Expression data from wildtype and unc-37 mutant A-class motor neurons in C. elegans. Caenorhabditis elegans

In Caenorhabditis elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, we have employed a cell-specific microarray strategy that has now identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. We show that expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord. Overall design: We employ the mRNA-tagging method to isolate poly(A) RNA from wildtype and unc-37 mutant A-class motor neurons by expressing a 3X FLAG-tagged poly(A) binding protein PAB-1 in DA/VA neurons under control of the unc-4 promoter. A 2-round IVT protocol (modified from the Affymetrix small-sample protocol) was used to convert starting RNA into biotinylated aRNA.

在秀丽隐杆线虫（Caenorhabditis elegans）中，VA与VB运动神经元为谱系来源的姐妹细胞，但二者通过与不同中间神经元形成突触来调控运动功能。VA神经元的特异性输入由UNC-4同源域蛋白（UNC-4 homeoprotein）及其转录共抑制因子UNC-37/Groucho决定，二者在VA神经元中发挥作用，阻断VA与原本专供VB神经元结合的中间神经元形成化学突触和间隙连接。为揭示调控这一细胞命运选择过程的下游基因，我们采用了细胞特异性基因芯片（cell-specific microarray）策略，现已鉴定出受unc-4调控的转录本。其中一个基因为ceh-12，属于HB9同源域蛋白家族，其正常表达仅局限于VB神经元。我们发现，在unc-4突变体的VA运动神经元中异位表达CEH-12/HB9，可赋予其VB型突触输入。因此，本研究揭示了一种发育调控开关：运动神经元的输入特异性由差异表达的转录因子决定，这些转录因子可选择不同的突触前伴侣细胞。脊椎动物运动回路中UNC-4、HB9及Groucho的表达保守性提示，脊髓中的突触特异性调控可能存在相似的分子机制。实验设计：我们采用mRNA标记法（mRNA-tagging method），通过在DA/VA神经元中以unc-4启动子（unc-4 promoter）驱动表达3×FLAG标签的聚腺苷酸结合蛋白PAB-1，从野生型与unc-37突变体的A类运动神经元中分离聚腺苷酸RNA（poly(A) RNA）。采用经修改自Affymetrix小样本实验方案的两轮体外转录（IVT）流程，将起始RNA转化为生物素标记的反义RNA（aRNA）。