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. 2017 Jan 23;27(2):199-209.
doi: 10.1016/j.cub.2016.11.045. Epub 2017 Jan 5.

Sexually Dimorphic Differentiation of a C. Elegans Hub Neuron Is Cell Autonomously Controlled by a Conserved Transcription Factor

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Sexually Dimorphic Differentiation of a C. Elegans Hub Neuron Is Cell Autonomously Controlled by a Conserved Transcription Factor

Esther Serrano-Saiz et al. Curr Biol. .
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Functional and anatomical sexual dimorphisms in the brain are either the result of cells that are generated only in one sex or a manifestation of sex-specific differentiation of neurons present in both sexes. The PHC neuron pair of the nematode C. elegans differentiates in a strikingly sex-specific manner. In hermaphrodites the PHC neurons display a canonical pattern of synaptic connectivity similar to that of other sensory neurons, but in males PHC differentiates into a densely connected hub sensory neuron/interneuron, integrating a large number of male-specific synaptic inputs and conveying them to both male-specific and sex-shared circuitry. We show that the differentiation into such a hub neuron involves the sex-specific scaling of several components of the synaptic vesicle machinery, including the vesicular glutamate transporter eat-4/VGLUT, induction of neuropeptide expression, changes in axonal projection morphology, and a switch in neuronal function. We demonstrate that these molecular and anatomical remodeling events are controlled cell autonomously by the phylogenetically conserved Doublesex homolog dmd-3, which is both required and sufficient for sex-specific PHC differentiation. Cellular specificity of dmd-3 action is ensured by its collaboration with non-sex-specific terminal selector-type transcription factors, whereas the sex specificity of dmd-3 action is ensured by the hermaphrodite-specific transcriptional master regulator of hermaphroditic cell identity tra-1, which represses the transcription of dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner.


Fig. 1
Fig. 1. Dimorphic synaptic connectivity and function of PHC
A: Schematic synaptic connectivity. Edge weights were collected from [8]. Only connections that directly involve PHC are shown. B: PHC is required in hermaphrodites for response to harsh (picking-force) touch to the tail. Bar graph indicates the mean percentage of animals responding to harsh touch, with error bars indicating the minimum and maximum from two experimental replicates. Significance was calculated using Fisher’s exact test. ** p<0.01, n.s. p>0.05. “n” in each column indicates the total number of animals assayed. C: PHC is required for a specific step of the male mating behavior, vulva search behavior. Mutant and Histamine-silenced animals tested for the male’s vulva location efficiency. Box plot representation of the data is shown, with whiskers pointing from min to max. Median and mean values are indicated by horizontal line and “+”, respectively. Statistics was calculated using Kruskal-Wallis test. ****P < 0.0001, ***P < 0.0005. “n” above graphs indicates the total number of animals assayed.
Fig. 2
Fig. 2. Sexually dimorphic extension of the PHC axon
A: PHC axon and dendrite morphology at different developmental stages. PHC was visualized with the transgene otEx6776, which expresses gfp under the control of the eat-4prom11Δ12 driver, which expresses in PHC of both males and hermaphrodites (described in more detail in Fig. 5). As landmark, the PHA and PHB neurons, which do not noticeably change morphology between the sexes, are filled with DiD (termination point of PHA/B marked with orange arrowhead, PHC with white arrowhead). PHC dendrites are marked with purple arrowhead. Scale bar: 50 μm. Dashed box indicates the area that is magnified in the right column. Scale bar in magnified panels: 10 μm. B: Summary of PHC axon extension. Male specific synaptic contacts (referring to both inputs and outputs) are schematically indicated with blue arrows. C: Male-specific axon extension is controlled cell-autonomously as determined by cell-specific sex change via manipulation of the sex-determination pathway. Transgenic array names: otIs520 (eat-4prom11::gfp) was crossed with PHCp::fem-3 (lines otEx6879 and otEx6980) and PHCp::tra-2ic (lines otEx6881 and oEx6882) strains. PHCp = eat-4prom11Δ11 driver (Fig. 5B). Significance was calculated using student t-test, ***P < 0.0005, **P < 0.005 and *P < 0.01. n.s. >0.1
Fig. 3
Fig. 3. Scaling of synaptic vesicle components in PHC
A: eat-4/VGLUT fosmid reporter expression (otIs518) in the adult male tail. The complete set of all male-specific, eat-4/VGLUT-expressing neurons will be reported elsewhere. Scale bar: 20μm. B: eat-4/VGLUT fosmid (otIs518) reporter expression measured by absolute fluorescence (right panel) and normalized to expression of the mel-28 nuclear envelope protein (bq5). otIs520 reporter expression measured by absolute fluorescence in him-8(e1489) background and dmd-3(tm2863);him-8(e1489) (left panel). C: Temporal dynamics of scaling of eat-4/VGLUT fosmid (otIs518) reporter expression. D: smFISH analysis of endogenous eat-4/VGLUT expression in young adult animals. PHC was marked with otIs520 (green). See Fig. S2 for eat-4 smFISH probe specificity. Scale bar: 20μm. E,F: Scaling of transcription of other vesicular markers, as assessed by SL2-based fosmid reporter expression [33]. Temporal dynamic of rab-3 fosmid (otIs498) expression in L4 vs. adult animals (F). G: eat-4/VGLUT scaling is controlled cell-autonomously as shown by masculinization and feminization experiments. otIs520 (eat-4prom11::gfp) was crossed with PHCp::fem-3 (lines otEx6879 and otEx6980) and with PHCp::tra-2ic (lines otEx6881 and oEx6882) strains. PHCp (eat-4prom11Δ11 driver). Wildtype data showing in this plot is same as in panel B (left panel), genotypes were scored in parallel. Significance was calculated using student t-test, ***P < 0.001, **P < 0.01. See Fig. S1 for more regulators of eat-4 expression.
Fig. 4
Fig. 4. Sexually dimorphic neuropeptide expression in PHC
A: flp-11 reporter expression (ynIs40) in wildtype hermaphrodites and males, in animals in which the sex of PHC was changed via fem-3 or tra-2ic expression and in dmd-3(tm2863) mutants. The table indicates the percentage of neurons that show expression of the flp-11 reporter in the different conditions assayed. N=number of animals. Scale bar: 50 μm. B: Temporal dynamics of flp-11 reporter gene expression in larval and adult male stages. Cell bodies are labeled with white arrow, dendritic projections with orange arrow. White asterisks indicate ray projections. Scale bar: 50 μm.
Fig. 5
Fig. 5. Analysis of the cis-regulatory control elements of the eat-4 locus
A: Dissection of the eat-4prom5 promoter. In the analysis of expression between two and three lines (n > 10) were scored for expression. The different shades of gray indicate the relative fluorescence intensity. B: Deletion analysis of the eat-4prom11 promoter. The pie charts indicate the percentage of neurons that express the array with the four different shades denoting the overall fluorescence intensity in AIM and PHC in both sexes (white: no expression; black: strongest expression observed in any construct; light and dark grey: intermediate levels that are clearly distinct from each other and from maximum or no expression). At least two lines were analyzed for each deletion construct. Color bars in the promoters refer to putative homeodomain binding sites: blue bar indicates the CEH-14 binding site according to the ModEncode consortium; purple bar indicates the putative UNC-86 binding site. See Methods for sequences. See Fig. S1 for effect of CEH-14 and UNC-86 on eat-4 expression. C: eat-4prom11 and eat-4prom11Δ18 reporter expression in the head and tail of young adults. Scale bar: 25 μm. D: Summary schematic of eat-4/VGLUT regulatory logic. The same elements are required to achieve activator and repressor effects in the PHC and AIM neurons, but with opposite sexual specificity. In the AIM neurons, unknown factors “A” (for activator) and “R” (for repressor) may be different DMD transcription factors.
Fig. 6
Fig. 6. dmd-3 expression and function
A: smFISH analysis of endogenous dmd-3 expression in young adult animals. No expression is observed in hermaphrodites; in males, expression is observed in multiple cells including PHC, marked with the transgenic array otIs520. Masculinization of PHC (via PHCp::fem-3) in otherwise hermaphroditic animals activates dmd-3 transcription. The inset shows the PHC nucleus stained with DAPI. More than 10 animals were scored for presence of dots in each condition and all animals showed similar staining patterns relative to one another. See Fig. S2 for dmd-3 smFISH probe specificity. Scale bar: 50 μm. B: PHC axons fail to extend in dmd-3(tm2863) mutant males and these defects are rescued by PHC-specific expression of dmd-3 [oExt6908 (eat-4p11Δ11::dmd-3)]. Scale bars: 50 μm. Wildtype and PHCp::fem-3 data showing in this plot is the same as in Fig. 2B for the adult stage. See panel D for quantification. C: Ectopic expression of dmd-3 in PHC of hermaphrodites is sufficient to scale eat-4/VGLUT expression (4th panel) and dmd-3 is required in hermaphrodites for the axon extension conferred by masculinization of PHC (2nd and 3rd panel). Transgenic array names: otEx6879, otEx6880 (eat-4p11Δ11::fem-3); otEx6908(eat-4p11Δ11::dmd-3). See panel D for quantification. D: Quantification of the axon extension and eat-4/VGLUT scaling in the different conditions showed in previous panels. Significance was calculated using student t-test, ***P < 0.005, **P < 0.05.

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