Supplementary Material for: Hedgehog signaling during gut formation in the freshwater leech, Helobdella austinensis

Introduction: The hedgehog signaling pathway plays a crucial role in inducing segment polarity through cell-cell interactions in various metazoans, including arthropods and annelids. However, its involvement in organogenesis and segmentation among lophotrochozoans remains inconsistent. This study aimed to explore the role of the hedgehog gene during gut development in the freshwater leech, Helobdella austinensis. Methods: Developmental RT-PCR and in situ hybridization were performed to examine the expressions of hedgehog genes. In addition, embryos were treated with Cyclopamine (a hedgehog signaling antagonist) and Purmorphamine (a Smo agonist) to examine the potential interactions between Helobdella orthologs to hedgehog and two NKL genes: Hau-NK2 and Hau-NK4. Results: We examined the expressions of four core pathway members - Hedgehog (Hh), Patched (Ptc), Smoothened (Smo), and the downstream transcription factor Gli - spatiotemporally during the embryonic stages of H. austinensis. All four genes were expressed in the developing gut and proboscis during organogenesis but not during the segmentation stage. Additionally, the treatment of embryos with Cyclopamine and Purmorphamine revealed that NK genes are regulated by hedgehog signaling. Furthermore, NK2 and NK4 were expressed in the developing gut rather than in a segmental stripe pattern. Conclusions: This study confirms that the hedgehog signaling pathway is associated with gut development in the freshwater leech, Helobdella austinensis. The expression patterns of hedgehog pathway genes and their interaction with NK genes suggest a role for hedgehog signaling in regulating gut development rather than segmentation in the freshwater leeches.

引言：刺猬信号通路（hedgehog signaling pathway）可通过细胞间相互作用在包括节肢动物、环节动物在内的多种后生动物中诱导体节极性，发挥关键调控作用。但目前学界对于该通路在冠轮动物的器官发生与体节形成过程中的参与情况仍存在争议。本研究旨在探究刺猬基因（hedgehog genes）在淡水水蛭奥斯汀水蛭（Helobdella austinensis）肠道发育过程中的功能。
方法：本研究采用发育型逆转录聚合酶链反应（developmental RT-PCR）与原位杂交（in situ hybridization）技术，对刺猬基因的表达模式进行检测。此外，本研究分别使用环巴胺（Cyclopamine，一种刺猬信号通路拮抗剂）与普摩拉明（Purmorphamine，一种Smo激动剂）处理胚胎，以探究奥斯汀水蛭刺猬同源基因与两个NKL家族基因Hau-NK2、Hau-NK4之间的潜在相互作用。
结果：本研究对奥斯汀水蛭胚胎发育阶段的四大核心通路成员——刺猬蛋白（Hedgehog，Hh）、Patched蛋白（Patched，Ptc）、Smoothened蛋白（Smoothened，Smo）以及下游转录因子Gli——进行了时空表达谱分析。结果显示，四大核心基因均在器官发生阶段的发育中肠道与吻结构中表达，但体节形成阶段无相关表达。此外，使用环巴胺与普摩拉明处理胚胎的实验表明，NKL家族基因的表达受刺猬信号通路调控。进一步研究发现，NK2与NK4基因仅在发育中的肠道中表达，而非呈体节条纹模式分布。
结论：本研究证实，刺猬信号通路与淡水水蛭奥斯汀水蛭的肠道发育密切相关。刺猬信号通路基因的表达模式及其与NKL家族基因的相互作用，提示该通路在淡水水蛭中主要调控肠道发育而非体节形成过程。