Axial patterning of C. elegans male sensilla identities by selector genes

The fan and rays of the C. elegans male tail constitute a compound sensory organ essential for mating. Within this organ, the individual sensilla, known as rays, have unique identities. We show that ray identities are patterned by a selector gene mechanism in a manner similar to other serially homologous axial structures. One selector gene that promotes the identities of a subset of the rays is the Hox gene egl-5. Within EGL-5-expressing rays, further patterning is provided by a Pax-6 homolog and a signal of the TGFβ family. These genes and pathway coordinately specify multiple ray properties affecting all three terminal ray cell types. These properties include complex patterns of FMRFamide-like (FaRP) neuropeptides, serotonin (5HT) and dopamine expression, and ray morphology. Differences in these differentiated characteristics give each sensillum a unique identity and potentially endow the compound ray organ with a higher-order information gathering capacity.     

The Caenorhabditis elegans spalt-like gene sem-4 restricts touch cell fate by repressing the selector Hox gene egl-5 and the effector gene mec-3

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3. A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which we find is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; we show that a subset also express egl-5. We report: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. We conclude that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes, that sem-4 indirectly modulates mec-3 expression through its repression of egl-5 and that this negative regulation is required for proper determination of neuronal fates. We suggest that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes.     

The roles of an ephrin and a semaphorin in patterning cell-cell contacts in C. elegans sensory organ development

Ephrins and semaphorins regulate a wide variety of developmental processes, including axon guidance and cell migration. We have studied the roles of the ephrin EFN-4 and the semaphorin MAB-20 in patterning cell-cell contacts among the cells that give rise to the ray sensory organs of Caenorhabditis elegans. In wild-type, contacts at adherens junctions form only between cells belonging to the same ray. In efn-4 and mab-20 mutants, ectopic contacts form between cells belonging to different rays. Ectopic contacts also occur in mutants in regulatory genes that specify ray morphological identity. We used efn-4 and mab-20 reporters to investigate whether these ray identity genes function through activating expression of efn-4 or mab-20 in ray cells. mab-20 reporter expression in ray cells was unaffected by mutants in the Pax6 homolog mab-18 and the Hox genes egl-5 and mab-5, suggesting that these genes do not regulate mab-20 expression. We find that mab-18 is necessary for activating efn-4 reporter expression, but this activity alone is not sufficient to account for mab-18 function in controlling cell-cell contact formation. In egl-5 mutants, efn-4 reporter expression in certain ray cells was increased, inconsistent with a simple repulsion model for efn-4 action. The evidence indicates that ray identity genes primarily regulate ray morphogenesis by pathways other than through regulation of expression of semaphorin and ephrin.     

Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons

Mesencephalic dopaminergic (MesDA) neurons play crucial roles in motor and behavioral processes; their loss in Parkinson's disease (PD) results in striatal dopamine (DA) deficiency and hypokinetic movement disorder. The Pitx3 homeobox gene is expressed in the MesDA system. We now show that only a subset of MesDA neurons express Pitx3 and that in Pitx3-deficient aphakia mice, this subset is progressively lost by apoptosis during fetal (substantia nigra, SN) and postnatal (ventral tegmental area) development, resulting in very low striatal DA and akinesia. Similar to human PD, dorsal SN neurons (which are Pitx3 negative) are spared in mutant mice. Thus, Pitx3 defines a pathway for survival of neurons that are implicated in PD and that are required for spontaneous locomotor activity.     

Relaxed repression of herpes simplex virus type 1 genomes in Murine trigeminal neurons

The expression of herpes simplex virus (HSV) genomes in the absence of viral regulatory proteins in sensory neurons is poorly understood. Previously, our group reported an HSV immediate early (IE) mutant (d109) unable to express any of the five IE genes and encoding a model human cytomegalovirus immediate early promoter-green fluorescent protein (GFP) transgene. In cultured cells, GFP expressed from this mutant was observed in only a subset of infected cells. The subset exhibited cell type dependence, as the fractions of GFP-expressing cells varied widely among the cell types examined. Herein, we characterize this mutant in murine embryonic trigeminal ganglion (TG) cultures. We found that d109 was nontoxic to neural cultures and persisted in the cultures throughout their life spans. Unlike with some of the cultured cell lines and strains, expression of the GFP transgene was observed in a surprisingly large subset of neurons. However, very few nonneuronal cells expressed GFP. The abilities of ICP0 and an inhibitor of histone deacetylase, trichostatin A (TSA), to activate GFP expression from nonexpressing cells were also compared. The provision of ICP0 by infection with d105 reactivated quiescent genomes in nearly every cell, whereas reactivation by TSA was much more limited and restricted to the previously nonexpressing neurons. Moreover, we found that d109, which does not express ICP0, consistently reactivated HSV type 1 (KOS) in latently infected adult TG cultures. These results suggest that the state of persisting HSV genomes in some TG neurons may be more dynamic and more easily activated than has been observed with nonneuronal cells.     

The T-box gene tbx-2, the homeobox gene egl-5 and the asymmetric cell division gene ham-1 specify neural fate in the HSN/PHB lineage

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, we have been studying a Caenorhabditis elegans lineage that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. We have previously shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. In this study we examine tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, we identified the T-box protein TBX-2 as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage.     

Brain-derived neurotrophic factor modulates dopaminergic deficits in a transgenic mouse model of Huntington's disease

Dysfunction of dopaminergic neurons may contribute to motor impairment in Huntington's disease. Here, we study the role of brain-derived neurotrophic factor (BDNF) in alterations of the nigrostriatal system associated with transgenics carrying mutant huntingtin. Using huntingtin-BDNF+/- double-mutant mice, we analyzed the effects of reducing the levels of BDNF expression in a model of Huntington's disease (R6/1). When compared with R6/1 mice, these mice exhibit an increased number of aggregates in the substantia nigra pars compacta. In addition, reduction of BDNF expression exacerbates the dopaminergic neuronal dysfunction seen in mutant huntingtin mice, such as the decrease in retrograde labelling of dopaminergic neurons and striatal dopamine content. However, mutant huntingtin mice with normal or lowered BDNF expression show the same decrease in the anterograde transport, number of dopaminergic neurons and nigral volume. In addition, reduced BDNF expression causes decreased dopamine receptor expression in mutant huntingtin mice. Examination of changes in locomotor activity induced by dopamine receptor agonists revealed that, in comparison with R6/1 mice, the double mutant mice exhibit lower activity in response to amphetamine, but not to apomorphine. In conclusion, these findings demonstrate that the decreased BDNF expression observed in Huntington's disease exacerbates dopaminergic neuronal dysfunction, which may participate in the motor disturbances associated with this neurodegenerative disorder.     

Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification

The dopaminergic neurons of the basal ganglia play critical roles in CNS function and human disease, but specification of dopamine neuron phenotype is poorly understood in vertebrates. We performed an in vivo screen in zebrafish to identify dopaminergic neuron enhancers, in order to facilitate studies on the specification of neuronal identity, connectivity, and function in the basal ganglia. Based primarily on identification of conserved non-coding elements, we tested 54 DNA elements from four species (zebrafish, pufferfish, mouse, and rat), that included 21 genes with known or putative roles in dopaminergic neuron specification or function. Most elements failed to drive CNS expression or did not express specifically in dopaminergic neurons. However, we did isolate a discrete enhancer from the otpb gene that drove specific expression in diencephalic dopaminergic neurons, although it did not share sequence conservation with regulatory regions of otpa or other dopamine-specific genes. For the otpb enhancer, regulation of expression in dopamine neurons requires multiple elements spread across a large genomic area. In addition, we compared our in vivo testing with in silico analysis of genomic regions for genes involved in dopamine neuron function, but failed to find conserved regions that functioned as enhancers. We conclude that regulation of dopaminergic neuron phenotype in vertebrates is regulated by dispersed regulatory elements.     

Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans

We designed an automatic system to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big, and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. We show that the egl-4 gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in the egl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-beta and insulin pathways, respectively.     

Analysis of dopamine transporter gene expression pattern -- generation of DAT-iCre transgenic mice

The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon-improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome-dopamine transporter-iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome-dopamine transporter-iCre transgene and a construct expressing the beta-galactosidase gene after Cre-mediated recombination. In situ studies showed that beta-galactosidase (5-bromo-4-chloroindol-3-yl beta-D-galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of beta-galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome-dopamine transporter-iCre transgenic line is a unique tool for targeting Cre/loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins.     

Temporal expression of mutant LRRK2 in adult rats impairs dopamine reuptake

Parkinson's disease (PD) results from progressive degeneration of dopaminergic neurons. Most PD cases are sporadic, but some have pathogenic mutation in the individual genes. Mutation of the leucine-rich repeat kinase-2 (LRRK2) gene is associated with familial and sporadic PD, as exemplified by G2019S substitution. While constitutive expression of mutant LRRK2 in transgenic mice fails to induce neuron death, transient expression of the disease gene by viral delivery causes a substantial loss of dopaminergic neurons in mice. To further assess LRRK2 pathogenesis, we created inducible transgenic rats expressing human LRRK2 with G2019S substitution. Temporal overexpression of LRRK2(G2019S) in adult rats impaired dopamine reuptake by dopamine transporter (DAT) and thus enhanced locomotor activity, the phenotypes that were not observed in transgenic rats constitutively expressing the gene throughout life time. Reduced DAT binding activity is an early sign of dopaminergic dysfunction in asymptomatic subjects carrying pathogenic mutation in LRRK2. Our transgenic rats recapitulated the initiation process of dopaminergic dysfunction caused by pathogenic mutation in LRRK2. Inducible transgenic approach uncovered phenotypes that may be obscured by developmental compensation in constitutive transgenic rats. Finding in inducible LRRK2 transgenic rats would guide developing effective strategy in transgenic studies: Inducible expression of transgene may induce greater phenotypes than constitutive gene expression, particularly in rodents with short life time.