POTENTIAL REGULATORY ROLES OF G PROTEIN-COUPLED RECEPTORS AND HOMEOBOX GENES IN THE CEREBRAL GANGLION OF ARTEMIA DURING EARLY DEVELOPMENT
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摘要:
为揭示卤虫变态发育过程中的神经调控机制, 研究报告了孤雌生殖卤虫三个典型发育阶段脑神经节的转录动态, 鉴定了各阶段特异表达的基因并预测了其功能。结果表明: 第1龄期的高表达基因(HEG)涉及大量的有机物代谢和免疫调节, 如含硫化合物转运及鸟氨酸代谢; 第3龄期的HEGs与细胞呼吸、甲基化和嘌呤代谢相关; 第8龄期的HEGs涉及神经信号传递和细胞间识别。其中, G蛋白偶联受体(GPCR)和homeobox基因在第3和第8龄期显著高表达, 继而探讨这些基因的亚家族表达特征及功能, 如视蛋白受体、神经活性配体受体和嗅觉受体这类GPCRs在幼体发育的相对后期阶段显著高表达, homeobox基因家族中的Unc-42、Arx和Ceh-14在第3龄期高表达, Arx、Ceh-14、Nkx2和Phox在第8龄期高表达。总之, 第1龄期幼体通过增强代谢和物质运输来支持其快速生长, 第3龄期到第8龄期是细胞分化和神经系统成熟的关键阶段, GPCR和homeobox的高表达提示卤虫视觉发育及神经系统的进一步特化。研究不仅提供了一组脑神经节转录组数据, 还为认识卤虫的发育机制及深入理解神经调控在甲壳动物变态发育过程中的作用提供了新线索。
Abstract:Artemia, a small aquatic crustacean widely distributed in saline and alkali environments, represents as a valuable model for investigating gene regulation mechanisms in crustacean development. Under favorable conditions, Artemia larvae, complete metamorphosis through multiple molts within 2—4 weeks, but the neural regulation mechanisms involved in individual development are currently unclear. This study reports on the transcriptional dynamics of the cerebral ganglia across three developmental stages, identifying genes specifically expressed at each stage and predicting their functions. In the instar I stage, highly expressed genes (HEGs) are primarily involved in the metabolism of organic compounds and immune regulation, including sulfur compound transport and ornithine metabolism. In instar Ⅲ, HEGs are related to cellular respiration, methylation, and purine metabolism, while in instar Ⅷ, they are associated with neural signal transmission and cell-cell recognition. Notably, G protein-coupled receptors (GPCRs) and homeobox genes are significantly highly expressed in the instar Ⅲ and instar Ⅷ stages. The functions of these gene subfamilies were further explored: GPCRs, such as opsin receptors, neuroactive ligand receptors, and olfactory receptors, are highly expressed in the relatively later stages of larval development. Homeobox genes, including Unc-42, Arx, and Ceh-14 are highly expressed in instar Ⅲ stage, while Arx, Ceh-14, Nkx2, and Phox are highly expressed in instar Ⅷ stage. The results indicate that instar Ⅰ larvae grow efficiently by enhancing metabolism and substance transport, while instar Ⅲ to Ⅶ is an important stage for cell differentiation and nervous system maturation. The increased expression of GPCRs and homeobox genes during this time points to further visual development and nervous system specialization in Artemia. This study provides a comprehensive transcriptome dataset of cerebral ganglion and offers insights into the developmental mechanisms of Artemia, shedding light on the neural regulatory roles in crustacean development.
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Keywords:
- Development /
- Cerebral ganglion /
- G protein-coupled receptors /
- Homeobox /
- Artemia
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图 1 孤雌生殖卤虫发育过程中脑神经节的基因表达特征
A. 第1、3和8龄期卤虫幼体形态; B. Transcripts和unigenes的长度分布, transcripts代表无参组装得到的所有转录本序列, unigenes为所有转录本聚类和去冗余后的最终参考基因; C. 样本间基于fpkm值的相关性分析热图(上)及fpkm值标准化后的表达谱热图(下); D. 各发育阶段比较组的差异表达基因数量柱状图和相互关系弦图
Figure 1. The gene expression signature of cerebral ganglion during the development of A. parthenogenetica
A. Morphological images of the instar Ⅰ, instar Ⅲ, and instar Ⅷ larvae of Artemia; B. Length distribution of transcripts and unigenes, where transcripts represent all assembled transcript sequences from the de novo assembly, and unigenes are the final reference genes obtained by clustering and deduplication of all transcripts; C. Heatmap of sample correlation analysis based on fpkm values (top) and expression profile heatmap after fpkm value normalization (bottom); D. Bar plot showing the number of differentially expressed genes across developmental stage comparisons and chord diagram representing their interrelationships
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图 2 差异表达基因GO富集结果
红色/蓝色节点代表在前者/后者中差异高表达的unigenes, 绿色节点代表通路或功能, 连线表示二者存在相互关系; 黄色/蓝色区域分别表示其中的unigenes主要在前者/后者中显著高表达
Figure 2. GO enrichment results of DEGs
Red/blue nodes represent unigenes that are differentially upregulated in the former/latter, green nodes represent pathways or functions, and the connections indicate relationships between them; The yellow/blue areas highlight unigenes that are predominantly upregulated in the former/latter
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图 3 差异表达基因KEGG富集结果
横轴表示该通路的富集因子, 气泡大小表示富集到该通路的unigenes数量, 黑色/白色用于区分该比较组中前者/后者差异高表达基因集的富集结果
Figure 3. KEGG enrichment results of DEGs
The x-axis represents the rich factor of the pathway, the bubble size indicates the number of unigenes enriched in the pathway, and black/white distinguishes the enrichment results of upregulated gene sets in the former/latter group within the comparison
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图 4 差异表达基因转录因子富集结果
红色/蓝色节点代表在前者/后者中差异高表达的unigenes, 绿色节点代表转录因子; 黄色/蓝色区域分别表示其中的unigenes主要在前者/后者中显著高表达
Figure 4. Transcription factor enrichment results of DEGs
Red/blue nodes represent unigenes that are differentially upregulated in the former/latter and green nodes represent transfactors; The yellow/blue areas highlight unigenes that are predominantly upregulated in the former/latter
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图 5 高表达基因GO富集结果
左侧为各阶段高表达基因的fpkm值标准化后的表达谱热图; 右侧展示了相应各阶段高表达基因的GO通路或功能的相互关系及模块划分: 节点代表通路或功能, 连线粗细表示两个通路或功能之间共享unigenes的数量, 同一模块中通路或功能的富集结果具有高度相关性
Figure 5. GO enrichment results of HEGs
The left panel shows the heatmap of normalized fpkm values for highly expressed genes at each stage, while the right panel displays the relationships and module divisions of GO pathways or functions corresponding to these highly expressed genes. Nodes represent pathways or functions, and the width of the edges indicates the number of shared unigenes between two pathways or functions. Pathways or functions within the same module have highly correlated enrichment results
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图 6 高表达基因KEGG富集结果
通过Ward.D方法基于富集结果相关性对各阶段高表达基因富集到的KEGG通路进行聚类和分组, 同组内通路具有高相关性
Figure 6. KEGG enrichment results of HEGs
Clustering and grouping of KEGG pathways enriched by HEGs at each stage are performed using the Ward.D method, based on the correlation of enrichment results. Pathways within the same group exhibit high correlation
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图 7 Homeobox基因家族的转录动态与系统发育分析
Figure 7. Transcriptional dynamics and phylogenetic analysis of the homeobox gene family
表 1 样本测序数据汇总
Table 1 Summary of sample sequencing data
样本名称
Name原始Reads 质控后Reads 错误率
Error rate (%)Q20 (%) Q30 (%) GC含量
GC percentage (%)Unigene比对率
Mapped to unigenes (%)1st 21463430 20153560 0.03 97.01 92.03 39.11 85.56 3rd 21617734 20742657 0.03 97.31 92.75 39.27 84.85 8th 23498595 22545548 0.03 96.62 91.2 39.19 83.63 
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表 2 基因注释统计结果
Table 2 Statistical results of gene annotation
数据库
DatabaseUnigene数量
Number of Unigenes比例Percentage(%) NR 12751 28.52 GO 12077 27.02 KEGG 7213 16.13 NT 3700 8.27 KOG 5572 12.46 SwissProt 9712 21.72 PFAM 12309 27.54 至少被以上1个数据库注释 17501 39.15 被以上7个数据库注释 1378 3.08 其他数据库: AnimalTFDB 1030 2.30 其他数据库: STRING 7230 16.18 Unigene总数 44694 100
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表 3 第3龄期或第8龄期中差异高表达GPCR基因的功能或亚类
Table 3 Functions or subclasses of differentially highly expressed GPCR genes in instar Ⅲ or instar Ⅷ stage
频次
Frequency功能
Function上、下游关系
Upstream-downstream relationship19 7跨膜受体视紫红质家族 上游/功能 8 神经活性配体-受体相互作用 上游/功能 6 嗅觉受体 上游/功能 6 嗅觉受体活动 上游/功能 6 嗅觉感知 上游/功能 4 3种7跨膜甜味受体GPCR 上游/功能 4 cAMP信号通路 下游 3 钙信号通路 下游 3 复眼感光蛋白BCRH1 上游/功能 3 复眼感光蛋白BCRH2 上游/功能 3 G蛋白RECEP-F1-2结构域 上游/功能 3 蛇形7跨膜GPCR化学感受器 上游/功能 3 神经肽信号通路 上游/功能 注: 频次指该功能词条在所有数据库中合并和去重后出现的总次数Note: Frequency refers to the total number of the functional term occurrences after merging and deduplication across all databases
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表 4 三个发育阶段差异表达基因和高表达基因的主要功能
Table 4 Main functions of DEGs and HEGs across the three developmental stages
发育阶段
Developmental stageDEGs和HEGs主要功能
Main functions of DEGs and HEGs第1龄期 免疫反应激活及调节 糖苷、醇和谷胱甘肽合成代谢 葡萄糖跨膜转运及调节 鸟氨酸代谢 硫酸盐及含硫化合物转运 第3龄期 细胞呼吸与电子传递 甲基化相关催化调节 嘌呤代谢与响应外界刺激 神经系统过程 GPCR信号传导: 嗅觉受体、视蛋白受体、神经活性配体受体、cAMP信号通路和钙信号通路 Homeobox相关基因: Arx、Ceh-14和Unc-42 第8龄期 转运与定位调节 受精与细胞间识别 神经系统过程、突触信号释放、神经递质分泌与调节 GPCR信号传导: 嗅觉受体、视蛋白受体、神经活性配体受体、cAMP信号通路和钙信号通路 Homeobox相关基因: Arx、Ceh-14、Nkx-2.1和Phox-2
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