The Transcription Factor Cux1 Regulates Dendritic Morphology of Cortical Pyramidal Neurons

Citation: Li N, Zhao C-T, Wang Y, Yuan X-B ( The Transcription Factor Cux1 Regulates Dendritic Morphology of Cortical Pyramidal Neurons Ning Li 0 Chun-Tao Zhao 0 Ying Wang 0 Xiao-Bing Yuan 0 Lin Mei, Medical College of Georgia, United States of America 0 1 Institute of Neuroscience and State Key Laboratory of Neurobiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China , 2 Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences , Shanghai , China , 3 School of Lab Medicine and Life Science, Wenzhou Medical College , Wenzhou , China In the murine cerebral cortex, mammalian homologues of the Cux family transcription factors, Cux1 and Cux2, have been identified as restricted molecular markers for the upper layer (II-IV) pyramidal neurons. However, their functions in cortical development are largely unknown. Here we report that increasing the intracellular level of Cux1, but not Cux2, reduced the dendritic complexity of cultured cortical pyramidal neurons. Consistently, reducing the expression of Cux1 promoted the dendritic arborization in these pyramidal neurons. This effect required the existence of the DNA-binding domains, hence the transcriptional passive repression activity of Cux1. Analysis of downstream signals suggested that Cux1 regulates dendrite development primarily through suppressing the expression of the cyclin-dependent kinase inhibitor p27Kip1, and RhoA may mediate the regulation of dendritic complexity by Cux1 and p27. Thus, Cux1 functions as a negative regulator of dendritic complexity for cortical pyramidal neurons. - Funding: This work was supported by National Basic Research Program of China (2006CB943903, 2006CB943903) and grants from the National Natural Science Foundation of China (30625023) and the Chinese Academy of Sciences (KSCX2-YW-R-103). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The Cux (also known as Cut and CDP) proteins are a family of homeobox transcription factors identified in all metazoans and implicated in the regulation of cell proliferation and differentiation in many organisms (reviewed in reference [1,2]). In higher vertebrates, two Cux genes, Cux1/CDP and Cux2, have been identified [3,4,5]. The murine Cux1 is expressed in most tissues, including the brain [6], while Cux2 is enriched primarily in the nervous system [5]. In the mouse cerebral cortex, both Cux1 and Cux2 are expressed in postmitotic pyramidal neurons of upper cortical layers from embryonic stages to adulthood and in precursor cells of the proliferative ventricular and subventricular zones (VZ/SVZ) [7,8,9,10]. However, the function of Cux genes in the mammalian central nervous system remains largely unknown. Cux2 was reported to control the proliferation of neural progenitor cells at the cortical SVZ area and the spinal cord, although with opposite effects in these two regions [11,12]. Beyond the regulation of cell proliferation, the persistent expression of Cux genes in postmitotic pyramidal neurons in upper layers of the cortex suggests that they may have specific functions in these differentiated cells [8,10]. Cortical pyramidal neurons have well-defined dendritic trees [13]. Considering that Cut regulates the dendritic branching patterns of Drosophila multidendritic da sensory neurons [14], we examined the potential function of Cux proteins in the development of dendritic trees of cortical pyramidal neurons. We found that Cux1, but not Cux2, could regulate the dendritic complexity of cultured cortical pyramidal neurons. The expression of the cyclin-dependent kinase inhibitor, p27Kip1, was specifically suppressed by Cux1, and co-expression of p27 with Cux1 could compensate Cux1s effect on dendritic arborization of cortical pyramidal neurons. Furthermore, the small GTPase RhoA may mediate the regulation of dendritic complexity by Cux1 and p27. Thus, our results indicate that Cux1 can regulate dendritic morphology of cortical pyramidal neurons. Cux1 can regulate dendritic morphology of cultured cortical pyramidal neurons To examine the function of Cux proteins in postmitotic cortical pyramidal neurons, we cloned the Cux1 and Cux2 genes from the rat brain and transiently transfected them into cortical neurons after culturing in vitro for 3 days (DIV3). Comparing to cells transfected with the control vector, Cux1-overexpressing pyramidal neurons showed a reduced dendritic complexity (Figure 1A). At DIV5 and DIV6, the dendritic arborization of Cux1-tansfected cells was markedly reduced, as indicated by the total dendritic branch length (Length; Figure 1B, D) and the total dendritic branch tip number (Branch Tips; Figure 1C, E). The dendritic complexity was not significantly changed at DIV4 (Figure 1DE), suggesting that the regulation of dendritic complexity by Cux1 is not a fast process. In contrast to Cux1, the dendritic complexity of Cux2-transfected cells was not affected at all time points examined (Figure 1). Together, these data showed that increasing the intracellular level of Cux1, but not Cux2, could reduce the dendritic complexity of cortical pyramidal neurons in culture. To further examine the function of Cux1, we designed two RNA-interference (RNAi) constructs targeting different regions of the Cux1 ORF. Consistent with previous reports [15,16], both of the RNAi constructs, Cux1-RNAiA and Cux1-RNAiB, could effectively down-regulate Cux1 expression (Figure 2A). When we transfected the RNAi constructs into cortical pyramidal neurons in culture, they increased the complexity of dendritic arborization (Figure 2CE). In contrast, RNAi-knockdown of Cux2 did not affect dendritic arborization of cortical pyramidal neurons in culture (Figure 2B, F, G). Therefore, Cux1 has a regulatory effect on the dendritic morphology of cortical pyramidal neurons. Transcriptional repressor activity of Cux1 is required in regulating dendritic morphology Cux1 is generally regarded as a transcriptional repressor with four conserved DNA-binding domains: three Cut repeats (CR1 to CR3) and one Cut homeodomain (HD) (Figure 3A). To examine whether transcriptional repressor activity of Cux1 is required to regulate dendritic morphology of cortical pyramidal neurons, we constructed three different deletions of Cux1: Cux1-DB contained all four DNA-binding domains, while Cux1-NT and Cux1-CT referred to the remaining N- and C-terminal fragments, respectively (Figure 3A). Comparing to the control, Cux1-NT and Cux1-CT did not reduce the dendritic complexity of cortical pyramidal neurons in culture (Figure 3BD). In contrast, overexpressing Cux1-DB resulted in a significant decrease in dendritic complexity, comparable to the effect of the full-length Cux1 (Figure 3BD). In addition, we constructed Cux1-CR1CR2 and Cux1-CR3HD, each has half of the four DN (...truncated)