Neurons have highly asymmetric cellular morphologies and polarized cellular functions that are necessary for establishing neural circuitry and for proper functioning of the nervous system. Specialized processes, called dendrites, are used by neurons for reception of stimuli, while axons function in the transmission of signals. In neurons, mRNA localization and translational repression are used to change the protein composition of various regions of the cell, allowing for distinct axonal and dendritic morphologies and environments that are equipped for their various cellular tasks. A significant portion of the eukaryotic genome encodes for RNA-binding proteins (RBPs), which play important roles in localizing and translationally regulating RNAs. Since studies have shown that a large number of mRNAs are localized within dendrites, this suggests that the RBPs contribute broadly to neuronal development and function by localizing and regulating mRNAs. Based on a previous screen of RBP-encoding genes that affect dendrite morphogenesis in dendritic arborization neurons (da neurons) in Drosophila that identified 89 genes (Olesnicky, Killian, and Gavis; in preparation), I extended this screen to determine if any of these evolutionarily conserved RBP genes are important for dendrite morphogenesis in C. elegans PVD neurons as well. A significant decrease in dendritic arborization was found in dcr-1 mutants and preliminary results suggest that sup-26 and mtr-4 mutants may have decreased 3rd and 4th order dendritic branching. In addition, several other candidate genes are currently being investigated. Thus far, the results suggest that DCR-1/Dicer, an RBP involved in the microRNA pathway, SUP-26/Alan Shepard, an RBP implicated in translational control of mRNAs, and MTR-4/L(2)35Df, a component of the eukaryotic RNA exosome play an evolutionarily conserved role in dendrite development in flies and worms.
A neuron is a specialized cell that transmits nerve impulses. Dendrites of a neuron receive signals from other cells or the environment and transmit them to the soma. Dendrites branch out to cast a wide receptive field and establish neural connections that govern behavior, learning, and memory; therefore regulation of dendritic branching is essential for sensory reception. It is important to understand how these different processes form because many neurological diseases show atypical dendritic and axonal phenotypes (Kulkarni and Firestein 2012). RNA-binding proteins (RBPs) have become of particular interest as many RBPs have recently been implicated in the process of dendrite formation. RBPs are known to be important in post-transcriptional regulation of gene expression. A previous study showed that an RBP named Shep regulates dendrite development in Drosophila (Mapes et al., 2010). To determine if Shep is evolutionarily conserved in its role in dendrite development we tested its C. elegans ortholog (sup-26) for a role in dendrite development in the multidendritic PVD sensory neuron. Loss of sup-26 activity results in a significant reduction of terminal dendrites in the PVD neuron. Furthermore, time course analysis of dendrite development revealed sup-26 mutants have a dendrite maintenance defect. sup-26 is expressed in many cells including the PVD neuron and SUP-26 protein localizes to the cytoplasm, consistent with its role as a potential translational regulator. Dendrite defects are partially rescued by PVD-specific expression of the SUP-26::GFP fusion protein, which suggests that SUP-26 functions cell-autonomously within the PVD to regulate dendrites. We hypothesize that sup-26 acts as a translational repressor of mRNAs that are important for dendrite regulation. Because sup-26/shep functions in dendrite development in fly and worm, and has three human homologs expressed in the brain, it suggests that sup-26 orthologs may be important in the development of dendrites in humans as well.
PAL-1 is a protein that regulates posterior development of Caenorhabditis elegans embryos. Although pal-1 mRNA is present throughout the entire embryo, the PAL-1 protein is only transcribed in the posterior end of the nematode worm. MEX-3, a RNA binding protein, binds to the pal-1 mRNA, preventing its translation in the anterior section of the embryo. The MEX-3 protein is essential to maintaining embryo polarity and ensuring that posterior features develop only in the posterior end of the worm. During development, MEX-3 is present throughout the 1-cell and 2-cell embryo stage. MEX-3 is then degraded in the posterior end during the 4-cell stage, allowing the expression of PAL-1 in the two posterior blastomeres. By the 8-cell stage, MEX-3 is depleted from the entire embryo with remnants remaining in germline cells. The ubiquitination pathway is hypothesized to mark MEX-3 for degradation, localizing the protein at various stages of embryo development. This study screened various E3 ubiquitin ligases to determine which ligases are specifically used to mark MEX-3 for degradation during embryo development. Double-stranded RNA was created for selected E3 ubiquitin ligases and then injected into adult worms. This invoked RNA interference (RNAi) of these ubiquitin ligases in the embryos of the adult worms. Knockout of genes D2089.2, F46A9.5, F59B2.6, and Y82E9BR.15 resulted in embryonic lethality. Fluorescence microscopy of GFP::MEX-3 (green fluorescent protein labeled MEX-3) revealed that only F59B2.6 (zif-1 gene) and Y82E9BR.15 (elc-1 gene) knockouts affected MEX-3 localization. Double knockouts of zif-1 and another developmental gene, mex-5, support the hypothesis that zif-1 acts after other regulatory events in MEX-3 localization.
Dendrites establish proper neural networks, allowing normal brain functions such as social networking, learning, and memory. Thus, it is no surprise that dendritic defects are associated with many neurological disorders. Accordingly, understanding the molecular mechanisms behind dendrite formation and maintenance is an important research goal. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the post-transcriptional level. The CPEB class of RNA-binding proteins mediates many post-transcriptional mechanisms, and homologs of this protein have previously been identified as important in synaptic plasticity and dendrite morphogenesis. Here, we identify the Caenorhabditis elegans CPEB homolog CPB-3 as necessary for typical dendritic branching in the PVD multidendritic sensory neuron. This study also points to a previously undescribed function of a CPEB; loss of CPB-3 causes gene expression profile changes in touch neurons. Thus, we believe that CPB-3 is a very strong candidate for regulating the transport and translation of target mRNAs within dendrites. Furthermore, the CPB-3 homolog CPEB1 is expressed in the human brain suggesting that this RNA-binding protein is a candidate regulator of dendrite development in humans.
The protein ubiquitination system is a targeted protein degradation pathway that is an essential component of cell cycle progression in mitosis and meiosis. Recent evidence indicates that the ubiquitin system is required for the degradation of zinc finger proteins that play important roles in embryogenesis. It is possible that the ubiquitin system regulates other proteins involved in early embryonic development by controlling which proteins are degraded, and thereby influencing cell fates. In Caenorhabditis elegans (C. elegans), there is a single ubiquitin activating enzyme, which has a well-understood function. The twenty-two ubiquitin conjugating enzymes in C. elegans have been researched to a moderate extent. Finally, there are believed to be about six hundred ubiquitin-protein ligases. Most of these ubiquitin ligases’ exact functions, the proteins they target, remain unknown. Ubiquitin ligases are perhaps the most interesting enzymes in the ubiquitin system because they determine which proteins are targeted for degradation. Two important proteins involved in the embryonic development of C. elegans are posterior alae defective 1 (PAL-1) and muscle excess 3 (MEX-3). PAL-1 is a homeodomain transcription factor protein that is required to specify posterior cell fates. MEX-3 is an RNA-binding protein that binds to pal-1 mRNA in the anterior cells and restricts the translation of PAL-1 to the posterior cells of the embryo, and thereby influences anterior cell fates. Both pal-1 mRNA and MEX-3 protein are present throughout newly fertilized embryos, but by the four-cell stage MEX-3 is depleted in posterior cells and can only bind to pal-1 mRNA in anterior cells, preventing the translation of PAL-1 in these cells. It is thought that MEX-3 depletion in the posterior cells is due to it being targeted by unknown ubiquitin ligases and degraded by the 26S proteasome. Research shows that two homologous mRNA binding proteins (MEX-5 and MEX-6) protect MEX-3 from inactivation and degradation in the anterior, allowing for the repression of PAL-1 translation. One major unanswered question is the identity of the ubiquitin ligase(s) that targets MEX-3 for degradation in the posterior of the embryo. This study attempts to answer that question. RNA interference screening of ubiquitin ligases that are expressed during embryonic development has permitted the identification of 20 ubiquitin ligases that do not target MEX-3 for degradation. Screening of additional ubiquitin ligases may lead to a better understanding of the regulation of many key proteins. By understanding more about the interactions between MEX-3 protein and pal-1 mRNA and how they are regulated, we will learn more about how embryonic development unfolds and what can potentially go wrong.
The Caenorhabditis elegans male and hermaphrodite nervous systems display sexually dimorphic development characterized, in part, by the presence of 8 hermaphrodite-specific neurons and 89 male-specific neurons. We are interested in identifying the genes and molecular mechanisms that govern sex-specific neural development in C. elegans. Through a mutagenesis screen using a pkd-2::GFP reporter to label male-specific neurons, we recovered several mutants that display defects in sex-specific neural development. Males carrying the sm129 mutation lack pkd-2::GFP expression in the male-specific CEM neurons that are involved in mate finding. Genetic epistasis experiments suggest that CEM neurons are improperly specified or differentiated. We cloned the sm129 mutation and determined that it is an allele daf-19 based on three pieces of evidence: (1) RNAi of daf-19 phenocopies sm129, (2) sm129 fails to complement a daf-19 null mutation, and (3) we found a mutation in daf-19 that likely affects splicing. We are also testing to see if sm129 mutants can be rescued by adding a wild type copy of daf-19. daf-19 encodes an RFX transcription factor that activates genes required for sensory cilia function in ciliated neurons such as the CEMs. daf-19 null mutants lack all sensory cilia, have sensory defects, and display a constitutive dauer phenotype (worms enter an alternative part of the lifecycle associated with starvation survival). We are currently investigating how this mutation affects ciliated neurons such as CEMs but does not affect dauer formation.