Mechanisms for Maintaining Cell Identity in C.elegans Olfactory Neurons

Maintenance of cell identity is a complex process that depends on developmentally determined transcriptional states and on environmental input. In neurons, which are both highly differentiated and highly sensitive to external stimuli, maintenance of identity is especially challenging. In this thesis, I describe the isolation and characterization of several genes involved in maintaining the identities of two olfactory neuron subtypes, AWCON and AWCOFF, in the nematode Caenorhabditis elegans. The AWCON and AWCOFF identities are specified by a stochastic decision during embryogenesis, but several of the genes involved in this embryonic decision are subsequently downregulated. Additional mechanisms must therefore act to maintain the AWCON and AWCOFF cell fates. I cloned and characterized a transcription factor, nsy-7, that was required to maintain expression of the AWCON marker str-2. nsy-7 mutants also misexpressed the AWCOFF marker srsx-3 in both AWCs. The chemotaxis phenotype of these mutants indicated that their defects in str-2 and srsx-3 expression corresponded to a more general change in cell identity from AWCON to AWCOFF. nsy-7 expression was restricted to AWCON by the signaling pathway that controls the initial fate decision. I collaborated with the Bulyk lab to discover a specific seven-nucleotide DNA sequence bound by the NSY-7 protein. This sequence was present in the srsx-3 promoter and required for NSY-7-mediated repression of srsx-3 in AWCON. Using this sequence, I was also able to predict new transcriptional targets of NSY-7. In a screen to identify additional genes affecting maintenance of str-2 and srsx-3 expression, I isolated nineteen mutants, including the uncharacterized conserved transcription factor hmbx-1 and components of the TGF! pathway. I showed that TGF! signaling is required continuously in adults to maintain srsx-3 expression and depends on dauer pheromone, an external sensory cue. I then identified new AWCON- and AWCOFF-specific markers and examined the extent to which changes in expression of str-2 or srsx-3 correlate with larger-scale changes in gene expression in the two AWC neurons. Together, my results indicate that several interlocking genetic pathways combine to maintain the AWCON and AWCOFF cell identities, including several factors not previously known to be involved in this process

The Ligand Binding Domain of GCNF Is Not Required for Repression of Pluripotency Genes in Mouse Fetal Ovarian Germ Cells

In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E) 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (2R01HG00257-20)National Human Genome Research Institute (U.S.) (2R01HG00257-20

The homeodomain protein hmbx-1 maintains asymmetric gene expression in adult C. elegans olfactory neurons

Differentiated neurons balance the need to maintain a stable identity with their flexible responses to dynamic environmental inputs. Here we characterize these opposing influences on gene expression in Caenorhabditis elegans olfactory neurons. Using transcriptional reporters that are expressed differentially in two olfactory neurons, AWCON and AWCOFF, we identify mutations that affect the long-term maintenance of appropriate chemoreceptor expression. A newly identified gene from this screen, the conserved transcription factor hmbx-1, stabilizes AWC gene expression in adult animals through dosage-sensitive interactions with its transcriptional targets. The late action of hmbx-1 complements the early role of the transcriptional repressor gene nsy-7: Both repress expression of multiple AWCOFF genes in AWCON neurons, but they act at different developmental stages. Environmental signals are superimposed onto this stable cell identity through at least two different transcriptional pathways that regulate individual chemoreceptor genes: a cGMP pathway regulated by sensory activity, and a daf-7 (TGF-β)/daf-3 (SMAD repressor) pathway regulated by specific components of the density-dependent C. elegans dauer pheromone

Transcriptional regulation and stabilization of left–right neuronal identity in C. elegans

At discrete points in development, transient signals are transformed into long-lasting cell fates. For example, the asymmetric identities of two Caenorhabditis elegans olfactory neurons called AWCON and AWCOFF are specified by an embryonic signaling pathway, but maintained throughout the life of an animal. Here we show that the DNA-binding protein NSY-7 acts to convert a transient, partially differentiated state into a stable AWCON identity. Expression of an AWCON marker is initiated in nsy-7 loss-of-function mutants, but subsequently lost, so that most adult animals have two AWCOFF neurons and no AWCON neurons. nsy-7 encodes a protein with distant similarity to a homeodomain. It is expressed in AWCON, and is an early transcriptional target of the embryonic signaling pathway that specifies AWCON and AWCOFF; its expression anticipates future AWC asymmetry. The NSY-7 protein binds a specific optimal DNA sequence that was identified through a complete biochemical survey of 8-mer DNA sequences. This sequence is present in the promoter of an AWCOFF marker and essential for its asymmetric expression. An 11-base-pair (bp) sequence required for AWCOFF expression has two activities: One region activates expression in both AWCs, and the overlapping NSY-7-binding site inhibits expression in AWCON. Our results suggest that NSY-7 responds to transient embryonic signaling by repressing AWCOFF genes in AWCON, thus acting as a transcriptional selector for a randomly specified neuronal identity

<i>Gcnf^Δ/Δ</i> mutants fail to down-regulate <i>Oct4</i> and display morphological defects.

<p>(A) Quantitative RT-PCR for <i>Oct4</i> on E9.5 embryos. Plotted here are average fold changes (relative to <i>Gcnf^+/+</i> whole embryo; all values normalized to <i>Actb</i>) of at least four independent biological replicates. Error bars show standard deviations among biological replicates. (B) Images of <i>Gcnf^+/+</i> wild-type and <i>Gcnf^Δ/Δ</i> mutant embryos at E9.5. All embryos are oriented with head facing left. Scale bar, 1 mm.</p

<i>Gcnf</i> expression in fetal ovarian germ cells.

<p>(A) Levels of <i>Gcnf</i> transcript in female (XX) and male (XY) gonads from wild-type and germ-cell-depleted (<i>W/W^v</i>) mutant embryos at E12.5, E14.5, and E16.5, as determined by Illumina sequencing of gonadal RNA. Plotted here are average numbers of reads of <i>Gcnf</i> per million total reads from two individual biological replicates. (B) Single molecule fluorescence in situ hybridization for <i>Gcnf</i> mRNA (red) in sections of XX genital ridges or gonads, with germ cells marked by SSEA1 (green), and nuclei marked by DAPI staining (blue). Large red-orange spots in the E14.5 image are auto-fluorescent blood cells. Boxes indicate areas shown in higher magnification below each image. Scale bar, 20 um.</p

Gcnf mutants undergo meiosis normally.

<p>(A–D) Immunofluorescence for SYCP3 (red) and GCNA (green) in <i>Gcnf^+/+TAM</i> wild-type (A,B) and <i>Gcnf^fl/fl;Ubc-Cre-ERT2^TAM</i> mutant (C,D) ovaries at E16.5; insets show higher magnification of areas boxed in white, SYCP3 staining alone. 100% of GCNA+ cells in both wild type (60/60) and mutant (84/84) were also SYCP3+. B and D, Deconvolution microscopy images demonstrating thread-like staining of SYCP3. Single-channel images showing SYCP3 alone are shown to the right of two-channel images. (E and F) Immunofluorescence for γH2AX (red) and GCNA (green) in <i>Gcnf^+/+TAM</i> wild-type and <i>Gcnf^fl/fl;Ubc-Cre-ERT2^TAM</i> mutant ovaries at E16.5. Single-channel images are shown to the right. Anterior is to the left. Scale bars, 50 um (A,C,E,F) or 5 um (B,D).</p

<i>Gcnf</i>-mutant germ cells down-regulate OCT4 and NANOG similarly to wild type.

<p>Immunostaining for OCT4 (A, B, and C) and NANOG (D, E, and F) proteins in ovary sections. At E13.5 OCT4 and NANOG are readily detectable in <i>Gcnf^+/+</i> wild-type ovarian germ cells (A and D, respectively). At E16.5, as expected, OCT4 and NANOG have been down-regulated and are undetectable in <i>Gcnf^+/+TAM</i> wild-type germ cells (B and E). E16.5 <i>Gcnf^fl/fl;Ubc-Cre-ERT2^TAM</i> mutant germ cells do not express OCT4 and NANOG and are indistinguishable from wild type (C and F). Anterior is to the left. Scale bar, 50 um.</p

Exon 7 of <i>Gcnf</i> is efficiently deleted in mutants.

<p>(A) Quantitative RT-PCR for exon 7 of <i>Gcnf</i> in ovaries of <i>Gcnf^fl/fl; Ubc-Cre-ERT2^TAM</i> mutants and <i>Gcnf^+/+TAM</i> wild-type littermate controls at both E15.5 and E16.5. Plotted here are average fold changes (relative to <i>Gcnf^+/+TAM</i> ovaries; all values normalized to <i>Hprt</i>) for two (at E15.5) or three (at E16.5) independent biological replicates. Error bars show standard deviations among biological replicates. (B) Immunostaining for germ cell marker MVH in <i>Gcnf^+/+TAM</i> wild-type and <i>Gcnf^fl/fl; Ubc-Cre-ERT2^TAM</i> mutant ovaries at E16.5. Anterior is to the left. Scale bar, 50 um.</p

A set of genes critical to development is epigenetically poised in mouse germ cells from fetal stages through completion of meiosis

In multicellular organisms, germ cells carry the hereditary material from one generation to the next. Developing germ cells are unipotent gamete precursors, and mature gametes are highly differentiated, specialized cells. However, upon gamete union at fertilization, their genomes drive a totipotent program, giving rise to a complete embryo as well as extraembryonic tissues. The biochemical basis for the ability to transition from differentiated cell to totipotent zygote is unknown. Here we report that a set of developmentally critical genes is maintained in an epigenetically poised (bivalent) state from embryonic stages through the end of meiosis. We performed ChIP-seq and RNA-seq analysis on flow-sorted male and female germ cells during embryogenesis at three time points surrounding sexual differentiation and female meiotic initiation, and then extended our analysis to meiotic and postmeiotic male germ cells. We identified a set of genes that is highly enriched for regulators of differentiation and retains a poised state (high H3K4me3, high H3K27me3, and lack of expression) across sexes and across developmental stages, including in haploid postmeiotic cells. The existence of such a state in embryonic stem cells has been well described. We now demonstrate that a subset of genes is maintained in a poised state in the germ line from the initiation of sexual differentiation during fetal development and into postmeiotic stages. We propose that the epigenetically poised condition of these developmental genes is a fundamental property of the mammalian germ-line nucleus, allowing differentiated gametes to unleash a totipotent program following fertilization.Howard Hughes Medical Institut