Introduction

• The study examines how left/right patterning signals determine the orientation (situs) of organs in chick embryos.
• It focuses on understanding the independent regulation of different aspects of laterality such as heart position, gut rotation, and embryonic rotation.
• This builds on previous findings that genes like Sonic hedgehog (Shh) play a key role in establishing left/right asymmetry.

Key Concepts and Terms

• Left/Right (LR) Asymmetry: The natural difference in the placement of organs on the left versus right side (e.g., the heart is usually on the left).
• Situs: The overall arrangement or position of the internal organs.
• Heterotaxia: A condition in which different organs show mixed or inconsistent left/right characteristics.
• Isomerism: A state where an embryo develops two similar sides (for example, two “left” sides), losing the normal asymmetry.
• Sonic hedgehog (Shh): A gene normally expressed on the left side of Hensen’s node that helps direct left-sided development.
• Nodal: A gene activated by Shh that is crucial for determining heart orientation and other asymmetries.
• Activin and Follistatin: Proteins that regulate Shh expression; Activin can repress Shh while Follistatin blocks Activin’s effect.
• In situ hybridization: A laboratory technique used to visualize where specific genes are expressed in tissues.

Methods and Experimental Approach

• Chick embryos were used in both in vitro (culture) and in ovo (within the egg) experiments.
• Researchers manipulated gene expression by:
  • Implanting beads soaked with proteins (such as Shh and follistatin) to locally alter signaling.
  • Using retroviral vectors to misexpress genes like nodal in specific areas.
  • Performing surgical removal of the prospective heart region to test its role in laterality.
• Whole-mount in situ hybridization was employed to detect gene expression patterns.
• The experiments tested whether altering signals at Hensen’s node affects heart, gut, and overall embryonic rotation.

Key Findings (Results)

• Misexpression of Shh on the right side led to:
  • Bilateral (both sides) expression of Shh instead of the normal left-only pattern.
  • Disruption of normal heart orientation (heart situs) and gut rotation.
  • A heterotaxia-like condition where different organs showed independent alterations in their left/right patterning.
• Ectopic (misplaced) expression of nodal on the right side altered heart positioning, supporting its role in heart asymmetry.
• Application of Activin on the left repressed Shh expression, whereas Follistatin beads eliminated the normal asymmetry of Shh expression.
• Surgical removal of the heart-forming region affected heart looping but did not consistently alter other aspects of laterality, indicating independent regulation.
• The experiments suggest a cascade where early Shh signals trigger nodal expression, which then directs heart development.
• Results indicate that left/right patterning is established later in development (in a streak-autonomous manner), rather than by an early fixed prepattern.

Conclusions and Implications

• Different aspects of organ laterality (heart, gut, embryonic rotation) can be independently influenced by key signaling molecules.
• Nodal is confirmed as a crucial gene for heart orientation, likely acting downstream of Shh.
• An activin-like signal on the right side of Hensen’s node is important for repressing Shh and establishing normal asymmetry.
• The findings support a step-by-step (recipe-like) gene cascade that determines body patterning.
• This research provides insights into congenital defects in humans related to abnormal organ positioning.

Step-by-Step Summary (Like a Cooking Recipe)

• Step 1: In early chick embryos, Shh is produced on the left side of Hensen’s node – the first ingredient that sets the developmental stage.
• Step 2: A right-side activin signal keeps Shh restricted to the left, much like keeping salt on one side of a dish.
• Step 3: Shh then triggers the expression of nodal, the next key ingredient that specifically influences heart positioning.
• Step 4: Altering these signals (by adding extra Shh or nodal) disrupts normal organ orientation, similar to a recipe going awry when ingredients are mis-measured.
• Step 5: Blocking activin with follistatin confirms that a proper balance of signals is essential for correct organ placement.

Additional Observations

• Multiple signaling pathways work together in a coordinated yet independent manner to regulate organ positioning.
• This independence explains why some organs may develop abnormally while others remain normal.
• The study enhances our understanding of how body asymmetry is established during development, much like assembling a complex puzzle.

引言

• 本研究探讨了左右定位信号如何决定鸡胚中器官的排列（位点）。
• 重点在于理解心脏位置、肠道旋转和胚胎整体旋转等各个方面左右不对称的独立调控。
• 研究基于先前发现的Sonic hedgehog (Shh)等基因在左右不对称中起关键作用的基础上展开。

关键概念与术语

• 左右不对称：指器官在身体左右两侧排列的自然差异（例如，心脏通常位于左侧）。
• 位点（Situs）：器官在体内的整体排列方式。
• 异位症（Heterotaxia）：不同器官出现混合左右特征的情况。
• 同形症（Isomerism）：胚胎出现两侧相同（如两侧都表现为左侧）的情况，失去正常的不对称性。
• Sonic hedgehog (Shh)：一种通常在Hensen节点左侧表达的基因，帮助引导左侧发育。
• Nodal：由Shh激活的重要基因，对心脏定位及其他不对称性起关键作用。
• Activin与Follistatin：调控Shh表达的蛋白质；Activin能抑制Shh，而Follistatin则能阻断Activin的作用。
• 原位杂交：一种用于检测组织中特定基因表达位置的实验技术。

方法和实验设计

• 研究使用鸡胚在体外培养和蛋内实验两种模式进行。
• 通过以下方法操控基因表达：
  • 植入浸泡了蛋白质（如Shh或Follistatin）的微珠，改变局部信号环境。
  • 利用逆转录病毒载体在特定区域使Nodal基因错误表达。
  • 通过手术去除预定形成心脏的区域，以研究其在左右定位中的作用。
• 采用全胚原位杂交技术观察基因表达模式。
• 实验检验了在Hensen节点改变信号是否会影响心脏、肠道及胚胎整体旋转的正常定位。

主要发现

• 在右侧错误表达Shh会导致：
  • Shh由原本仅在左侧表达变为双侧表达。
  • 心脏和肠道的正常排列受到扰乱。
  • 出现类似异位症的情况，各器官的左右定位出现独立改变。
• 在右侧外源表达Nodal改变了心脏的位置，支持其在心脏左右定位中的关键作用。
• 施用Activin能在左侧抑制Shh表达，而Follistatin微珠则消除了Shh表达的左右差异。
• 手术切除心脏形成区影响了心脏环状发育，但对其他左右定位方面影响不大，表明各部分调控独立。
• 实验表明，一个信号级联中，早期的Shh信号触发Nodal表达，进而指导心脏发育。
• 结果显示左右定位不是由早期胚盘预先设定，而是在后期通过“原条自主”机制逐步建立，就像按菜谱操作一样。

结论及意义

• 关键信号分子可以独立影响心脏、肠道和胚胎旋转等各个器官的左右定位。
• Nodal被证实为决定心脏定位的重要基因，可能在Shh之后发挥作用。
• 右侧Hensen节点的Activin信号对于抑制Shh、建立正常左右不对称至关重要。
• 研究支持了一种逐步（如同烹饪菜谱）的基因级联模型来确定体内器官的排列。
• 这些发现为理解人类器官位置异常的先天缺陷提供了新的线索。

逐步总结（如同烹饪菜谱）

• 步骤1：在早期鸡胚中，Hensen节点左侧产生Shh信号，就像加入第一种调料奠定基础。
• 步骤2：右侧的Activin信号确保Shh仅限于左侧，类似于将盐只撒在菜的一边。
• 步骤3：Shh激发Nodal表达，成为影响心脏定位的关键原料。
• 步骤4：改变这些信号（如额外加入Shh或Nodal）会扰乱器官正常排列，就像原料比例失调导致菜品味道异常。
• 步骤5：用Follistatin阻断Activin进一步证明，维持信号平衡对器官正确定位至关重要。

额外观察

• 多个信号通路协同作用，各自独立调控器官的左右定位。
• 这种独立调控解释了为何部分器官可能出现异常而其他器官依然正常。
• 研究增进了我们对胚胎左右不对称形成过程的理解，就像拼凑复杂拼图一样。