Research Article

Direct Conversion of C. elegans Germ Cells into Specific Neuron Types

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Science  21 Jan 2011:
Vol. 331, Issue 6015, pp. 304-308
DOI: 10.1126/science.1199082

Abstract

The ability of transcription factors to directly reprogram the identity of cell types is usually restricted and is defined by cellular context. Through the ectopic expression of single Caenorhabditis elegans transcription factors, we found that the identity of mitotic germ cells can be directly converted into that of specific neuron types: glutamatergic, cholinergic, or GABAergic. This reprogramming event requires the removal of the histone chaperone LIN-53 (RbAp46/48 in humans), a component of several histone remodeling and modifying complexes, and this removal can be mimicked by chemical inhibition of histone deacetylases. Our findings illustrate the ability of germ cells to be directly converted into individual, terminally differentiated neuron types and demonstrate that a specific chromatin factor provides a barrier for cellular reprogramming.

There is much interest in developing methods to direct cell differentiation to a specific cell fate. However, the transcription factors required to induce the identity of specific cell types in a multicellular organism are remarkably ineffective in imposing their fate-inducing activity when ectopically expressed, resulting in the generally accepted paradigm that transcription factors can exert their activities only in specific cellular contexts (1, 2). Classic examples for such context dependency are the cell type–restricted ability of human MyoD to induce muscle cell features (3), the region-restricted ability of ectopically expressed Drosophila Eyeless to induce ectopic eyes (4), or, as a more recent example, the restricted ability of a cocktail of transcription factors to directly reprogram the identity of mouse pancreatic cell types (5). Our understanding of the mechanistic basis of the context dependency of transcription factor activity is limited. Overcoming such context dependency would have major implications for a variety of different applications. For example, the generation of specific cell types through transcription factor–mediated reprogramming strategies may allow the establishment of in vitro disease models for basic research or the provision of source material for cellular replacement therapies.

Context dependency of CHE-1 activity. We sought to establish a system in which we could study the mechanistic basis of the context dependency of transcription factor activity. To this end, we used a genetic approach in the nematode C. elegans, using the zinc finger transcription factor CHE-1, which is required to induce the identity of a specific class of gustatory neurons called ASE neurons (6, 7). CHE-1 exerts this activity through binding directly to a cis-regulatory motif (termed the “ASE motif”) present in many ASE-specific terminal differentiation genes, such as those encoding chemoreceptors, neurotransmitter receptors, signaling proteins, and neurotransmitter transporters (6). Like CHE-1, several other invertebrate and vertebrate transcription factors are also known to co-regulate many terminal features of differentiated neurons in such a manner and have been termed “terminal selectors” (8).

To test whether CHE-1 is not only required but also sufficient to induce ASE fate, we ectopically expressed CHE-1 throughout the entire animal in either larval or adult stages, using an inducible heat shock promoter. Such misexpression results in broad ectopic expression of an artificial reporter of CHE-1 transcription factor activity, which is composed of a multimerized ASE motif (“8x ASE motif reporter”) (6) (Fig. 1). This indicates that, in principle, CHE-1 can exert its biochemical activity of DNA binding and transcriptional activation without spatial or temporal constraints. In contrast, postembryonic ectopic expression of che-1 during larval or adult stages is able to induce markers for terminal ASE fate (the gcy-5 chemoreceptor and the ceh-36 homeobox gene; see table S1 for list of markers) only in a small number of head sensory neurons but nowhere else in the animal (Fig. 1 and table S2). This result illustrates the context dependency of the ASE fate–inducing activity of CHE-1.

Fig. 1

Context dependency of CHE-1 induction of target genes. Ectopic expression of CHE-1 can induce an artificial reporter of CHE-1 activity (a multimerized binding site called the 8x ASE motif) throughout the animal (top row; observed in all 50 of the heat-shocked animals), but can induce ASE cell fate markers such as the gcy-5 chemoreceptor (exclusively expressed in ASER) or the ceh-36 homeobox gene (expressed in ASEL/R and AWCL/R) only in one to three types of other sensory neurons (blue arrows), consistent with previous reports (6, 7). Heat shock induction of CHE-1 was done at late larval stages. Large boxes show magnified views of small boxes in the same panels. Table S1 contains details on transgenic reporters; table S2 shows data quantification. All images that show entire worms are at 160× magnification; all images showing parts of the worm are at 630× magnification.

The chromatin factor lin-53 controls context dependency of CHE-1. To test the hypothesis that the mechanistic basis for such context dependency lies in inhibitory, perhaps chromatin-based mechanisms that may prevent CHE-1 from reprogramming the identity of other cells, we established and screened through an RNA interference (RNAi) library that targets all genes in the C. elegans genome with predicted roles in chromatin regulation, based on the presence of characteristic protein domains (9) (table S3). We found that RNAi-mediated knockdown of lin-53, the C. elegans ortholog of the phylogenetically conserved, WD40 domain–containing retinoblastoma binding protein RbAp46/48 (10), permits ectopically expressed CHE-1 to induce the ASE neuronal fate markers gcy-5 and ceh-36 in a large number of normally non-neuronal cells in the midbody region of larval and adult animals (Fig. 2). Up to 52% of animals (n = 227) showed this effect (table S4), which could be observed using distinct, nonoverlapping double-stranded RNA clones that target lin-53 and all six of the tested transgenic che-1 lines (see supporting online material).

Fig. 2

Removal of lin-53 permits che-1 to induce ASE fate markers in the germ line. (A) ASE cell fate markers (gcy-5 and ceh-36), a transformation of nuclear morphology (from germ line with large nucleolus to neuronal with characteristic intranuclear speckles), and outgrowth of cellular extensions [blue arrows; also see (B)] are induced in the gonad of lin-53(–) animals upon ubiquitous induction of che-1 expression. Noninduced lin-53(+) controls are shown in Fig. 1. This phenotype is observed in the germ line of up to 52% of heat-shocked animals (see tables S4 and S5 and fig. S3 for quantification) and in all six transgenic che-1heat-shock lines; the line shown here is otIs284. Heat shock of lin-53(–) animals that do not carry the che-1heat-shock array do not show any ectopic marker induction. (B) Higher-magnification image of axonal extensions induced by ectopic che-1 expression in lin-53(RNAi) animals. The protruding vulva (Pvul) phenotype is a characteristic pleiotropy of loss of lin-53. Movies S1 to S3 show additional examples of axonal projections.

Germ cells are the target of reprogramming. Closer examination of the animals revealed that the cells showing gcy-5 and ceh-36 induction are located within the gonad (Fig. 2). Differential interference contrast (DIC) microscopy showed that germ cells of che-1heat-shock; lin-53(RNAi) animals lose their characteristic, fried egg–shaped nuclear and nucleolar morphology and adopt a speckled neuronal nuclear morphology (Fig. 2). Moreover, these cells grow cellular extensions resembling axo-dendritic projections (Fig. 2 and movies S1 to S3). These neuron-like cells indeed originate from the germ line, as they were not observed after ectopic che-1 expression and lin-53 removal in a glp-4 mutant background (fig. S1), in which no germ line is formed (11). Genetic removal of sperm [fem-3lf mutants (12)] or oocyte [fem-3gf mutants (13)] or prevention of entry into meiosis [glp-1gf mutants (14)] does not affect neuron induction, whereas severe reduction of the mitotic pool [glp-1lf mutants (14)] does, which suggests that it is the mitotic germ cells that become neuron-like (fig. S1). This is further supported by the position of the converted cells relative to mitotic and meiotic markers (fig. S2) and the observation that animals in the second and third larval stages, which contain mostly mitotic but no meiotic cells, show germ cell–neuron conversion upon ectopic che-1 expression and lin-53 knockdown (table S4). The conversion to neuron-like cells is efficient and fast; as many as 60 germ cells (of a total of about 200 mitotic germ cells) undergo neuronal induction (fig. S3), and morphological changes and marker induction first occur 6 hours after che-1 induction (table S5). For comparison, the induction of the 8x ASE motif, an indicator of CHE-1 transcriptional activity occurs 4 hours after che-1 induction in the gonad. The induction of the 8x ASE motif in the gonad of wild-type animals, as well as antibody staining conducted in both wild-type and lin-53(RNAi) animals, rules out the possibility that lin-53(RNAi) merely results in germline derepression of the che-1 transgene. [We also note that lin-53(RNAi) does not result in germline derepression of a previously described transgenic array, let-858::gfp, known to be derepressed after loss of several different chromatin factors (15).]

Extent of germ cell–neuron conversion. We assessed the nature of these che-1–induced, neuron-like cells with a number of fate markers. Through antibody staining of a marker that labels specific germ cell structures, the P-granules, we confirmed that this germ cell feature is indeed lost upon ectopic che-1 expression and lin-53 knockdown (fig. S4). Moreover, the reprogrammed cells express all five tested pan-neuronal reporter genes: the rab-3/Rab3 and snb-1/synaptobrevin genes, which encode presynaptic proteins normally exclusively expressed in all cells of the nervous system; the pan-neuronal axonal regulators unc-33/CRMP and unc-119; and the pan-neuronal signaling factor rgef-1 (Fig. 3A; see table S1 for transgenic reporters). Antibody staining against endogenous proteins also shows ectopic expression of presynaptic synaptobrevin/SNB-1 and RIM/UNC-10 proteins; these proteins appear to cluster in presynaptic specialization in the axonal extensions of the reprogrammed germ cells (Fig. 3B and fig. S5). The reprogrammed cells also express a ciliated marker gene, osm-6, a member of the intraflagellar transport particle, normally expressed exclusively in all ciliated sensory neurons, including ASE (Fig. 4A). Green fluorescent protein (GFP)–tagged OSM-6 protein also appears to cluster in particles in the induced neurons (Fig. 4A), which suggests that these cells express and cluster intraflagellar transport particles. All tested components of the gene battery that combinatorially define ASE identity are also expressed in the induced neurons. That is, aside from the above-mentioned chemoreceptor gcy-5 and the transcription factor ceh-36 (shown again in Fig. 4A), the putative chemoreceptor gcy-7, which is normally exclusively expressed in ASE, and the vesicular glutamate transporter eat-4, normally expressed in ASE and a restricted number of additional head ganglia neurons, are also expressed in the reprogrammed germ cells (Fig. 4A). Induction of eat-4 demonstrates that the reprogrammed cells are glutamatergic. The reprogrammed ASE-like cells do not express a battery of markers that are normally expressed in other neuron types, such as dopaminergic, serotonergic, cholinergic, and GABAergic markers, among others (Fig. 4B). This argues that the reprogrammed cells are not merely generic and/or misspecified neurons but closely resemble normally differentiated ASE neurons. Taken together, animals ectopically expressing che-1 and lacking lin-53 not only contain their normal set of two ASE gustatory neurons in the head, but contain a gonad filled with dozens of ASE-like neurons.

Fig. 3

Reprogrammed germ cells express pan-neuronal markers and cluster presynaptic proteins. (A) Induction of pan-neuronal reporter genes in che-1heat-shock; lin-53(RNAi) animals. (B) Induction of the presynaptic SNB-1 protein, as assessed by antibody staining. An individual ASE-like neuron (labeled with gcy-5::gfp) is shown in higher magnification to show punctate staining of SNB-1 in red marker along the extension of the process (white arrowheads). che-1 expression was induced in late (L3/L4) larval stages of animals exposed to lin‑53(dsRNA) starting in the parental generation. Adult midbody regions are shown. White stippled lines indicate outline of the animal; yellow stippled lines indicate outline of the gonad. The penetrance of induction of the markers shown here is the same as for the gcy-5::gfp marker shown in Fig. 2 and table S4. See table S1 for information about transgenes and their wild-type expression pattern.

Fig. 4

Reprogrammed germ cells express markers for ASE, but not other neuronal fates. Experimental conditions and image labeling are as described in Fig. 3. (A) ASE-specific markers expressed in che-1heat-shock; lin-53(RNAi) animals. osm-6::gfp is a full-length protein fusion. (B) Other neuronal fates are not expressed in che-1heat-shock; lin-53(RNAi) animals. The left column shows an overall view of the entire animal to illustrate the expression of non-ASE markers (all mCherry-based reporters) in other neuron types, and the right column shows an enlarged view (white box) of the gonad region, illustrating that these markers do not become induced in the che-1 reprogrammed germ cells, labeled with gcy-5::gfp.

Specificity of germ cell–neuron conversion. Removal of RNA-binding gene regulatory factors has been shown to result in the formation of teratomas (i.e., cells of various origins and types) in the germ line (16). The effects that we observed upon loss of lin-53 are not a reflection of teratoma formation, because we observed no expression of various neuronal fate markers other than those of ASE fate in the gonad of che-1heat-shock; lin-53(RNAi) animals. However, the very same neuronal markers (GABA neuron marker, cholinergic neuron marker) were expressed in the gonad of animals in which the translation regulator gld-1, a previously described repressor of teratoma formation (16), is knocked down (fig. S6). Therefore, removal of lin-53 does not by itself trigger alternative developmental programs but primes germ cells to be responsive to a neuronal fate inducer such as che-1.

Germ cell conversion to other neuron types. To test whether lin-53 removal also permits the conversion of germ cells into other neuron types, we tested two other terminal selector genes (8): the phylogenetically conserved Pitx-type homeobox gene unc-30, a terminal selector required for the generation of GABAergic motor neurons in the ventral nerve cord (17, 18), and the EBF-like transcription factor unc-3, required for the generation of two types (A- and B-type) of cholinergic motor neurons in the ventral nerve cord (19, 20). When ectopically misexpressed, neither unc-30 nor unc-3 was able to induce GABAergic or cholinergic neuron fate in the germ line, respectively. However, upon removal of lin-53, heat-shock induction of either unc-30 or unc-3 resulted, like che-1 induction, in germ cells losing their characteristic morphology and instead adopting neuron-like nuclear morphology and growing axonal projections (Fig. 5, A and B). In the case of unc-3, the ectopic neurons expressed a marker characteristic of cholinergic A/B-type ventral cord motor neurons (acr-2; Fig. 5B), whereas ectopic expression of unc-30 resulted in the expression of the GABAergic marker unc-47 (Fig. 5A). In neither case did we observe any ASE marker expression to be induced (>100 animals scored); also, neither cholinergic nor GABAergic markers were induced by che-1 (Fig. 4B). We conclude that upon loss of lin-53, germ cells acquire the ability to be reprogrammed into distinct neuron types through the activity of neuron type–specific terminal selector transcription factors.

Fig. 5

Reprogramming activity of other selector genes in lin-53(–) animals. (A) Heat-shock induction of the unc-30 terminal selector induces GABAergic, D-type motor neuron fate (as assessed by neuronal morphology and unc-47 vesicular GABA transporter expression) in the germ line of lin-53(RNAi) animals. White asterisks indicate ventral cord neurons that normally express the marker and are located in proximity to the gonad. Six of 32 unc-30heat-shock; lin-53(RNAi) animals (line 1, otEx4442), 4 of 21 animals (line 2, otEx4443), and 5 of 23 animals (line 3, otEx4444) show germline unc-47::gfp (oxIs12) expression. Without heat shock and RNAi, none of >100 animals show the effect. (B) Heat-shock induction of the unc-3 terminal selector induces A/B-type motor neuron fate in the germ line of lin-53(RNAi) animals. White asterisks indicate ventral cord neurons that normally express the marker and are located in proximity to the gonad. The white arrows indicate axonal-like extensions. Five of 24 unc-3heat-shock; lin-53(RNAi) animals show expression of a gfp reporter for the autoreceptor acr-2 expression; none of 20 unc-3heat-shock animals [no lin-53(RNAi)] show this effect. Without heat shock and RNAi, none of >100 animals show the effect.

Does lin-53 removal prime germ cells to respond only to factors that induce neuronal fates, or can they now respond to other factors as well? To address this question, we ectopically expressed a selector gene, the C. elegans MyoD homolog hlh-1 that was previously shown to be able to ectopically induce muscle fate in early embryos (21). We found that hlh-1 is unable to convert the germ cells of lin-53(RNAi) animals into muscle cells (fig. S7). These negative results need to be cautiously interpreted, but may represent a first hint toward a target selectivity of lin-53. That is, lin-53 may only restrict the developmental potential specifically toward a neuronal developmental program, whereas other factors may serve to prevent the induction of other, non-neuronal differentiation programs.

LIN-53, which is ubiquitously expressed (10), is one of several phylogenetically conserved histone chaperones that are thought to assist in the recruitment of various distinct types of histone modifiers or remodelers (including histone methyltransferases, histone acetylases, and histone deacetylases) to histone H3 and histone H4 (22, 23). LIN-53 orthologs in various species have been found to be integral components of at least six different protein complexes, each displaying diverse biochemical and biological roles: the NURD and NURF nucleosome remodeling complexes, the CAF-1 chromatin assembly factor complex, the Sin3A transcriptional repressor complex, the PRC2 histone methyltransferase complex, and the HAT1 histone acetyltransferase complex (22, 23) (table S6). RNAi of representative members of each of these complexes either did not phenocopy the lin-53(RNAi)–induced reprogramming ability of ectopic CHE-1 expression or could not be interpreted because of early lethality induced by RNAi (table S6). However, two of the LIN-53–associated complexes (NURD and Sin3a) each contain at least two histone deacetylase (HDAC) components and the reprogramming role of lin-53 may involve several complexes; for these reasons, we sought to broadly inhibit HDAC function by using two distinct chemical inhibitors, valproic acid and trichostatin A (24). At sublethal doses, animals treated with either drug survived and permitted heat shock–induced che-1 to induce ASE fate in the germ line (and no other cell type) even in the presence of functional lin-53 (Fig. 6). Even though these drug effects may be unrelated to normal lin-53 function, these results nonetheless provide a strong indication that histone modifications are key players in restricting the ability of a transcription factor to reprogram cellular identity.

Fig. 6

Inhibitors of HDACs also permit che-1 to reprogram germ cells. Animals were grown on plates containing the respective drug, che-1 was induced in late larval stages, and 1 day later, adult P0 animals were scored for ectopic gcy-5::gfp expression in the gonad. Relative to lin-53(RNAi), the drug’s effects appear to be weaker (5 of 40 animals affected show ASE-like neurons in valproic acid–treated animals, 3 of 40 in TSA-treated animals; none of 50 mock-treated animals show an effect) and not as many germ cells appear to be transformed, possibly because of drug dosage issues [too high a dose results in no viable progeny, a phenotype similar to RNAi of at least one C. elegans HDAC (29)]. Heat shock in the absence of the che-1heat-shock transgene does not have an effect on drug-treated animals (40 animals scored). TSA, trichostatin A.

Conclusions. Our finding that the removal of a single chromatin factor, together with the induction of single transcription factors, can produce distinct and specific neuron types in a heterologous cellular context is a testament to the simplicity of programs that control neuronal differentiation. The role of complex, multistage neuronal developmental programs may mainly lie in orchestrating the activation of a terminal regulatory routine—that is, one or more terminal selectors that directly control terminal differentiation genes. This notion may apply to more complex systems as well, because recent work has shown that it takes as few as two transcription factors to drive a differentiated fibroblast toward a specific neuronal fate (25). Our findings are also a testament to the totipotency of germ cells (26). This totipotency is normally kept in check by a variety of transcriptional and posttranscriptional mechanisms (26), but is unleashed either spontaneously in pathological situations (germ cell tumors) (27) or upon culturing germ stem cells under specific conditions, which transform these cells into cells indistinguishable from pluripotent embryonic stem cells (28).

We have shown that the ability of germ cells to be directly reprogrammed into neurons can be unleashed, even in the adult animal, through removal of a single gene that we speculate to be involved in rendering neuronal differentiation genes inaccessible to transcriptional induction by, for example, contributing to the formation of facultative (i.e., conditional and developmentally regulated) heterochromatin. Seen in a broader context, our results indicate that the reprogramming of cellular identity may critically depend not just on providing the correct transcription factor(s) that induce a specific fate, but also on the removal of inhibitory mechanisms that restrict transcription factor activity. We anticipate that the disablement of such inhibitory mechanisms may provide an efficient strategy to ectopically generate neuron types in other organisms, or perhaps even in cell culture, using isolated germ cells.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1199082/DC1

Materials and Methods

Figs. S1 to S7

Tables S1 to S6

Movies S1 to S3

References

References and Notes

  1. Supported by NIH grants R01NS039996-05 and R01NS050266-03. O.H. is an Investigator of the HHMI and B.T. is a Francis Goelet Research Scientist. We thank T. Schedl, M. Krause, J. McGhee, P. Sengupta, Y. B. Qi, Y. Jin, M. Nonet, S. Strome, and members of the Hobert lab for reagents; I. Greenwald and members of the Hobert lab for comments on the manuscript; and Q. Chen for expert assistance in strain generation.
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