Chromatin modification of Notch targets in olfactory receptor neuron diversification

Journal name:
Nature Neuroscience
Year published:
Published online


Neuronal-class diversification is central during neurogenesis. This requirement is exemplified in the olfactory system, which utilizes a large array of olfactory receptor neuron (ORN) classes. We discovered an epigenetic mechanism in which neuron diversity is maximized via locus-specific chromatin modifications that generate context-dependent responses from a single, generally used intracellular signal. Each ORN in Drosophila acquires one of three basic identities defined by the compound outcome of three iterated Notch signaling events during neurogenesis. Hamlet, the Drosophila Evi1 and Prdm16 proto-oncogene homolog, modifies cellular responses to these iteratively used Notch signals in a context-dependent manner, and controls odorant receptor gene choice and ORN axon targeting specificity. In nascent ORNs, Hamlet erases the Notch state inherited from the parental cell, enabling a modified response in a subsequent round of Notch signaling. Hamlet directs locus-specific modifications of histone methylation and histone density and controls accessibility of the DNA-binding protein Suppressor of Hairless at the Notch target promoter.

At a glance


  1. Differential Notch signaling and Ham and Svp expression delineates ORN identity.
    Figure 1: Differential Notch signaling and Ham and Svp expression delineates ORN identity.

    (a) 3-ORN sensillum lineage. Cut marks non-neural pIIa progeny. In the neural lineage, Pon is asymmetric at each stage (N, high Notch activity). Pan-neuronal Elav marks three ORNs, each differentially expressing Ham, Svp and Pon. pNa divides before pNb, and pOa and pOb cells also divide asynchronously, producing transient five- and seven-cell stages (not shown). (bf) Wild-type MARCM clones derived from a single SOP, immunostained for GFP (green) and other cell type–specific markers. Rows in each panel represent different apico-basal focal planes of the pupal antenna. Each individual cell in the immunostained MARCM clone is indicated with a number and is represented by the same number in the accompanying summary diagram. Scale bars represent 5 μm. (b) Seven-cell stage clone. Pon is shown in red and Elav in blue. Pon was expressed on one Elav-positive and one Elav-negative cells. pIIa progeny do not form neurons and are not shown. Cell identity was assigned by stereotypic position along the apico-basal axis and by integrating information from cf and Supplementary Figure 1. (c) Four-cell stage clone. Cut is shown in red and Ham in blue. Cut marks the outer cells derived from pIIa. Ham was expressed in one of the Cut-negative pIIb daughters. (d) Four-cell stage clone. Pros is shown in red and Ham in blue. Ham was expressed in the Pros-negative pNa cell. (e) Six-cell stage clone. Pon is shown in red and Ham in blue. Ham was expressed in one Pon-positive and one Pon-negative cell. (f) Eight-cell stage clone. Svp is shown in red and Ham in blue. Svp was expressed in the cell that showed weaker Ham staining than its sibling. *One pIIa progeny is out of the focal plane.

  2. Naa, Nab and Nba identities in the ORN lineage.
    Figure 2: Naa, Nab and Nba identities in the ORN lineage.

    (a) Axonal projections of the wild-type ORN clones labeled by GAL4-NP0724. Left, different focal planes of an antennal lobe along the antero-posterior axis. Only five glomeruli DA4l, VA1d, DL1, VL1 and VM6 were innervated by the clone. Each of these glomeruli was the projection target of one of the ORNs in the 3-ORN and 4-ORN sensilla, as shown in the schematics on the right. Scale bar represents 20 μm. (bd) Axonal projection of wild-type clones that were labeled by pan-neuronal Gal4-C155 and induced at a late stage of the ORN lineage. Panels in each column represent different focal planes of an antennal lobe. Only two of the three target glomeruli of ORNs in the at3 sensillum (DA4m and DA4l) and two of the four target glomeruli of ORNs in the ab1 sensillum (VA2 and DL1 or DM1 and V) were co-innervated by the labeled clone, as shown in the schematics at the bottom. (e) Representation of the ORN lineage for ab10, at3 and ab1 sensilla. The odorant receptors expressed and glomerulus innervated by each ORN in the sensilla are indicated.

  3. ham mutant ORNs switch axonal projection identities.
    Figure 3: ham mutant ORNs switch axonal projection identities.

    (a,b) Axonal projection of the ham clones labeled by pan-neuronal Gal4-C155. The panels in each row represent different focal planes of an antennal lobe. The mutant clones failed to project to DA4l and DA4m (a) and to DL1 and VA2 (b), the target glomeruli of Naa and Nab ORNs, of the at3 sensillum and of the ab1 sensillum, respectively. (c) ham mutant clones failed to project DM6, the target glomerulus of Nab ORN in the ab10 sensillum. (d) Axonal projection of the wild-type clones of the Gal4-AM29–labeled ORNs in ab10 sensillum to DM6 and DL4. (e) ham clones failed to arborize at the DM6 glomerulus and projected further toward DL4. Scale bar represents 20 μm.

  4. Ham acts as a switch between Nab and Nba odorant receptor expression identity.
    Figure 4: Ham acts as a switch between Nab and Nba odorant receptor expression identity.

    (a) A bar chart showing the percentage change in a given ORN class in ham mutants or in flies with ectopic ham expression (neur>ham). Data is shown as mean ± s.e.m. For each experiment, a probability was determined by unpaired, two-tailed Student's t tests. For ham loss-of-function, we examined Or43a (wild type (WT), n = 11; ham, n = 8; P = 0.14), Or2a (wild type, n = 5; ham, n = 15; P = 2.1 × 10−24), Or19a (wild type, n = 6; ham, n = 6; P = 5.8 × 10−7), Or67a (wild type, n = 6; ham, n = 3; P = 4.2 × 10−8) and Or49a (wild type, n = 6; ham, n = 5; P = 0.00033) expression. For ham ectopic expression, we examined Or67a (wild type + heat shock, n = 4; neur>ham + heat shock, n = 16; P = 0.00043) and Or49a (wild type + heat shock, n = 11; neur>ham + heat shock, n = 16; P = 0.00023). (b,c) Whole-mount antennae of wild-type and ham1/Df(2L)ED1195 flies immunostained for the neuronal marker Elav (magenta) and GFP (green) to show the expression of a given Or-mCD8::GFP reporter. Scale bar represents 50 μm and applies to both b and c. Or2a-mCD8::GFP–expressing neurons were lost (b) and Or19a-mCD8::GFP–expressing neurons were gained in the in b ham−/− antenna (c). (d) Or19a-mCD8::GFP expression in a single sensillum, showing duplication of Or19a-expressing neurons in the ham−/− antenna. Scale bar represents 5 μm.

  5. Modification of Notch activity by a Ham-CtBP complex.
    Figure 5: Modification of Notch activity by a Ham-CtBP complex.

    (a) Ectopic ham expression suppresses Notch signaling in the cell-fate decision between notal-microchaete hair and socket cells. Black arrows indicate normal hair or socket cells. Ectopic expression of ham caused either a partial fate conversion (recognized by an ectopic hair-like protrusions on the socket, gray arrow) or a full conversion from socket to hair (white arrows). Scale bar represents 20 μm. (b) Bar charts quantifying the fraction of each phenotype on the notum (mean ± s.e.m.). The conversion of socket to hair phenotype caused by ectopic Ham was enhanced by removing one copy of Notch (N55e11) (wild type, n = 10 nota, 500 microchaete sensory organs; N+/−, n = 10 nota, 500 microchaete sensory organs;109-68>ham, n = 8 nota, 248 microchaete sensory organs; N+/−; 109-68>ham, n = 7 nota, 69 microchaete sensory organs). (c) Fate conversions caused by ectopic Ham were suppressed by removing the Ham CtBP-binding site (hamΔCtBP) and/or by reducing CtBP levels (CtBP87De−10) (CtBP+/−, n = 10 nota, 500 microchaete sensory organs; 109-68>ham, n = 13 nota, 444 microchaete sensory organs; CtBP+/−; 109-68>ham, n = 13 nota, 900 microchaete sensory organs; 109-68 >hamΔCtBP, n = 10 nota, 1,134 microchaete sensory organs; CtBP+/−; 109-68>hamΔCtBP, n = 8, 1,411 microchaete sensory organs). (d) The domain structure of Ham, mammalian homologs Evi1, Prdm16 and C. elegans homolog Egl43. PR, PRDI-BF1 and RIZ homology. (e) A CtBP GST pulldown revealed an interaction between Ham and CtBP. (f) Mutation of the PLDLS sequence to a nonfunctional PLASS sequence led to the loss of this interaction. Lower panels show input controls probed with antibody to V5 (10%) or stained with Coomassie Brilliant Blue (CBB). Full-length blots and gels are presented in Supplementary Figure 6.

  6. Ham drives chromatin modification at Notch-target loci.
    Figure 6: Ham drives chromatin modification at Notch-target loci.

    (a) mRNA levels for E(spl)m3 and a control gene (cbx). Ham blocked induction of the E(spl)m3 locus by Notch signaling. A two-way ANOVA analysis highlighted an interaction between Notch signaling state and Ham induction state (P = 0.00012). The significance of the effect of Ham induction on the Notch activated state was P = 0.00023, as determined by a post-hoc Tukey test. Data are presented as mean ± s.e.m. of target gene expression and chromatin modifications in the absence and presence of Ham (without Notch activation or 30 min after Notch activation). (b,c) Trimethylation of H3-K4 (b) and H3-K27 (c) at E(spl)m3 and E(spl)m6 loci measured by ChIP. Independent of Notch, Ham activity suppressed H3-K4 trimethylation (P = 0.0015, Tukey test) and increased H3-K27 trimethylation at the E(spl)m3 locus (P = 0.00023, Tukey test). Data are presented as in a. (d) Occupancy of histone H3 at the E(spl)m3 and E(spl)m6 enhancers (m3s and m6s) or ORFs (m3o and m6o) measured by ChIP. Ham specifically increased H3 occupancy at the E(spl)m3 enhancer independently of Notch (P = 0.00022, Tukey test). The levels in the ORF remain unchanged. Data are presented as in a. (e) Occupancy of Su(H) at the E(spl)m3 and E(spl)m6 promoters as measured by ChIP. At the E(spl)m3 promoter, Ham prevented the increase in Su(H) occupancy that usually occurs after Notch signaling activation. A two-way ANOVA analysis highlighted an interaction between Notch signaling state and Ham induction state (P = 0.011). The effect of Ham on the Notch activated state was P = 0.0018 (Tukey test). Data are presented as in a.

  7. Ham controls ORN lineage Notch target dynamics.
    Figure 7: Ham controls ORN lineage Notch target dynamics.

    (a) Notch mediates the outcome of ectopic Ham expression on ORN identity (mean ± s.e.m.). A two-way ANOVA analysis highlighted an interaction between Notch genotype and ectopic Ham expression state (P = 0.036). The Notch genotype altered ORN identity in a Ham overexpression (P = 0.00055, Tukey test), but not a wild-type (P = 0.70, Tukey test), background (wild type, n = 6; N+/−, n =7; neur>ham, n = 11; N+/−; neur>ham, n = 9). (b) Quantification of wild-type E(spl)m8-lacZ expression in Naa (gray) and Nab (black) clones (n = 4, 6, 5 and 5, for the four-, six-, seven- and eight-cell stages, respectively; mean ± s.e.m.). (cg) Wild-type (ce) and ham (f,g) clones immunostained for β-galactosidase (E(spl)m8-lacZ, red), GFP (green) and Ham (blue). In e and g, four and two pIIa progeny are out of the focal planes, respectively. Scale bar represents 5 μm. (h,i) Quantification of E(spl)m8-lacZ expression in wild-type (black) and ham (white) clones (mean ± s.e.m.). At the four-cell stage, E(spl)m8-lacZ levels were the same between wild-type (n = 4) and ham mutants (n = 7; P = 0.43, unpaired two-tailed Student's t test; h). In the wild type at the six-cell stage, E(spl)m8-lacZ levels dropped in both Naa and Nab (i). In ham mutants, E(spl)m8-lacZ levels did not drop; they were 56% higher in wild type than in Naa (P = 0.016, unpaired two-tailed Student's t test; wild type, n = 8 wild type; ham, n = 7) and 52% higher in Nab (P = 0.032, unpaired two-tailed Student's t test; wild type, n = 8; ham, n = 7).


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Author information

  1. These authors contributed equally to this work.

    • M Rezaul Karim &
    • Hiroaki Taniguchi


  1. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

    • Keita Endo &
    • Kei Ito
  2. Disease Mechanism Research Core, RIKEN Brain Science Institute, Wako, Saitama, Japan.

    • M Rezaul Karim,
    • Hiroaki Taniguchi,
    • Emi Kinameri,
    • Matthias Siebert &
    • Adrian W Moore
  3. Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, UK.

    • Alena Krejci &
    • Sarah J Bray
  4. Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic.

    • Alena Krejci
  5. Institute of Entomology, Biology Centre of the Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic.

    • Alena Krejci


K.E., M.R.K., E.K., M.S., S.J.B. and A.W.M. carried out genetic analyses. H.T., A.K., E.K. and M.S. carried out molecular biology. K.E., K.I., S.J.B. and A.W.M. wrote the paper. A.W.M. conceived and coordinated the study.

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