Convergent Transcriptional Programs Regulate cAMP Levels in C. elegans GABAergic Motor Neurons.

Both transcriptional regulation and signaling pathways play crucial roles in neuronal differentiation and plasticity. Caenorhabditis elegans possesses 19 GABAergic motor neurons (MNs) called D MNs, which are divided into two subgroups: DD and VD. DD, but not VD, MNs reverse their cellular polarity in a developmental process called respecification. UNC-30 and UNC-55 are two critical transcription factors in D MNs. By using chromatin immunoprecipitation with CRISPR/Cas9 knockin of GFP fusion, we uncovered the global targets of UNC-30 and UNC-55. UNC-30 and UNC-55 are largely converged to regulate over 1,300 noncoding and coding genes, and genes in multiple biological processes, including cAMP metabolism, are co-regulated. Increase in cAMP levels may serve as a timing signal for respecification, whereas UNC-55 regulates genes such as pde-4 to keep the cAMP levels low in VD. Other genes modulating DD respecification such as lin-14, irx-1, and oig-1 are also found to affect cAMP levels.

Meis/UNC-62 isoform dependent regulation of CoupTF-II/UNC-55 and GABAergic motor neuron subtype differentiation.

Gene regulatory networks orchestrate the assembly of functionally related cells within a cellular network. Subtle differences often exist among functionally related cells within such networks. How differences are created among cells with similar functions has been difficult to determine due to the complexity of both the gene and the cellular networks. In Caenorhabditis elegans, the DD and VD motor neurons compose a cross-inhibitory, GABAergic network that coordinates dorsal and ventral muscle contractions during locomotion. The Pitx2 homologue, UNC-30, acts as a terminal selector gene to create similarities and the Coup-TFII homologue, UNC-55, is necessary for creating differences between the two motor neuron classes. What is the organizing gene regulatory network responsible for initiating the expression of UNC-55 and thus creating differences between the DD and VD motor neurons? We show that the unc-55 promoter has modules that contain Meis/UNC-62 binding sites. These sites can be subdivided into regions that are capable of activating or repressing UNC-55 expression in different motor neurons. Interestingly, different isoforms of UNC-62 are responsible for the activation and the stabilization of unc-55 transcription. Furthermore, specific isoforms of UNC-62 are required for proper synaptic patterning of the VD motor neurons. Isoform specific regulation of differentiating neurons is a relatively unexplored area of research and presents a mechanism for creating differences among functionally related cells within a network.

HES-Mediated Repression of Pten in Caenorhabditis elegans.

The hairy/enhancer-of-split (HES) group of transcription factors controls embryonic development, often by acting downstream of the Notch signaling pathway; however, little is known about postembryonic roles of these proteins. In Caenorhabditis elegans, the six proteins that make up the REF-1 family are considered to be HES orthologs that act in both Notch-dependent and Notch-independent pathways to regulate embryonic events. To further our understanding of how the REF-1 family works to coordinate postembryonic cellular events, we performed a functional characterization of the REF-1 family member, HLH-25. We show that, after embryogenesis, hlh-25 expression persists throughout every developmental stage, including dauer, into adulthood. Like animals that carry loss-of-function alleles in genes required for normal cell-cycle progression, the phenotypes of hlh-25 animals include reduced brood size, unfertilized oocytes, and abnormal gonad morphology. Using gene expression microarray, we show that the HLH-25 transcriptional network correlates with the phenotypes of hlh-25 animals and that the C. elegans Pten ortholog, daf-18, is one major hub in the network. Finally, we show that HLH-25 regulates C. elegans lifespan and dauer recovery, which correlates with a role in the transcriptional repression of daf-18 activity. Collectively, these data provide the first genetic evidence that HLH-25 may be a functional ortholog of mammalian HES1, which represses PTEN activity in mice and human cells.

A transcriptional program promotes remodeling of GABAergic synapses in Caenorhabditis elegans.

Although transcription factors are known to regulate synaptic plasticity, downstream genes that contribute to neural circuit remodeling are largely undefined. In Caenorhabditis elegans, GABAergic Dorsal D (DD) motor neuron synapses are relocated to new sites during larval development. This remodeling program is blocked in Ventral D (VD) GABAergic motor neurons by the COUP-TF (chicken ovalbumin upstream promoter transcription factor) homolog, UNC-55. We exploited this UNC-55 function to identify downstream synaptic remodeling genes that encode a diverse array of protein types including ion channels, cytoskeletal components, and transcription factors. We show that one of these targets, the Iroquois-like homeodomain protein, IRX-1, functions as a key regulator of remodeling in DD neurons. Our discovery of irx-1 as an unc-55-regulated target defines a transcriptional pathway that orchestrates an intricate synaptic remodeling program. Moreover, the well established roles of these conserved transcription factors in mammalian neural development suggest that a similar cascade may also control synaptic plasticity in more complex nervous systems.

A C. elegans homolog of huntingtin-associated protein 1 is expressed in chemosensory neurons and in a number of other somatic cell types.

Huntingtin-associated protein 1 (HAP1) is a binding partner for huntingtin, the protein responsible for Huntington's disease. In mammals, HAP1 is mostly found in brain where it is expressed in neurons. Although several functions have been proposed for HAP1, its role has not yet been clearly established. In this paper, we report on the identification of a HAP1 Caenorhabditis elegans homolog called T27A3.1. T27A3.1 shows conservation with rat and human HAP1, as well as with Milton, a Drosophila HAP1 homolog. To determine the cellular expression of T27A3.1 (multiple isoforms; a-e), we generated several transgenic worm lines expressing a fluorescent reporter protein [green fluorescent protein (GFP) and DsRed2] under the control of the promoter for T27A3.1. We have found that T27A3.1 is expressed in many cell types including a subset of chemosensory neurons in the head and tail. These include the amphid chemosensory neurons ASKL and R, ASIL and R, ADFL and ASEL, the phasmid neurons PHBL and R, and the CAN neurons that are required for worm survival.