not the sole determinant.
• Tight Junctions: Cell–cell junctions that maintain tissue integrity and help establish proper signaling gradients.
• 5HT (Serotonin): A signaling molecule that becomes asymmetrically localized and is important for LR patterning.
• Xnr-1: A gene normally expressed on the left side of the embryo, serving as a marker for LR asymmetry.

Experimental Methods and Approach

• Dominant negative (DN) constructs for Par6 and aPKC were microinjected into one-cell stage Xenopus embryos to disrupt apical–basal polarity.
• Vangl2 morpholinos and additional constructs (diversin, disheveled, RSG1) were used to inhibit planar cell polarity.
• Injections were targeted to specific blastomeres to distinguish effects in cells that contribute to the GRP from those that do not.
• The researchers analyzed organ placement (situs), cilia positioning, tight junction integrity, and the expression of asymmetry markers (Xnr-1 and 5HT).
• Conjoined twin experiments were performed by inducing secondary organizers (using XSiamois injections) to test the “big brother effect” in LR instruction.

What Were the Results? (Findings)

• Disruption of ABP proteins (Par6 and aPKC) resulted in randomized organ placement (heterotaxia).
• Inhibition of PCP proteins (through Vangl2 MO and others) similarly led to randomization of the LR axis.
• These effects occurred even when the disruption was limited to cells not contributing to the GRP, indicating a cilia-independent mechanism.
• Altered expression of the LR marker gene Xnr-1 and mislocalization of 5HT were observed upon interference with polarity pathways.
• Tight junction integrity was compromised, suggesting that proper cell adhesion is necessary for LR patterning.
• In conjoined twin experiments, both the primary and induced organizers required intact polarity signals for correct LR orientation.

Experimental Steps (Step-by-Step Approach)

• Step 1: Microinject DN constructs for Par6 and aPKC into one-cell stage embryos to disrupt apical–basal polarity.
• Step 2: Inject Vangl2 morpholinos and other PCP-disrupting reagents to inhibit planar cell polarity.
• Step 3: Target specific blastomeres to differentiate between GRP-contributing and non–GRP cells.
• Step 4: Assess cilia positioning in the GRP and examine the localization of asymmetry markers such as 5HT and Xnr-1.
• Step 5: Use a biotin-labeling assay to evaluate tight junction integrity.
• Step 6: Induce conjoined twins by injecting XSiamois at the 16-cell stage and analyze heart situs in both twins.
• Step 7: Compare treated embryos with controls to determine the impact on LR patterning.

Key Conclusions (Discussion)

• Both apical–basal and planar cell polarity proteins are essential for proper LR asymmetry in vertebrate embryos.
• These proteins act upstream of asymmetric gene expression, affecting critical processes such as 5HT localization and tight junction formation.
• The study demonstrates that LR patterning can be established independently of ciliary flow, relying instead on cell–intrinsic polarity cues.
• Correct communication between early (primary) and later (secondary) organizers requires intact polarity signals, as shown by the conjoined twin experiments.

Significance and Broader Implications

• This research highlights a conserved, cilia-independent mechanism for establishing LR asymmetry in vertebrates.
• Understanding these polarity pathways may shed light on congenital defects related to organ misplacement (heterotaxia) in humans.
• The findings emphasize that cell polarity is a fundamental aspect of embryonic development, influencing the overall body plan.

Overall Summary (Step-by-Step Explanation)

• The study reveals that cell polarity proteins (both ABP and PCP) play a critical role in determining the left–right orientation of internal organs.
• Disrupting these proteins in frog embryos leads to random organ placement, altered gene expression, and mislocalization of key signaling molecules like 5HT.
• These effects are observed even in cells outside of the ciliated GRP, demonstrating a broader, cilia-independent role of polarity in LR patterning.
• Conjoined twin experiments confirm that early organizers must send proper orientation signals through intact polarity pathways to ensure normal LR development.
• Overall, the findings provide a detailed “recipe” for how conserved polarity mechanisms guide the establishment of the body’s left–right axis during development.

观察到的现象？（引言）

• 本研究探讨了细胞极性蛋白如何在非洲爪蟾胚胎早期发育中控制左右（LR）轴的定向。
• 研究比较了两种主要模型：一种依赖纤毛驱动的液体流动来决定左右不对称；另一种则依靠细胞内在的螺旋性（细胞极性）发挥作用。
• 结果表明，顶底极性（ABP）和面内极性（PCP）蛋白对于建立一致的左右不对称至关重要，这一过程独立于纤毛功能。

关键术语和概念

• 左右不对称：体内器官（如心脏、肝脏和胃）在体内有序地分布于左右两侧。
• 顶底极性（ABP）：细胞从顶端（apical）到基底（basal）的方向性；关键蛋白包括Par6和aPKC。
• 面内极性（PCP）：细胞在组织平面内的协调定向；涉及蛋白有Vangl2、diversin、disheveled和RSG1等。
• 器官错位（Heterotaxia）：器官排列随机或定向异常的情况。
• GRP（胃腔顶板）：非洲爪蟾胚胎中的一种具纤毛结构，通常参与左右不对称，但并非唯一决定因素。
• 紧密连接：维持细胞间粘附和组织完整性的结构，有助于建立正常的信号梯度。
• 5HT（血清素）：一种信号分子，其不对称定位对于左右模式化至关重要。
• Xnr-1：通常在胚胎左侧表达的基因，是左右不对称的标志。

实验方法和步骤

• 利用显性负（DN）构建体干扰Par6和aPKC，从而破坏胚胎的顶底极性。
• 采用Vangl2反义寡核苷酸及其他PCP干扰构建体（如diversin、disheveled、RSG1）抑制面内极性。
• 通过针对特定胚胎细胞进行微注射，区分贡献于GRP与不贡献于GRP的细胞效应。
• 检测器官位置、纤毛定位、紧密连接完整性以及不对称性标志（Xnr-1和5HT）的表达情况。
• 采用诱导双生胚实验（通过16细胞期注射XSiamois）来测试早期原始组织对后期组织的左右定向指令，即“大哥效应”。

结果（案例报告/发现）

• 干扰ABP蛋白（Par6和aPKC）导致器官排列随机化，出现器官错位。
• 抑制PCP蛋白（通过Vangl2 MO及其他构建体）同样使左右轴定向随机化。
• 即使仅在不构成GRP的细胞中进行干扰，也能观察到上述效应，表明这一机制独立于纤毛。
• 当极性通路受干扰时，左右标记基因Xnr-1的表达异常，5HT的定位也发生紊乱。
• 紧密连接受损，提示细胞间粘附对于左右模式化的重要性。
• 双生胚实验显示，原始组织和诱导组织均需要完整的极性信号才能实现正确的左右定向。

实验步骤（逐步操作方法）

• 步骤1：在单细胞阶段微注射DN-Par6和DN-aPKC构建体，破坏顶底极性。
• 步骤2：注射Vangl2反义寡核苷酸及其他PCP干扰试剂，抑制面内极性信号。
• 步骤3：将注射靶向特定胚胎细胞，区分GRP贡献细胞与非GRP细胞。
• 步骤4：检测GRP细胞中纤毛的定位，观察5HT和Xnr-1等不对称标记的分布情况。
• 步骤5：通过生物素标记实验评估紧密连接的完整性。
• 步骤6：在16细胞期注射XSiamois诱导双生胚，观察双生胚中两侧心脏的定向。
• 步骤7：将处理组与对照组进行比较，以确定极性干扰对左右模式化的影响。

主要结论（讨论）

• 顶底极性和面内极性蛋白对于脊椎动物胚胎的正常左右不对称建立至关重要。
• 这些蛋白在不对称基因表达之前发挥作用，影响5HT定位和紧密连接的形成。
• 研究证明，左右模式化可以在不依赖纤毛流动的情况下，通过细胞内在极性机制实现。
• 双生胚实验表明，早期原始组织必须通过完整的极性信号传递信息，指导后续组织实现正确的左右定向。

意义和更广泛的影响

• 本研究揭示了一种保守的、独立于纤毛机制的左右不对称建立途径。
• 深入了解这些极性通路有助于阐明与器官排列异常（器官错位）相关的先天性缺陷机制。
• 研究结果强调了细胞极性在胚胎发育和整体身体模式建立中的根本性作用。

总体摘要（逐步解释）

• 本研究显示，顶底极性（ABP）和面内极性（PCP）蛋白在确定胚胎内器官左右定位中发挥关键作用。
• 通过干扰这些蛋白，研究人员在非洲爪蟾胚胎中观察到器官排列随机化、基因表达异常以及关键信号分子（如5HT）定位紊乱。
• 这种干扰不仅影响构成GRP的细胞，也影响其他细胞，表明其机制超越了纤毛依赖。
• 双生胚实验进一步证明，早期原始组织必须通过完整的极性信号向后期诱导组织传递定向信息，确保左右模式化正常。
• 总体而言，该研究为我们提供了一份详细的“烹饪配方”，说明了保守的细胞极性机制如何在胚胎发育早期引导身体左右轴的建立，同时为解释人类器官排列异常提供了分子基础。