Modular Organization of Cis-regulatory Control Information of Neurotransmitter Pathway Genes in Caenorhabditis elegans

Here, Serrano-Saiz et al. describe the cis-regulatory logic of how neurotransmitter identity is imposed onto individual, distinct neuron types... We explore here the cis-regulatory logic that dictates gene expression in specific cell types in the nervous system. We focus on a set of eight genes involved in the synthesis, transport, and breakdown of three neurotransmitter systems: acetylcholine (unc-17/VAChT, cha-1/ChAT, cho-1/ChT, and ace-2/AChE), glutamate (eat-4/VGluT), and γ-aminobutyric acid (unc-25/GAD, unc-46/LAMP, and unc-47/VGAT). These genes are specifically expressed in defined subsets of cells in the nervous system. Through transgenic reporter gene assays, we find that the cellular specificity of expression of all of these genes is controlled in a modular manner through distinct cis-regulatory elements, corroborating the previously inferred piecemeal nature of specification of neurotransmitter identity. This modularity provides the mechanistic basis for the phenomenon of “phenotypic convergence,” in which distinct regulatory pathways can generate similar phenotypic outcomes (i.e., the acquisition of a specific neurotransmitter identity) in different neuron classes. We also identify cases of enhancer pleiotropy, in which the same cis-regulatory element is utilized to control gene expression in distinct neuron types. We engineered a cis-regulatory allele of the vesicular acetylcholine transporter, unc-17/VAChT, to assess the functional contribution of a “shadowed” enhancer. We observed a selective loss of unc-17/VAChT expression in one cholinergic pharyngeal pacemaker motor neuron class and a behavioral phenotype that matches microsurgical removal of this neuron. Our analysis illustrates the value of understanding cis-regulatory information to manipulate gene expression and control animal behavior.This work was supported by the Howard Hughes Medical Institute. E.S.-S. has been supported by the Ramon y Cajal program (RYC-2016-20537), and M.G. was supported by the European Molecular Biology Organization and Human Frontier Science Program postdoctoral fellowships.Peer reviewe

Renal Involvement in Patients with Mucolipidosis IIIAlpha/Beta: Causal Relation or Co-Occurrence?

Mucolipidosis IIIalpha/beta (MLIIIalpha/beta) is a rare lysosomal storage disorder characterized by childhood onset of flexion contractures of fingers, joint stiffness in the shoulders, hips, and knees, and mild short stature. Recessive mutations in the GNPTAB gene have been associated with MLIIIalpha/beta. We present five children aged 9-16 years from a large kindred family whose serum activities of several lysosomal enzymes were significantly elevated. Whole exome sequencing followed by confirmation by Sanger sequencing identified a novel homozygous missense mutation (c.22 A>G; p.R8G) in the GNPTAB gene in all affected subjects. The five patients initially presented with flexion contractures of fingers followed by stiffnes of large joints. Only two affected boys also had a nephrotic-range proteinuria. Renal biopsy showed focal segmental glomerulosclerosis and foamy appearance of glomerular visceral epithelial cells which were compatible with storage disease. No other known causes of proteinuria could be detected by both laboratory and biopsy findings. There was no known family history of hereditary kidney disease, and healthy siblings and parents had normal renal function and urinalysis. These findings suggest that the renal involvement probably due to MLIIIalpha/beta, although it can still be present by coincidence in the two affected patients. (C) 2016 Wiley Periodicals, Inc

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification.

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems

Comparing expression patterns of CRISPR/Cas9-engineered reporter alleles with fosmid-based reporter transgenes.

Comparing expression patterns of CRISPR/Cas9-engineered reporter alleles with fosmid-based reporter transgenes.</p

<i>tab-1</i> regulates the differentiation of various neurons in the ABala lineage.

Fig 7A: In tab-1(ok2198) mutants, expression of both nlp-42(syb3238) and NeuroPAL reporters in AIN is lost. tab-1(ok2198) mutants also showed defects in unc-17(otIs576) reporter expression in AIN and AVD. No loss of reporter expression was observed in ttx-3(ot22) mutants. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Fig 7B: tab-1 is expressed in various neurons derived from the ABala lineage (adapted from Ma et al., 2021). In tab-1(ok2198) mutants, defects in NeuroPAL reporter expression, including ultrapanneuronal (UPN) reporter expression, are seen in neurons which express tab-1 embryonically. Representative images of wild type and mutant worms are shown with 10 μm scale bars. In all panels, neurons of interest are outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost. P-values were calculated by Fisher’s exact test.</p

Expression pattern of <i>ast-1(vlc19)</i> reporter allele.

ast-1 CRISPR/Cas9-engineered reporter allele, vlc19, [79] is expressed in the following head neuron classes: ADE, AIN, AIZ, ASG, AVG, CEPD, CEPV, I4, I5, M3, M5, RIV, RMD, RMDD, RMDV, SMBD, SMBV, SMDD, and SMDV. Expression in the midbody, ventral nerve cord, and tail was not examined. (EPS)</p

The HOX gene <i>egl-5</i> affects the differentiation of head and tail neurons.

Fig 8A:egl-5(u202) mutant animals show a loss of AWA marker expression, including NeuroPAL (otIs669) and an odr-10 reporter transgene (kyIs37). Representative images of wildtype and mutant worms are shown with 5 μm scale bars. Graphs compare expression in wildtype and mutant worms with the number of animals examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Fig 8B: egl-5(u202) mutant animals show changes in tail marker expression, including NeuroPAL (otIs669) in PDA, LUA, PVC and loss of eat-4 (otIs518) expression in LUA. Representative images of wildtype and mutant worms are shown with 5 μm scale bars. Graphs compare expression in wildtype and mutant worms with the number of animals examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. In all panels, neurons of interest are outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost.</p

Updated expression of the homeobox gene family with reporter alleles.

Fig 1A: Representative images of homeobox reporter alleles, generated by CRISPR/Cas9 genome engineering (see strain list in S5 Table) with different expression than previously reported fosmid-based reporter transgenes. Neuron classes showing expression not previously noted were identified by overlap with the NeuroPAL landmark strain, and are outlined and labeled in red. Neuron types in agreement with previous reporter studies are outlined in yellow. Head structures including the pharynx were outlined in white for visualization. Autofluorescence common to gut tissue is outlined with a white dashed line. An n of 10 worms were analyzed for each reporter strain. Scale in bottom or top right of the figure represents 5 μm. See also S1 Fig for more information on ceh-30 and ceh-31. Fig 1B: Summary of expression of all homeobox genes across the C. elegans nervous system, taking into account new expression patterns from panel A and all previously published data [6]. Black boxes indicate that a homeodomain transcription factor is expressed in that given neuron type and white boxes indicate that a homeodomain transcription factor is not expressed in that given neuron type. Neuron types along the x axis are clustered by transcriptomic similarity using the Jaccard index (see methods) and homeobox genes along the y axis are clustered similarly by their similar expression profiles in shared neuron types. See S3 Fig for numerical representation of homeoboxes per neuron.</p

<i>unc-39</i> controls differentiation of the AIA interneuron class.

unc-39R203Q mutant animals (either canonical e257 allele or CRISPR/Cas9 genome engineered ot1173 allele with identical nucleotide change) were analyzed. Fig 2A: unc-39 affects the cholinergic identity of the AIA interneuron class (unc-17 reporter allele syb4491 and a cho-1 promoter fragment which is part of the otIs653 array), and other AIA terminal identity markers: reporter alleles dmsr-2(syb4514), ins-1(syb5452) and flp-19(syb3278), and a mgl-1 promoter fragment otIs327. We did not quantify changes in AIA in the NeuroPAL color code, because it is variable in wild type. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression in wild type and mutant worms with the number of neurons examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Fig 2B: unc-39 affects the expression of the tagged eya-1 locus (nIs352 transgene) in AIA.</p