Cell-specific effects of the sole C. elegans Daughterless/E protein homolog, HLH-2, on nervous system development.

Are there common mechanisms of neurogenesis used throughout an entire nervous system? We explored to what extent canonical proneural class I/II bHLH complexes are responsible for neurogenesis throughout the entire Caenorhabditis elegans nervous system. Distinct, lineage-specific proneural class II bHLH factors are generally thought to operate via interaction with a common, class I bHLH subunit, encoded by Daughterless in flies, the E proteins in vertebrates and HLH-2 in C. elegans. To eliminate function of all proneuronal class I/II bHLH complexes, we therefore genetically removed maternal and zygotic hlh-2 gene activity. We observed broad effects on neurogenesis, but still detected normal neurogenesis in many distinct neuron-producing lineages of the central and peripheral nervous system. Moreover, we found that hlh-2 selectively affects some aspects of neuron differentiation while leaving others unaffected. Although our studies confirm the function of proneuronal class I/II bHLH complexes in many different lineages throughout a nervous system, we conclude that their function is not universal, but rather restricted by lineage, cell type and components of differentiation programs affected.

Piecemeal regulation of convergent neuronal lineages by bHLH transcription factors in Caenorhabditis elegans.

Cells of the same type can be generated by distinct cellular lineages that originate in different parts of the developing embryo ('lineage convergence'). Several Caenorhabditis elegans neuron classes composed of left/right or radially symmetric class members display such lineage convergence. We show here that the C. elegans Atonal homolog lin-32 is differentially expressed in neuronal lineages that give rise to left/right or radially symmetric class members. Loss of lin-32 results in the selective loss of the expression of pan-neuronal markers and terminal selector-type transcription factors that confer neuron class-specific features. Another basic helix-loop-helix (bHLH) gene, the Achaete-Scute homolog hlh-14, is expressed in a mirror image pattern relative to lin-32 and is required to induce neuronal identity and terminal selector expression on the contralateral side of the animal. These findings demonstrate that distinct lineage histories converge via different bHLH factors at the level of induction of terminal selector identity determinants, which thus serve as integrators of distinct lineage histories. We also describe neuron-to-neuron identity transformations in lin-32 mutants, which we propose to also be the result of misregulation of terminal selector gene expression.

Visualizing the organization and differentiation of the male-specific nervous system of C. elegans.

Sex differences in the brain are prevalent throughout the animal kingdom and particularly well appreciated in the nematode Caenorhabditis elegans, where male animals contain a little-studied set of 93 male-specific neurons. To make these neurons amenable for future study, we describe here how a multicolor reporter transgene, NeuroPAL, is capable of visualizing the distinct identities of all male-specific neurons. We used NeuroPAL to visualize and characterize a number of features of the male-specific nervous system. We provide several proofs of concept for using NeuroPAL to identify the sites of expression of gfp-tagged reporter genes and for cellular fate analysis by analyzing the effect of removal of several developmental patterning genes on neuronal identity acquisition. We use NeuroPAL and its intrinsic cohort of more than 40 distinct differentiation markers to show that, even though male-specific neurons are generated throughout all four larval stages, they execute their terminal differentiation program in a coordinated manner in the fourth larval stage. This coordinated wave of differentiation, which we call 'just-in-time' differentiation, couples neuronal maturation programs with the appearance of sexual organs.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity.

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor-encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an "ur-" bHLH gene.

Tetraspanin (TSP-17) protects dopaminergic neurons against 6-OHDA-induced neurodegeneration in C. elegans.

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling.

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

A nervous system-specific subnuclear organelle in Caenorhabditis elegans.

We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4-10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

The Prop1-like homeobox gene unc-42 specifies the identity of synaptically connected neurons.

Many neuronal identity regulators are expressed in distinct populations of cells in the nervous system, but their function is often analyzed only in specific isolated cellular contexts, thereby potentially leaving overarching themes in gene function undiscovered. We show here that the Caenorhabditis elegans Prop1-like homeobox gene unc-42 is expressed in 15 distinct sensory, inter- and motor neuron classes throughout the entire C. elegans nervous system. Strikingly, all 15 neuron classes expressing unc-42 are synaptically interconnected, prompting us to investigate whether unc-42 controls the functional properties of this circuit and perhaps also the assembly of these neurons into functional circuitry. We found that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides, and neuropeptide receptors. Anatomical analysis of unc-42 mutant animals reveals defects in axon pathfinding and synaptic connectivity, paralleled by expression defects of molecules involved in axon pathfinding, cell-cell recognition, and synaptic connectivity. We conclude that unc-42 establishes functional circuitry by acting as a terminal selector of functionally connected neuron types. We identify a number of additional transcription factors that are also expressed in synaptically connected neurons and propose that terminal selectors may also function as 'circuit organizer transcription factors' to control the assembly of functional circuitry throughout the nervous system. We hypothesize that such organizational properties of transcription factors may be reflective of not only ontogenetic, but perhaps also phylogenetic trajectories of neuronal circuit establishment.

Brn3/POU-IV-type POU homeobox genes-Paradigmatic regulators of neuronal identity across phylogeny.

One approach to understand the construction of complex systems is to investigate whether there are simple design principles that are commonly used in building such a system. In the context of nervous system development, one may ask whether the generation of its highly diverse sets of constituents, that is, distinct neuronal cell types, relies on genetic mechanisms that share specific common features. Specifically, are there common patterns in the function of regulatory genes across different neuron types and are those regulatory mechanisms not only used in different parts of one nervous system, but are they conserved across animal phylogeny? We address these questions here by focusing on one specific, highly conserved and well-studied regulatory factor, the POU homeodomain transcription factor UNC-86. Work over the last 30 years has revealed a common and paradigmatic theme of unc-86 function throughout most of the neuron types in which Caenorhabditis elegans unc-86 is expressed. Apart from its role in preventing lineage reiterations during development, UNC-86 operates in combination with distinct partner proteins to initiate and maintain terminal differentiation programs, by coregulating a vast array of functionally distinct identity determinants of specific neuron types. Mouse orthologs of unc-86, the Brn3 genes, have been shown to fulfill a similar function in initiating and maintaining neuronal identity in specific parts of the mouse brain and similar functions appear to be carried out by the sole Drosophila ortholog, Acj6. The terminal selector function of UNC-86 in many different neuron types provides a paradigm for neuronal identity regulation across phylogeny. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Invertebrate Organogenesis > Worms Nervous System Development > Vertebrates: Regional Development.

NDK-1, the homolog of NM23-H1/H2 regulates cell migration and apoptotic engulfment in C. elegans.

Abnormal regulation of cell migration and altered rearrangement of cytoskeleton are characteristic of metastatic cells. The first described suppressor of metastatic processes is NM23-H1, which displays NDPK (nucleoside-diphosphate kinase) activity. To better understand the role of nm23 genes in cell migration, we investigated the function of NDK-1, the sole Caenorhabditis elegans homolog of group I NDPKs in distal tip cell (DTC) migration. Dorsal phase of DTC migration is regulated by integrin mediated signaling. We find that ndk-1 loss of function mutants show defects in this phase. Epistasis analysis using mutants of the α-integrin ina-1 and the downstream functioning motility-promoting signaling module (referred to as CED-10 pathway) placed NDK-1 downstream of CED-10/Rac. As DTC migration and engulfment of apoptotic corpses are analogous processes, both partially regulated by the CED-10 pathway, we investigated defects of apoptosis in ndk-1 mutants. Embryos and germ cells defective for NDK-1 showed an accumulation of apoptotic cell corpses. Furthermore, NDK-1::GFP is expressed in gonadal sheath cells, specialized cells for engulfment and clearence of apoptotic corpses in germ line, which indicates a role for NDK-1 in apoptotic corpse removal. In addition to the CED-10 pathway, engulfment in the worm is also mediated by the CED-1 pathway. abl-1/Abl and abi-1/Abi, which function in parallel to both CED-10/CED-1 pathways, also regulate engulfment and DTC migration. ndk-1(-);abi-1(-) double mutant embryos display an additive phenotype (e. g. enhanced number of apoptotic corpses) which suggests that ndk-1 acts in parallel to abi-1. Corpse number in ndk-1(-);ced-10(-) double mutants, however, is similar to ced-10(-) single mutants, suggesting that ndk-1 acts downstream of ced-10 during engulfment. In addition, NDK-1 shows a genetic interaction with DYN-1/dynamin, a downstream component of the CED-1 pathway. In summary, we propose that NDK-1/NDPK might represent a converging point of CED-10 and CED-1 pathways in the process of cytoskeleton rearrangement.