DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE
215
Development 138, 215-225 (2011) doi:10.1242/dev.050161
© 2011. Published by The Company of Biologists Ltd
Notch regulates numb: integration of conditional and
autonomous cell fate specification
Mark Rebeiz*,†, Steven W. Miller* and James W. Posakony‡
SUMMARY
The Notch cell-cell signaling pathway is used extensively in cell fate specification during metazoan development. In many cell
lineages, the conditional role of Notch signaling is integrated with the autonomous action of the Numb protein, a Notch
pathway antagonist. During Drosophila sensory bristle development, precursor cells segregate Numb asymmetrically to one of
their progeny cells, rendering it unresponsive to reciprocal Notch signaling between the two daughters. This ensures that one
daughter adopts a Notch-independent, and the other a Notch-dependent, cell fate. In a genome-wide survey for potential Notch
pathway targets, the second intron of the numb gene was found to contain a statistically significant cluster of binding sites for
Suppressor of Hairless, the transducing transcription factor for the pathway. We show that this region contains a Notchresponsive cis-regulatory module that directs numb transcription in the pIIa and pIIIb cells of the bristle lineage. These are the
two precursor cells that do not inherit Numb, yet must make Numb to segregate to one daughter during their own division. Our
findings reveal a new mechanism by which conditional and autonomous modes of fate specification are integrated within cell
lineages.
KEY WORDS: Asymmetric cell division, Notch signaling, numb, Suppressor of Hairless, Default repression, Cis-regulatory module,
Drosophila
Division of Biological Sciences, Section of Cell and Developmental Biology, University
of California San Diego, La Jolla, CA 92093, USA.
*These authors contributed equally to this work
†
Present address: Department of Biological Sciences, University of Pittsburgh,
Pittsburgh, PA 15260, USA
‡
Author for correspondence ([email protected])
Accepted 15 November 2010
A previous report from our laboratory described the application
of a computational method called SCORE to identify statistically
significant clusters of transcription factor binding sites in the
genome (Rebeiz et al., 2002). We used this method in an attempt
to identify targets of Suppressor of Hairless [Su(H)], the
transducing transcription factor for the Notch pathway in
Drosophila. Besides recovering multiple genes already known to
be activated directly by Su(H), the SCORE technique identified as
potential targets several other core members of the Notch pathway,
including Delta (which encodes a ligand for the Notch receptor),
neuralized (which encodes an E3 ubiquitin ligase essential for
promoting endocytosis and activation of the Delta protein) and
numb. Here, we describe our identification and analysis of an
intronic cis-regulatory module that is responsible for the
transcriptional activation of numb in bristle precursor cells in
response to Notch signaling. Our findings illuminate a previously
unrecognized regulatory linkage that further intertwines the
conditional and autonomous modes of cell fate specification.
MATERIALS AND METHODS
Fly stocks
w1118 is a spontaneous partial deletion of the white locus that eliminates
gene function (Tweedie et al., 2009). sca-GAL4 and pnr-GAL4 are Pelement ‘enhancer trap’ insertions into the scabrous and pannier loci,
respectively, driving expression of GAL4 (Hinz et al., 1994; Calleja et al.,
1996). Nts1 is a temperature-sensitive point mutation of Notch induced by
ethyl methanesulfonate (EMS) mutagenesis (Shellenbarger and Mohler,
1975); N81k1 is an X-ray-induced deficiency lacking DNA from
chromosomal region 3C5-3C10, which overlaps the Notch locus (3C73C9) (Grimwade et al., 1985). UAS-numb flies carry a numb coding region
transgene (Wang et al., 1997) that can be misexpressed using the GAL4UAS system (Brand and Perrimon, 1993). numb1 is a strong hypomorphic
mutation caused by a P-element transposon insertion at 30B (Uemura et
al., 1989); numb2 is a diepoxybutane (DEB)-induced amorphic or strong
hypomorphic allele first described by Frise et al. (Frise et al., 1996);
DEVELOPMENT
INTRODUCTION
Metazoan development relies on two broad categories of
mechanisms for specifying cell fate: autonomous, in which fate is
determined by factors inherited by, or expressed in, the cell itself;
and conditional, in which fate is determined by external factors,
particularly cell-cell signaling. The cell lineage that gives rise to
Drosophila adult mechanosensory organs (Fig. 1) is well known
for its elegant combination of these two modes of fate specification
(Hartenstein and Posakony, 1990; Posakony, 1994; Rhyu et al.,
1994; Frise et al., 1996; Guo et al., 1996). At each of several
precursor cell divisions in this lineage, the two daughter cells signal
to each other via the Notch pathway. The fate of one daughter is
specified by this signal. The other daughter inherits the Notch
pathway antagonist Numb, asymmetrically segregated from the
precursor cell. This renders the second daughter immune to the
reciprocal Notch signal, ensuring that it adopts the alternative,
Notch-independent, cell fate. The fly sensory organ lineage thus
embodies a universal strategy for generating cell fate asymmetry
during development.
In this lineage, the fates of two of the precursor cells (pIIa and
pIIIb) are specified by Notch signaling (Fig. 1, blue arrowheads).
It is essential, therefore, that these two cells do not inherit
substantial amounts of Numb from their respective mother cells.
However, each must make Numb to distribute to its own Notchindependent daughter cell. The solution to this regulatory problem
has been a lingering question (Rhyu et al., 1994).
216
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Development 138 (2)
CA, USA). The final product was confirmed by sequencing (GENEWIZ,
South Plainfield, NJ, USA) and inserted into the germline of w1118 embryos.
Five independent insertion lines were obtained; when tested using pnrGAL4 as the driver, all five lines gave the same ‘balding’ phenotype shown
in Fig. S4 in the supplementary material.
Reduction of Notch pathway activity
Su(H) short-hairpin (sh) RNAi expression
UAS-Su(H)shRNA flies were crossed to CD2-GFP flies, and the progeny
crossed to pnr-GAL4/TM6c flies. Tubby (Tb)+ pupae at 0-3 hours after
puparium formation (APF) were collected from this latter cross and aged
at 25°C for 13-15 hours before dissection and staining. Tissue genotypes
were distinguished by the cellular phenotype at scutellar macrochaete
positions (which are transformed when both driver and responder are
present), and by the detection of GFP expression.
Notch loss of function
numb796 is a strong loss-of-function allele induced by EMS mutagenesis
(Buescher et al., 1998). Df(2L)30A-C is an X-ray-induced deficiency
lacking DNA from chromosomal region 30A3-C5, which overlaps the
numb locus (30B3-B5) (Uemura et al., 1989; Tweedie et al., 2009).
Reporter gene constructs
Fragments tested in reporter constructs were amplified by PCR on genomic
DNA templates; primer sequences are supplied in Table S1 in the
supplementary material. Binding site mutants were created by overlapextension PCR (Ho et al., 1989); see Table S1 in the supplementary
material for mutagenesis primer sequences. The CD2 fragment and all
mutant variants thereof were PCR cloned from the Celera (Alameda, CA,
USA) sequencing strain and are fully compliant with the Celera genome
sequence (Adams et al., 2000). Fragments were inserted into the KpnI and
BglII restriction sites of the GFP reporter vector pH-Stinger (Barolo et al.,
2000).
Germline transformation
P element-mediated germline transformation was performed as previously
described (Rubin and Spradling, 1982) using w1118 as the recipient strain.
Generation of flies bearing an inducible Su(H) short-hairpin RNAi
construct
A GAL4-inducible RNAi construct for reducing Su(H) activity was
designed according to Haley et al. (Haley et al., 2008). This transgene
expresses a 71-nucleotide stem-loop sequence within a pre-miR-1 scaffold
that leads to the production of 21-nucleotide silencing RNAs; these
recognize a complementary sequence within the second exon of Su(H).
Two synthetic oligonucleotides (IDT, Coralville, IA, USA; see Table S1 in
the supplementary material) were annealed in 1⫻ TE (Tris-EDTA) buffer
and cloned into the pUAST-based vector pNE3 (Ben Haley, UC Berkeley,
numb misexpression experiments
Flies of the genotype y w; sca-GAL4/Cyo were crossed to flies of the
genotype w1118; numbCD2-GFP; UAS-numb/TM6B. Progeny lacking either
the sca-GAL4 driver or the UAS-numb responder were used as controls.
numb rescue constructs
Cloning of a 19.5 kb numb genomic DNA rescue construct was carried
out in three steps. First, a 538 bp fragment upstream of the proximal
promoter was PCR amplified (primers: forward 5⬘-ttgcggccgcaaGGTAAATCTGAAGGCGAAGCCATG-3⬘, reverse 5⬘-gcggccgcgaccgccgggtcGTGTCCCCGGGATAAGGTTGCAGATGCAA-3⬘; uppercase,
genomic sequence; lowercase, bases added for cloning purposes) and
cloned into pCR2.1-TOPO. Both primers contain synthetic NotI sites at
their 5⬘ ends (bold). Internal to the 3⬘ NotI site, a synthetic DrdI and an
endogenous SmaI site (both underlined) were used to clone a 19.5 kb
SmaI/DrdI fragment containing the entire numb proximal promoter and
transcription unit (see Fig. 2A), digested from BAC clone RP98-6D23
(Hoskins et al., 2000) into the pCR2.1-TOPO clone containing the abovementioned 538 bp fragment. The 19.5 kb fragment was then cloned into
CaSpeR(NotI) (Malicki et al., 1993) using the flanking NotI sites. In clones
used for both wild-type and CD2 deletion rescue constructs, the DrdI/SmaI
fragment insertion occurred in the reverse orientation, yielding a fully
functional rescue transgene, with a small portion of 5⬘ noncoding sequence
inserted downstream of the gene, which should have no effect on the
outcome of our experiments.
To delete the CD2 enhancer, overlap-extension PCR (Ho et al., 1989)
was used to generate a 1.6 kb fragment that overlapped endogenous PmeI
and SacI sites (external primers: 5⬘-GAATCCACAAGTATTCGCCAGATG-3⬘,
5⬘-TGAACTGTATCTGTGTGCTCGGAG-3⬘;
internal
primers: 5⬘-TTTGCAGTATTTAAAATAGTAATTAATAGATGGTAAAAACTTTAAAACTT-3⬘, 5⬘-AAGTTTTAAAGTTTTTACCATCTATTAATTACTATTTTAAATACTGCAAA-3⬘). This 1.6 kb fragment was
cloned via PmeI/SacI sites into a 5.3 kb PmeI/SacII fragment contained in
pH-Stinger (Barolo et al., 2000). Next, the 5.3 kb PmeI/SacII fragment
harboring a deletion of the CD2 enhancer was cloned into the wild-type
numb rescue fragment contained in pCR2.1-TOPO, and subsequently
inserted into CaSpeR(NotI). Transgenic fly lines carrying either the wildtype (numbRCwt) or CD2 deletion (numbRC2del) rescue constructs were
generated by Genetic Services (Cambridge, MA, USA).
numb rescue experiments
Flies of the genotype w; Df(2L)30A-C/CyO or w; numb1/CyO were crossed
to flies of the genotype w; numb796/CyO; numbRCX, where X refers to
either the wild-type (wt) or CD2 enhancer deletion (2del) rescue construct
DEVELOPMENT
Fig. 1. Cell fate specification in the Drosophila mechanosensory
bristle lineage. The adult mechanosensory bristle lineage in
Drosophila originates from a single sensory organ precursor cell (SOP or
pI) and consists of a series of asymmetric cell divisions in which one
daughter (yellow) adopts a Notch-dependent, and the other daughter
(purple) a Notch-independent, cell fate. These outcomes are the result
of the repeated use of a combination of conditional and autonomous
cell fate specification mechanisms. At each division, the two daughter
cells signal to each other via the Notch pathway (horizontal arrows),
and one daughter is rendered immune to this signal by its inheritance
of the Numb protein (red), segregated asymmetrically in the dividing
precursor cell. The Notch-dependent precursor cells in the lineage, pIIa
and pIIIb, are indicated with blue arrowheads.
Males of the genotype Nts1/Y; CD2-GFP/+ were crossed to wa N81k1/FM7,
Kr-GAL4>UAS-GFP females at 18°C. Pupae at 0-4 hours APF were
collected, aged at 18°C for an additional 24 hours and separated on the
basis of Kr>GFP expression under an epifluorescence dissection
microscope. Immediately prior to dissection, pupae were exposed to a
temperature of 37°C for 2 hours. Presence of the CD2-GFP chromosome
was detected by GFP expression at macrochaete positions.
Notch regulates numb
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217
Fig. 2. A cluster of conserved Su(H) binding sites in the Drosophila numb gene. (A)Diagram of the numb locus and genomic DNA
fragments used in this study. The diagram is drawn to scale, and shows the position of high-affinity Su(H) binding motifs (S). The upper transcript
isoform is zygotically expressed; the lower isoform is maternally expressed (Uemura et al., 1989). Boxed area marks the cluster of five predicted
Su(H) binding sites found by SCORE analysis (Rebeiz et al., 2002). Genomic DNA fragments used for numb mutant rescue experiments (numbRC)
are indicated, as are fragments tested for enhancer activity in GFP reporter transgenes. Red boxes indicate numb coding sequences.
(B)Electrophoretic mobility shift assay (EMSA) analysis of predicted Su(H) binding sites in the boxed region shown in A. Sites are numbered as in C.
All five sites are efficiently bound in vitro by a purified GST-Su(H) fusion protein; the binding of a positive control Su(H) site [m4 S3 (+); S3 from the
E(spl)m4 gene (Bailey and Posakony, 1995)] is shown for comparison. A single-base substitution in numb site S2 (S2mut, red) almost completely
abolishes binding. (C)Patterns of evolutionary conservation of Su(H) binding sites in the identified cluster. Solid lines indicate site orthology, based
on the presence of a conserved site motif in a comparable location and on shared sequences adjacent to individual sites. Dotted line connects a pair
of site occurrences (10/10 match) in D. pseudoobscura and D. mojavensis only. Data for D. simulans, D. sechellia and D. persimilis are omitted for
clarity; all five sites are conserved in all three of these species, except site 1b, which is absent in D. persimilis. See Fig. S1 in the supplementary
material for sequence alignments. Diagrams in A and C were generated using the GenePalette software tool (Rebeiz and Posakony, 2004).
Transgene rescue of the numb2 mutant phenotype in mosaic
clones
To assay phenotypic rescue in numb2 mutant clones, females of the
genotype y w Ubx-FLP; FRT40A were crossed to males of the genotype
w/Y; y+ numb2 ck FRT40A/CyO; numbRCX/Sb, where X is either ‘wt’ or
‘2del’. Flies not carrying the CyO balancer were sorted for presence of the
rescue construct (numbRC). Because of the dark eye color provided by the
Ubx-FLP transgene, Sb was used to identify flies that did not receive the
rescue construct chromosome, and annotated as ‘no RC’; Sb+ flies were
assumed to contain a copy of the rescue construct. Forty macrochaete
bristle positions on the dorsal head and thorax per fly were assayed for the
ck mutant phenotype, indicating numb2 mutant territories, and the bristle
phenotype in these territories was scored as shown in Table S2 in the
supplementary material.
Immunohistochemistry
Staining of pupal nota was performed on animals incubated at 25°C and
dissected at timed stages measured in hours APF. Timed pupae were fixed
in 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington,
PA, USA) with 0.1% Triton X-100 (Sigma) in PBS. For experiments using
the anti-Hamlet antibody, the Triton X-100 concentration in the fixative
solution was raised to 0.3% to increase tissue permeability. Before both
primary and secondary antibody incubations, samples were blocked for 1
hour in a 1:10 solution of western blocking reagent (Roche) in PBS with
0.1% Triton X-100. Stained tissues were imaged on a Leica confocal
microscope.
DEVELOPMENT
variant. Curly+ progeny were collected and scored for the ‘double socket’
phenotype at macrochaete positions along the dorsal head and thorax, in
postorbital bristle rows and at bristle positions around the circumference of
the T1 femur. Three independent insertion lines were analyzed for each
rescue construct variant; the statistical significance of phenotypic differences
between the variant groups was evaluated by the Mann-Whitney U test.
To evaluate pIIIb daughter cell fates, the same cross was performed
except that CyO, Kr-GAL4 and UAS-GFP balancers were substituted and
rescue construct lines wt-A and 2del-B were used. Pupae lacking Kr>GFP
expression were dissected and stained at 24-30 hours APF. For each
genotype, confocal images of the microchaete field from five nota were
collected. Only positions for which a Su(H)-positive cell(s) could be easily
matched with a Pros/Pros or Pros/Elav pair were marked and counted.
RESEARCH ARTICLE
Primary antibodies used in this study were: mouse-anti-Cut [1:100;
Developmental Studies Hybridoma Bank (DSHB)] (Blochlinger et al.,
1990), mouse anti-Prospero (1:10; DSHB) (Spana and Doe, 1995), guinea
pig anti-Hamlet (1:1000) (Moore et al., 2002), guinea pig anti-Senseless
(1:2000) (Nolo et al., 2000), guinea pig anti-Numb (1:2000) (O’ConnorGiles and Skeath, 2003), rabbit anti-Su(H) (1:1000; Santa Cruz
Biotechnology), rabbit anti-CG3227 (1:2000), rabbit anti-GFP (1:500;
Invitrogen) and rat anti-Elav (1:200; DSHB). Secondary antibodies used
(all from Invitrogen) were: Alexa 555-conjugated goat anti-mouse
(1:1000), Alexa 647-conjugated goat anti-mouse (1:400), Alexa 555conjugated goat anti-guinea pig (1:400), Alexa 647-conjugated goat antiguinea pig (1:1000), Alexa 647-conjugated goat anti-rabbit (1:1000), Alexa
647-conjugated goat anti-rat (1:1000) and Alexa 488-conjugated chicken
anti-rabbit (1:1000). Hoechst stain (1:1000; Invitrogen) was used as a DNA
marker.
In situ hybridization
A 1.8 kb PCR-cloned genomic DNA fragment overlapping the last exon of
numb was cloned into EcoRI and NotI sites of pBluescript SK I and used
to transcribe a digoxygenin-labeled riboprobe (see Table S1 in the
supplementary material for PCR primer sequences). In situ hybridization
was performed as previously described (Reeves and Posakony, 2005) with
the modification that instead of the normal 1:1500 dilution of 10 mg/ml
proteinase K, a 1:25,000 dilution was used to protect protein epitopes for
detection of the Cut protein by antibody stain (above) after the in situ
hybridization step.
Mobility shift assays
Electrophoretic mobility shift assays (EMSAs) using purified GST-Su(H)
were performed as described previously (Bailey and Posakony, 1995).
Oligonucleotide probe sequences are listed in Table S1 in the
supplementary material.
Scanning electron microscopy (SEM)
Adult flies were collected for SEM and prepared as described previously
(Miller et al., 2009).
RESULTS
Experimental verification and evolutionary
conservation of computationally identified Su(H)
binding sites in numb
To obtain a preliminary assessment of the functionality of the
putative Su(H) binding site cluster identified in Drosophila
melanogaster numb by the SCORE computational method (Fig.
2A) (Rebeiz et al., 2002), we carried out two additional analyses:
an electrophoretic mobility shift assay (EMSA) and phylogenetic
footprinting (Fig. 2B,C).
The SCORE survey of the genome used a stringent definition of
the DNA-binding specificity of Su(H), requiring a sequence that
matches the motif YGTGDGAA (TGTGTGAA omitted). From
previous studies of the interactions of Su(H) with its target genes,
such sites are predicted to be bound by Su(H) protein with high
affinity (Tun et al., 1994; Bailey and Posakony, 1995; Nellesen et
al., 1999). Indeed, we found by EMSA that Su(H) efficiently bound
all five predicted sites in the numb cluster in a sequence-specific
manner (Fig. 2B).
Sequence comparisons revealed that, of the five Su(H) sites in
the cluster (designated 1a, 1b, 2, 3 and 4), three (sites 2, 3 and 4)
are precisely conserved in all 12 Drosophila species with
sequenced genomes, with the exception of a single nucleotide
change (CGTGGGAA to CGTGAGAA) in site 3 of D. virilis,
which remains compatible with high-affinity binding by Su(H)
(Fig. 2C; see Fig. S1 in the supplementary material for sequence
alignments). Site 1a is exactly conserved in 10 of the 12 species,
being absent only in the sister pair D. virilis and D. mojavensis.
Development 138 (2)
Finally, the fifth site (1b) shows conservation only among members
of the melanogaster subgroup. This site lies only eight bp away
from site 1a, a distance that is likely to preclude simultaneous
occupancy of both sites. We have previously observed similar
evolutionary instability of adjacent Su(H) sites (Castro et al., 2005).
Overall, the phylogenetic footprinting analysis indicated that four
of the five sites in the numb cluster have withstood selection
pressure for 40-60 million years, implying a functional role for
these motifs.
Enhancer activity of the Su(H) binding site cluster
region in numb
To test directly whether the Su(H) binding site cluster in the numb
intron identifies one or more functional cis-regulatory modules, we
incorporated into a GFP reporter transgene a 2.6 kb genomic DNA
fragment bearing all five of the Su(H) sites identified in silico (see
Fig. 2A). In multiple independent transgenic lines carrying this
construct, we observed GFP expression in the embryonic CNS and
PNS (data not shown); in the CNS, optic lobes, retinal field and
Johnston’s organ primordium of late third-instar larvae (data not
shown); and in pupal-stage precursor cells for the microchaete
bristles of the notum (Fig. 3A). We next tested the activity of two
smaller fragments from this 2.6 kb region: CD1, bearing sites 1a,
1b and 2; and CD2, containing sites 3 and 4 (see Fig. 2A). The 387
bp CD1 fragment directed expression in the embryonic CNS (see
Fig. S2 in the supplementary material), but displayed no activity in
the microchaete field (Fig. 3B). However, the CD2 fragment,
spanning 682 bp and containing two conserved Su(H) binding sites,
directed strong GFP expression in developing microchaetes (Fig.
3C). Like the 2.6 kb fragment, it was also active in the embryonic
CNS (see Fig. S2 in the supplementary material), the retinal field,
the larval brain and the Johnston’s organ primordium of the eyeantenna disc (data not shown). We conclude that the SCORE
method has indeed identified regions of the numb gene that display
transcriptional regulatory activity in vivo.
Cell-type specificity of the CD2 cis-regulatory
module during bristle development
To define the cell-type specificity of the numb CD2 enhancer
activity in the bristle lineage (Fig. 3K), we performed a detailed
analysis of GFP accumulation from the reporter transgene in pupal
nota at 16-18 hours APF. We used a monoclonal antibody against
the Cut protein to fluorescently label the nuclei of all cells in
developing microchaetes (Blochlinger et al., 1993).
No GFP was detected in the sense organ precursor (SOP), even
when its division was imminent (Fig. 3D). At the two-cell stage of
the lineage, however, many developing bristle organs expressed
nuclear GFP in the posterior daughter of the SOP, the pIIa cell (Fig.
3E). Other two-cell positions displayed no GFP, consistent with a
lag between the birth of the pIIa cell and the appearance of
detectable GFP fluorescence. Because we never observed GFP in
the anterior cell of a two-cell pair (pIIb), we conclude that reporter
gene transcription is first stimulated in pIIa and not in the SOP. Fig.
3F shows a later-stage two-cell position, in which the pIIb cell was
about to divide. Here, the intensity of GFP in pIIa was increased in
comparison to the early two-cell position shown in Fig. 3E. By
contrast, even as pIIb was dividing, no GFP was detectable in this
cell (Fig. 3F). Fig. 3G shows a three-cell position, in which pIIb
had already divided to yield its pIIIb and pIIIb sib daughters, and
the posterior, GFP-positive pIIa cell was about to divide, as shown
by diffuse fluorescence of both GFP and the anti-Cut antibody. In
the four-cell position shown in Fig. 3H, the pIIa cell had divided,
DEVELOPMENT
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and both of its progeny, the presumptive socket and shaft cells,
exhibited what is likely to be inherited GFP fluorescence, though
we cannot rule out the possibility that the CD2 enhancer continued
to be active in one or both of these cells. Also at the four-cell stage,
an anterior Cut-positive cell had also begun accumulating GFP
(Fig. 3H). Based on its anterior location, size and apical
disposition, this cell is putatively pIIIb. The transcription factor
Hamlet (Ham) is first expressed in the microchaete lineage in pIIIb,
and has been shown to be a crucial regulator of the fates of its
progeny (Moore et al., 2004). Staining pupal nota at 17 hours APF
with anti-Ham antibody confirmed that the newly GFP-positive cell
was indeed pIIIb (Fig. 3J). Finally, once pIIIb divides, GFP is
inherited by its progeny, the presumptive sheath cell and neuron
(though, again, we cannot exclude continued enhancer activity in
these cells). Thus, when the microchaete lineage divisions had been
completed, a total of four GFP-positive cells were observed (Fig.
3I), representing the initial activation of the numb CD2 enhancer
in the pIIa and pIIIb cells, and (at least) the perdurance of GFP in
their postmitotic progeny. It is also clear from our analysis that the
pIIIb sib cell and its precursors within the lineage (pIIb, SOP) do
not activate the CD2 enhancer, as no GFP was ever observed in
these cells. The cells that did activate novel transcription directed
by the enhancer (pIIa and pIIIb) were Notch-responsive cells,
consistent with the presence of two conserved Su(H) binding sites
in the CD2 fragment.
numb transcript accumulation in Notch-responsive
cells of the bristle lineage
The foregoing analysis of the activity of the CD2 enhancer
fragment suggests that numb might be subject to selective
transcriptional activation in the Notch-responsive precursor cells
of the bristle lineage. To investigate this question, we carried out
DEVELOPMENT
Fig. 3. The numb CD2 enhancer is active in the pIIa and pIIIb cells of the mechanosensory bristle lineage in Drosophila. (A-C)Thoracic
microchaete field at 24 hours after puparium formation (APF), after all cell divisions in the bristle lineage have been completed. GFP reporter gene
activity is shown in green. (A)A 2.6 kb genomic DNA fragment containing the Su(H) binding site cluster (Fig. 2A) displays enhancer activity in the
bristle lineage. (B)CD1, a 387 bp subfragment containing three Su(H) sites (Fig. 2A), fails to direct reporter gene expression in the bristle lineage.
(C)CD2, a 682 bp subfragment containing two Su(H) sites (see Fig. 2A), recapitulates the activity of the parent 2.6 kb fragment. (D-J)Temporal
analysis of CD2 reporter gene activity (GFP, green) in the bristle lineage (see K). Anti-Cut antibody (blue) labels all cells of the lineage. Schematic
diagrams show the inferred identity of each cell (dotted lines). (D)No reporter gene expression is evident at the one-cell (SOP) stage (D). Following
division of the SOP, GFP is expressed in the posterior pIIa cell (E,F), but is absent from the anterior pIIb cell, even as it nears division (div) (F). As the
pIIa cell divides (G), GFP is distributed to its daughters, the presumptive socket (so) and shaft (sh) cell pair (H). GFP then accumulates in one of the
anterior cells, presumably pIIIb, based on its larger size relative to the pIIIb sib cell (H). When pIIIb divides, GFP is distributed to its progeny, the
presumptive neuron (ne) and sheath (st) cells (I). Anti-Hamlet labeling of a four-cell microchaete position, demonstrating that the anterior GFPpositive cell at this stage is pIIIb, which specifically expresses Hamlet (J, red). (K)Schematic of the cell lineage of notum microchaetes. The pIIa and
pIIIb cells are highlighted in red. See also Fig. S2 in the supplementary material.
RESEARCH ARTICLE
in situ hybridization assays on pupal nota at 16 hours APF, when
most microchaete organs were at the two- to three-cell stage of
bristle development (Fig. 4). We found that, at this time, evenly
spaced cells or cell clusters specifically accumulated elevated
levels of numb transcript (Fig. 4A; see Fig. S3 in the
supplementary material). The spacing of these strongly
expressing cells was reminiscent of the microchaete pattern in
the notum. numb transcript was also observed at low levels
across the epidermal field (Fig. 4A; see Fig. S3 in the
supplementary material).
To assess whether the strongly expressing cells belong to the
microchaete lineage, the in situ-hybridized tissue was also labeled
fluorescently with anti-Cut antibody. Cut-positive SOP positions
showed only the low epidermal levels of numb transcript
accumulation. However, a number of two-cell positions displayed
strong numb transcript signals in the more posterior Cut-labeled
cell, which is pIIa (Fig. 4B-B⬙). In a subset of three-cell positions,
a small apical anterior Cut-positive nucleus was seen to be
surrounded by cytoplasmic numb transcript (Fig. 4C-C⬙). Based on
the size and location of this cell, we interpret it to be pIIIb.
Therefore, strong transcript accumulation from the endogenous
numb gene was observed in the pIIa cell and probably the pIIIb
cell, but not in the SOP or pIIb cells. Our expression analysis thus
shows that elevated numb transcript levels appear in the bristle
lineage in a pattern consistent with the activity of the CD2
enhancer fragment, i.e. in those precursor cells with Notchspecified fates.
Development 138 (2)
sites resulted in the appearance of robust ectopic reporter gene
expression in pIIb, without severely affecting its expression in pIIa
(Fig. 5). This result suggests that the transcriptional activation
function of Su(H) does not provide the principal contribution to the
activity of the enhancer in pIIa; instead, expression is evidently
driven largely by other activators bound to the module.
Significantly, however, the experiment also reveals that Su(H) does
indeed act as a ‘default repressor’ of the CD2 enhancer (Barolo and
Posakony, 2002). This means that, immediately following the SOP
division, Su(H) in its repressive mode would act to prevent CD2
activation in both pIIa and pIIb. The repressive state would persist
in pIIb, but would be relieved in pIIa by the Notch signaling event
that specifies the pIIa fate, thus permitting the enhancer to drive
numb transcription specifically in the latter cell.
Default repression by Su(H) is required for the
normal pattern of numb CD2 enhancer activity
The presence of two highly conserved Su(H) binding sites within
the numb CD2 enhancer fragment suggests that the specific
activation of the enhancer in response to Notch signaling in pIIa and
pIIIb is dependent on Su(H). Introduction of single-base mutations
(YGTGDGAA to YGTGDCAA) into both of the Su(H) binding
Activation of the numb CD2 enhancer module
depends on relief of Su(H)-mediated repression by
Notch signaling
The data presented thus far are consistent with the model that
numb transcription is stimulated by the CD2 enhancer in
response to the asymmetric Notch signaling events that specify
the fates of the pIIa and pIIIb precursor cells. We sought to test
directly whether Notch signaling is necessary for activation of
the CD2 enhancer module.
We first wanted to show that Su(H), as the transducing
transcription factor for the Notch pathway, is required in trans for
the cell type-specific activity of the enhancer, as implied by the
Su(H)-binding-site-mutation experiment just described (Fig. 5). We
used a pannier (pnr)-GAL4 driver to express a Su(H) short-hairpin
RNAi construct in the central region of the pupal notum; the
flanking regions served as the control tissue (Fig. 6A). This
treatment resulted in a complete ‘balding’ phenotype in the adult
thorax (see Fig. S4 in the supplementary material) mimicking that
observed in Su(H)-mutant mosaic territories (Schweisguth, 1995),
indicating its effectiveness in reducing Su(H) gene activity.
Fig. 4. numb transcript accumulates at higher levels in specific
cells of the bristle lineage in Drosophila. (A)At 16 hours after
puparium formation (APF), elevated levels of transcript from the
endogenous numb gene accumulate at discrete positions within the
microchaete field of the developing notum, against a background of
low-level ubiquitous expression. Anterior is at the top; image is
centered on the notum midline and shows two symmetrical
microchaete rows. See Fig. S3 in the supplementary material for a
lower-magnification view of the full notum. (B-B⬙) numb transcript
(B⬘,B⬙) accumulates specifically in the posterior pIIa cell, identified by
anti-Cut staining of a developing microchaete at the two-cell stage (red
in B and B⬙; see Fig. 3K for a lineage diagram). (C-C⬙) numb transcript
accumulation (C⬘,C⬙) in the pIIIb cell, identified by anti-Cut staining of a
developing microchaete at the three-cell stage (red in C and C⬙). The
anterior pIIb cell has divided into the pIIIb and pIIIb sib cells. The larger
pIIIb cell accumulates numb transcript, whereas the smaller, apical pIIIb
sib cell does not (C⬘,C⬙).
Fig. 5. Mutation of the Su(H) binding sites in the numb CD2
enhancer yields ectopic activity in the Notch-independent pIIb
cell. (A-B⬙) Developing Drosophila microchaetes at the two-cell stage,
marked by Cut immunoreactivity (A,B; magenta in merge panels A⬙ and
B⬙). The posterior pIIa cell and anterior pIIb cell are indicated. (A⬘,A⬙)
The wild-type numb CD2 enhancer-reporter transgene (GFP; green in
A⬙) is active only in Notch-dependent pIIa. (B⬘,B⬙) numb CD2 reporter
gene bearing single-base mutations in its two Su(H) binding sites (GFP;
green in B⬙) is active in both pIIa and pIIb.
DEVELOPMENT
220
Notch regulates numb
RESEARCH ARTICLE
221
The numb CD2 enhancer is required for proper
specification of the shaft and neuron cell fates
The specific activation of the numb CD2 enhancer in the pIIa and
pIIIb precursor cells of the bristle lineage suggested to us that it
might have a role in specifying the fates of their Numb-inheriting
progeny cells, the shaft cell and neuron, respectively. We tested this
prediction by carrying out rescue experiments using genomic DNA
transgenes containing the numb locus. We found that a 20 kb
fragment that includes the proximal (zygotic) promoter and entire
coding region, along with substantial amounts of 5⬘ and 3⬘ flanking
sequence (Fig. 2A), efficiently rescued the lethality associated with
two different numb loss-of-function genotypes (Tweedie et al.,
2009). Moreover, adult mechanosensory organ development was
almost completely normal in numb mutant flies bearing the wildtype rescue fragment (Fig. 7F; see Table S2 in the supplementary
material). By contrast, mutant flies bearing a version of this
construct in which the numb CD2 enhancer had been deleted
(Fig. 2A) displayed a widespread ‘double socket’ phenotype, in
which the shaft cell was transformed into a second socket cell
(Fig. 7A-F; see Table S2 in the supplementary material). Similarly,
pupal-stage nota of CD2 enhancer-deleted animals showed frequent
transformation of the sensory neuron into a second sheath cell
(Fig. 7G,H). These results demonstrate that the function of the
CD2 enhancer module is required for the normal specification of
the numb-dependent, Notch-independent shaft and neuron cell
fates.
Fig. 6. Response of the numb CD2 enhancer to perturbations in
Notch signaling. (A-E⵮) CD2-GFP reporter gene expression (green) in
the genotype w; CD2-GFP/+; pnr-GAL4/UAS-Su(H)shRNA. (A,A’) Widefield view of pupal notum, with approximate boundary between GAL4expressing and non-expressing territories denoted by a dashed line, as
indicated above panel A. Positions enlarged in subsequent panels are
labeled. (B-C⵮) GFP expression in progeny cells following the SOP
division in either the GAL4-non-expressing (B-B⵮) or GAL4-expressing
(C-C⵮) territory. (D-E⵮) GFP expression in progeny cells following the
pIIb division in either the GAL4-non-expressing (D-D⵮) or GAL4expressing (E-E⵮) territory. (F-F⵮)Lack of GFP expression at the two-cell
stage in a Nts1/N81k1 background following temperature shift. Cells are
marked by anti-Sens (blue in A-F) and anti-Pros (red in A-F)
immunoreactivity. GFP is also absent from four-cell Pros-positive
positions (data not shown). Arrows in B-C⵮ and F-F⵮ indicate cells
adopting the pIIb fate. Arrowheads in D-E⵮ indicate cells adopting the
pIIIb sib fate.
DEVELOPMENT
At the two-cell stage, Su(H) RNAi caused both of the progeny
of the SOP to adopt the pIIb fate, owing to a failure of Notch signal
transduction in the pIIa cell (Fig. 6B,C). Nevertheless, as expected
from the result presented in Fig. 5, both of these cells expressed
GFP from the CD2 enhancer-reporter transgene (Fig. 6C⬙), as
Su(H) was no longer able to repress the enhancer in either cell.
This experiment also suggests that Su(H) does normally make
some contribution to the activation of the enhancer in pIIa, as the
Su(H) RNAi treatment appeared to reduce the level of GFP
accumulation in this cell relative to that observed in the wild-type
territory (compare Fig. 6B⬙ and 6C⬙). Later, following the division
of pIIb [which yields three cells in wild-type territory and four cells
in the Su(H) RNAi domain], GFP from the CD2 reporter appeared
only in pIIa and pIIIb at wild-type positions, but was expressed in
all four cells (two ‘pIIIb’ and two pIIIb sib cells) where Su(H)
RNAi was active (Fig. 6D,E).
We directly investigated the role of Notch signaling in activating
the CD2 enhancer by examining expression of the reporter gene in
notum tissue of female pupae bearing the temperature-sensitive
allele Nts1 in trans to a Notch null allele (Nts1/N81k1). Exposure of
these animals to 37°C for two hours at the appropriate time caused
a failure of the Notch signaling event that specifies the pIIa cell,
yielding two-cell positions in which both daughters of the SOP
have adopted the pIIb fate (Fig. 6F) (Hartenstein and Posakony,
1990; Posakony, 1994). Here, in contrast to what was observed
when Su(H) activity was reduced (Fig. 6C⬙), neither cell expressed
the CD2 reporter (Fig. 6F⬙). Combining the results presented in
Figs 5 and 6, we conclude that Notch signaling is indeed required
to activate the CD2 enhancer specifically in pIIa, and that it does
this, in part, by relieving Su(H)-mediated repression. We confirmed
the role of Notch signaling in CD2 enhancer activation in a
separate experiment in which overexpression of Numb was used to
abrogate Notch signaling activity throughout the sensory organ
lineage (Reddy and Rodrigues, 1999a) (see Figs S5 and S6 in the
supplementary material).
222
RESEARCH ARTICLE
Development 138 (2)
Fig. 7. Deletion of the numb CD2 enhancer causes
shaft-to-socket and neuron-to-sheath cell fate
transformations. (A-E)Scanning electron micrograph
images of numb796/Df(2L)30A-C; numbRC2del/+ flies,
showing the appearance of a ‘double socket’
phenotype at multiple bristle positions across the body.
(A)Head vibrissae; anterior is to the left. (B)Inner
vertical (left)/outer vertical (right) pair. (C)Bristle
position on the T1 femur. (D)Postorbital positions on
the posterior head. (E)Bristles on the anterior wing
margin also show the double socket phenotype. In A,
B, D and E, arrowheads point to bristle positions
showing a double socket phenotype; arrows indicate
positions at which the shaft fate has been properly
specified. (F)Quantification of bristle phenotypes
observed when either the numbRCwt (left half of the
graph) or the numbRC2del (right half of the graph)
rescue transgene is present in either a numb796/numb1
(‘796/1’) or a numb796/Df(2L)30A-C (‘796/Df’)
background. Displayed are the results for three
independent insertions of numbRCwt (lines A, C and
D) and numbRC2del (lines A, B and D). Bristles
positions scored were head and thorax dorsal
macrochaetes, postorbital bristles and T1 femur
positions, as indicated. numbRC2del results are highly
significantly different from the numbRCwt results by
the Mann-Whitney U test for both numb1 and
Df(2L)30A-C (P<0.001). See also Table S2 in the
supplementary material. (G,H)Illustration and
quantification of the effect of deleting the CD2
enhancer on the fates of the progeny of pIIIb.
Microchaete positions in pupal nota at 24-30 hours
APF are marked with anti-Su(H) (green; socket cell),
anti-Elav (blue; neuron) and anti-Pros (red; sheath cell)
antibodies. In numb796/Df(2L)30A-C animals bearing
one copy of the wild-type numb rescue construct
(RC-wt), no ‘double sheath’ positions are observed (G),
whereas those carrying one copy of the CD2 deletion
construct (RC-2del) show a significant prevalence of
this phenotype, reflecting a failure to specify the
numb-dependent neuron fate (H).
clusters. This wide range allows the detection of local maxima that
do not necessarily conform to the size expected for a canonical cisregulatory module. Judging from the present study, the unbiased
window-size approach might permit functional enhancer elements
to be detected owing to the proximity of multiple enhancers with
similar binding inputs. In any case, the SCORE technique
successfully identified a functional cis-regulatory module within
the ~50 kb of non-coding DNA within and surrounding numb.
Role of the Notch-activated numb CD2 enhancer in
the specification of the shaft and neuron cell
fates
We have shown here that a 20 kb genomic DNA fragment is
capable of nearly complete phenotypic rescue of two different
numb loss-of-function genotypes, and that deletion of the numb
CD2 enhancer from this fragment results in widespread ‘double
socket’ and ‘double sheath’ phenotypes, reflecting a failure to
specify the numb-dependent shaft and neuron cell fates. Thus,
transcriptional activation of numb in the pIIa and pIIIb precursor
cells, in response to the Notch signaling events that specify their
respective fates, plays an important role in the proper specification
of the Notch-independent progeny cell fate.
DEVELOPMENT
DISCUSSION
Successful computational identification of cisregulatory modules based on binding site
clustering
The transcriptional regulation of the numb gene has not previously
received much attention because most experimental efforts have
been focused on Numb protein localization, asymmetric
segregation and function as a Notch pathway inhibitor. The
motivation for the present study originated in a computational
search of the fly genome for new Notch pathway target genes
based on statistically significant clustering of Su(H) binding sites
(Rebeiz et al., 2002). Although it has been suggested that
homotypic site clustering is not a general property of cis-regulatory
modules in Drosophila, and therefore that this parameter is of
limited utility in computational prediction of enhancers (Li et al.,
2007), the data that we have presented here and in other reports
(Bailey and Posakony, 1995; Nellesen et al., 1999; Lai et al., 2000;
Rebeiz et al., 2002; Castro et al., 2005) indicate that this approach
can be quite effective in the case of Su(H) and other transcription
factors. One beneficial feature of our SCORE method (Rebeiz et
al., 2002) is the use of a largely unbiased window size (100-5000
bp) for the identification of statistically significant binding site
Given the high proportion of sensory organs in which the shaft
and neuron cell fates are correctly specified in the absence of the
CD2 enhancer, it seems clear that CD2 is not the only source of
Numb for pIIa and pIIIb. We confirmed this inference directly by
detecting Numb crescents in dividing pIIa cells in tissue lacking
CD2 function, having first demonstrated that the numb796 allele is
protein-null (see Fig. S7 in the supplementary material).
What might be the source of this additional Numb protein? It is,
of course, possible that numb is served by a second enhancer
module that also contributes to the transcriptional activation of the
gene in pIIa and pIIIb in response to Notch signaling; there is
substantial precedent for such ‘shadow’ or ‘secondary’ enhancers
in insects (Hong et al., 2008; Frankel et al., 2010). However, it is
very likely that the basal level of Numb protein that is detected in
all cells in the epidermis (Rhyu et al., 1994) also accumulates in
developing sensory organ cells, including pIIa and pIIIb,
independently of the CD2 enhancer. This protein would
presumably be segregated by the two precursor cells to their shaft
and neuron daughter cells, respectively, and might suffice, in most
cases, to inhibit Notch signaling in those cells.
What, then, would generate the need for the numb CD2 enhancer
activity? Integrating all of our findings, we currently favor the
following evolutionary scenario. Among the cells in the bristle
lineage, the pIIa and pIIIb precursors face a unique challenge:
because their own fates are specified by Notch signaling, it is
crucial that they do not inherit Numb, yet each must make
sufficient Numb to distribute asymmetrically to one of their
progeny cells (Rhyu et al., 1994). In an ancestral sensory organ
lineage, the ubiquitous basal level of Numb accumulation might
have been adequate to supply the needs of pIIa and pIIIb. But,
perhaps as the execution of the lineage became faster in some
rapidly developing insects [the time from birth to division for pIIa
and pIIIb is only 3-4 hours in Drosophila (Hartenstein and
Posakony, 1989; Reddy and Rodrigues, 1999b)], Numb
accumulation in these cells failed to meet the required threshold,
resulting in unacceptably high failure rates in shaft cell and neuron
specification. The emergence of the CD2 enhancer would then
have offered the selective advantage of supplementing the basal
Numb specifically in these two Notch-dependent precursor cells,
without elevating the global activity of the gene. In this scenario,
CD2 represents an evolutionary adaptation for ensuring the fidelity
of two cell fate decisions during mechanosensory organ
development.
Integration of conditional and autonomous
modes of cell fate specification
The Drosophila external sensory organ lineage has stood for
many years as an elegant example of the integration of
conditional and autonomous mechanisms of cell fate
specification (Posakony, 1994). The repeated use of a
combination of bi-directional Notch signaling between sister
cells and asymmetric segregation of the Notch pathway
antagonist Numb is a highly effective strategy for ensuring the
proper specification of cell fates in a succession of asymmetric
cell divisions. This is particularly so because the orientation of
the mitotic spindles and the segregation of Numb are tied to the
planar polarity system (Bellaiche et al., 2001), such that the
appropriate fate is assigned to the appropriate daughter with
extremely high fidelity. The results reported here bring this
Notch-Numb partnership full circle by demonstrating that a
reciprocal regulatory linkage also exists: Notch signaling
regulates numb (Fig. 8).
RESEARCH ARTICLE
223
Fig. 8. Model for the Notch-stimulated activation of numb
transcription in the pIIa precursor cell. (A)As the Notchindependent SOP prepares to divide, it segregates Numb protein (red)
in a crescent to its anterior side (right). Su(H) binds the CD2 enhancer
module and represses it (OFF). (B)Immediately after the division of the
SOP, pre-loaded transcriptional activator proteins (ACT) are bound to
the CD2 enhancer in both daughter cells. Su(H) in its repressor mode
keeps the enhancer OFF in both cells. The anterior daughter, the
presumptive pIIb, has inherited Numb from the SOP (red ring). (C)Bidirectional Notch (N) signaling between the two daughter cells then
specifies the pIIa fate (black horizontal arrow) and relieves ‘default
repression’ of the CD2 enhancer by Su(H) in this cell, permitting the
bound ACT proteins to activate numb transcription (ON) in partnership
with activated Su(H). Inherited Numb protein in pIIb renders this cell
unresponsive to the reciprocal Notch signal (gray horizontal arrow), and
Su(H) continues to repress the enhancer. (D-F)Interpretation of
experiments with the GFP reporter gene (see Figs 5, 6). (D)Mutation of
the two binding sites in the CD2 module prevents repression by Su(H),
leaving ACT-stimulated reporter activity in pIIa and permitting ectopic
activation of the reporter in pIIb. (E)Loss of Su(H) gene function causes
cell-autonomous failure of pIIa specification (so that both sisters adopt
the pIIb fate) and activation of the wild-type CD2 reporter gene in both
cells [due to loss of Su(H)-mediated repression]. (F)Loss of Notch
function likewise causes cell-autonomous failure of pIIa specification,
but the wild-type CD2 reporter remains off in both cells owing to
continued repression by Su(H).
DEVELOPMENT
Notch regulates numb
RESEARCH ARTICLE
We have shown that, although Notch signaling is essential to the
activation of the numb bristle enhancer, the transcriptional
activation function of Su(H) is not strictly required for enhancer
activity. Accordingly, we suggest that Notch signaling acts here in
large part as a trigger, relieving Su(H)-mediated ‘default
repression’ and permitting other activators bound to the enhancer
to drive numb transcription (Fig. 8). Some or all of these activators
are likely to be expressed in both pIIa and pIIb, as implied by the
nearly equivalent level of reporter gene activity observed in the two
cells when the Su(H) binding sites of the enhancer are mutated. We
further suggest that this regulatory strategy is relevant to the
question of timing raised earlier. Having Notch signaling act as a
trigger for the action of a pre-assembled complex of other
activators might help to ensure that the transcriptional response is
very rapid, allowing sufficient numb mRNA to be accumulated and
translated in pIIa and pIIIb before they divide.
Acknowledgements
We are grateful to Feng Liu for constructing and providing the UASSu(H)shRNA lines and to Ben Haley for providing the pNE3 vector. We thank
Tammie Stone for performing the EMSAs. Adrian Moore and the laboratory of
Yuh Nung Jan kindly provided anti-Hamlet antibody and fly stocks, respectively.
The SEM images were collected at San Diego State University with generous
help from Steve Barlow. S.W.M. received support from NIH predoctoral training
grant GM07240. This work was supported by NIH grant GM046993 to J.W.P.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.050161/-/DC1
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