Download Only a subset of the binary cell fate decisions

4435
Development 124, 4435-4446 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV8456
Only a subset of the binary cell fate decisions mediated by Numb/Notch
signaling in Drosophila sensory organ lineage requires Suppressor of
Hairless
Shuwen Wang, Susan Younger-Shepherd, Lily Y. Jan and Yuh Nung Jan*
Howard Hughes Medical Institute, and Departments of Physiology and Biochemistry, University of California, San Francisco, San
Francisco, California 94143-0724, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
In Drosophila, an adult external sensory organ (bristle)
consists of four distinct cells which arise from a sensory
organ precursor cell via two rounds of asymmetric
divisions. The sensory organ precursor cell first divides to
generate two secondary precursor cells, IIa and IIb. The
IIa cell then divides to produce the hair cell and the socket
cell. Shortly after, the IIb cell divides to generate the
neuron and the sheath cell. The membrane-associated
protein Numb has been shown to be required for the first
two asymmetric divisions. We now report that a new hypomorphic numb mutant not only displays a double-socket
phenotype, due to a hair cell to socket cell transformation,
but also a double-sheath phenotype, due to a neuron to
sheath cell transformation. This provides direct evidence
that numb functions in the neuron/sheath cell lineage as
well. Those results, together with our observation from
immunofluorescence analysis that Numb forms a crescent
in the dividing IIa and IIb cells suggest that asymmetric
localization of Numb is important for the cell fate determination in all three asymmetric cell divisions in the
sensory organ lineage. Interestingly, we found that in the
hair/socket cell lineage but not the neuron/sheath cell
lineage, a Suppressor of Hairless mutation acts as a
dominant suppressor of numb mutations whereas Hairless
mutations act as enhancers of numb. Moreover, epistasis
analysis indicates that Suppressor of Hairless acts downstream of numb, and results from in vitro binding analysis
suggest that the genetic interaction between numb and
Hairless may occur through direct protein-protein interaction. These studies reveal that Suppressor of Hairless is
required for only a subset of the asymmetric divisions that
depend on the function of numb and Notch.
INTRODUCTION
cell then divides to produce two outer support cells, the hair
cell and the socket cell. Shortly after the IIa cell division, the
IIb cell divides to produce two inner cells, the neuron and the
sheath cell (Fig. 5A).
Previous studies of the Drosophila embryonic and adult PNS
suggest that the gene numb plays a key role in generating
asymmetry in the SOP lineage (Uemura et al., 1989; Rhyu et
al., 1994). Immunocytochemical studies suggest that Numb is
a membrane-associated protein and is asymmetrically localized
in the dividing SOP cell. Upon SOP division, Numb is preferentially segregated into the IIb cell (Rhyu et al., 1994). In numb
loss-of-function mutants, the SOP cell divides symmetrically
to produce two IIa cells, which produce four outer support cells
(Uemura et al., 1989). Ectopic expression of Numb causes the
opposite cell fate transformation in embryos (Rhyu et al.,
1994). The generation of the four different cells of a sensory
bristle in adult flies also requires the function of numb. Mosaic
analysis reveals that the most extreme numb loss-of-function
phenotype is the formation of four socket cells. This foursocket phenotype arises from the abnormal symmetric division
of SOP to produce two IIa cells and the abnormal symmetric
Asymmetric cell division is a fundamental process for the generation of daughter cells with different fates. Two types of
mechanisms may be used in generating asymmetric cell
divisions, an extrinsic mechanism and an intrinsic mechanism.
The intrinsic mechanism involves the segregation of an
inherited determinant preferentially into one of the daughter
cells. The extrinsic mechanism may be executed through cellcell communication (reviewed by Horvitz and Herskowitz,
1992). In Drosophila, both mechanisms are used to generate
the distinct fates of the cells within an adult sensory organ
which, like the external sensory organ in the embryonic peripheral nervous system (PNS), is derived from a single sensory
organ precursor (SOP) cell (Hartenstein and Posakony, 1989).
A simple sensory organ comprises four cells with distinct fates:
a neuron, a sheath cell, a socket cell and a hair cell (Fig. 1A).
To generate these different cells of a sensory organ, the SOP
cell follows a stereotyped pattern of asymmetric cell divisions
(Hartenstein and Posakony, 1989). The SOP cell first divides
to generate two secondary precursor cells, IIa and IIb. The IIa
Key words: numb, Suppressor of Hairless, cell fate, neuron, sheath,
sensory organ, Drosophila
4436 S. Wang and others
Fig. 1. The numbSW allele exhibits
double-socket phenotype in the adult
sensory organs. (A) Schematic view of
a sensory bristle structure. (B) Macrochaete and microchaete bristles from a wild-type fly notum. The anterior (aDC) and posterior (pDC)
dorsal central macrochaete bristles are as indicated. (C) Macrochaete and microchaete bristles from a numbSW/numb2 fly notum. Note the
double-sockets of one aDC and two pDCs as indicated by the black arrowheads. (D,E) Wing margin bristles from a wild-type and a
numbSW/numb2 mutant fly, respectively. (F) Higher magnification of the boxed region in (C), showing a aDC double-socket (black arrowhead)
and a microchaete double-socket (white arrowhead).
division of each IIa cell to produce two socket cells (Rhyu et
al., 1994). The transformation of IIb to IIa has therefore
precluded the examination of the role of numb in the asymmetric division of the IIb cell in numb null mutants.
Besides the cell-intrinsic mechanism mediated by numb, a
cell-extrinsic mechanism mediated by Notch and Delta is also
used for asymmetric divisions in the SOP lineage (Hartenstein
and Posakony, 1990; Parks and Muskavitch, 1993). Analyses
of temperature-sensitive mutants of Notch and Delta reveal that
reduction of Notch or Delta function during the first SOP
division causes both daughter cells to assume the IIb cell fate,
which then divide symmetrically to produce four neurons
(Hartenstein and Posakony, 1990; Parks and Muskavitch,
1993). In addition, Notch is also involved in both the IIa cell
division and the IIb cell division, affecting the cell fates of both
the socket/hair lineage and the neuron/sheath lineage (Guo et
al., 1996). Notch and Delta probably mediate cell-cell communication through a receptor-ligand interaction in which
Notch acts as the receptor and Delta as a ligand (Heitzler and
Simpson, 1991; Rebay et al., 1991), and Numb acts by suppressing Notch activity during embryonic sensory organ development, probably via direct protein-protein interactions
between Numb and Notch (Guo et al., 1996).
In addition to their function in the lateral inhibition step
during the SOP formation, the genes Suppressor of Hairless
(Su(H)) and Hairless (H) help control the fates of adult sensory
organ cells (Lees and Waddington, 1942; Bang et al., 1991;
Bang and Posakony, 1992; Furukawa et al., 1992; Schweisguth
and Posakony, 1992; Schweisguth, 1995). Su(H) is a potent
dominant suppressor of H. Loss of function or overexpression
of Su(H) during SOP divisions causes the IIa cell to divide
symmetrically into two hair cells or two socket cells, respectively (Schweisguth and Posakony, 1992). Conversely, loss of
function or overexpression of H exhibits a two-socket or twohair phenotype, respectively (Bang et al., 1991; Bang and
Posakony, 1992). Shortly after the division of the IIa cell, the
Su(H) protein specifically accumulates in the nucleus of the
socket cell (Gho et al., 1996). These results suggest that Su(H)
is required for socket cell formation and H is required for hair
cell formation.
There is growing evidence suggesting that Su(H) functions
in the Notch signaling pathway. Both Su(H) and H show allelespecific genetic interactions with Notch and Delta during SOP
formation (Vaessin et al., 1985; de la Concha et al., 1988;
Fortini and Artavanis-Tsakonas, 1994). The Su(H) gene
encodes a protein that is highly homologous to the mammalian
RBP-J κ DNA-binding protein (Furukawa et al., 1992;
Schweisguth and Posakony, 1992), and by binding specifically
to sites in the promoter of the genes of the Enhancer of Split
(E(spl)) complex, acts as a transcriptional activator of these
genes (Bailey and Posakony, 1995; Lecourtis and Schweisguth,
1995). Activated Notch causes transcriptional activation of
these E(spl) genes in transgenic flies, and this activation
requires Su(H), providing further evidence for the participation
of Su(H) in Notch signaling (Bailey and Posakony, 1995;
Lecourtis and Schweisguth, 1995). A functional link between
Su(H) and Notch has also been established in cultured
Drosophila cells. When expressed in the S2 cells, the Su(H)
protein is localized to the nucleus. Coexpression of Su(H) and
Notch causes Su(H) protein to be retained in the cytoplasm,
probably due to direct binding of Su(H) to Notch. Upon activation of Notch by its ligand Delta, the Su(H) protein translocates to the nucleus (Fortini and Artavanis-Tsakonas, 1994).
This ability of activated Notch to cause translocation of Su(H)
protein is inhibited by Numb (Frise et al., 1996).
It has been proposed that Su(H) is involved in all three binary
decisions in the sense organ lineage (Posakony, 1994;
Schweisguth, 1995), implying that Notch signaling is transduced by Su(H) in all three binary decisions. However,
Schweisguth and Posakony (1994) did not observe a sheath-toneuron transformation in flies carrying extra copies of Su(H).
This result by itself does not prove but does raise the possibility that Su(H) is not required for the neuron/sheath cell fate
decisions. It is therefore important to resolve whether Su(H) is
indeed involved in all binary cell fate decisions mediated by
Notch and numb.
In this report, we first present the characterization of a hypomorphic mutant of numb, which exhibits both a hair-to-socket
transformation and a neuron-to-sheath cell transformation. We
then show that Su(H) is epistatic to numb and that a Su(H)
Interaction of Su(H) with numb 4437
mutation acts as a dominant suppressor of the numb
hypomorph while a H mutation acts as an enhancer of the numb
hypomorph. Moreover, the interactions of Su(H) and H with
numb only occur in the hair/socket cell lineage, indicating that
Su(H) and H are not required in determining the neuron/sheath
cell lineage. Further, in vitro binding analysis suggests that the
genetic interaction between numb and H probably occurs
through physical association of their protein products.
MATERIALS AND METHODS
Drosophila strains and genetics
Drosophila strains were raised on standard cornmeal-yeast agar
medium at 25°C or at room temperature. The three mutant alleles of
numb (numb1, numb2, and numb3) and the deficiency that uncovers
the numb locus (Df(2L)A-C) are described by Uemura et al. (1989).
The null mutation of Su(H) (Su(H)SF8) was as described by
Schweisguth and Posakony (1992). The four haploinsufficient alleles
of H (HB55, HDEB1, HB521 and HBD9) were isolated and characterized
by E. Grell (E. Grell, personal communication).
The numbSW allele was isolated through an F1 revertant screen of
the P-element-induced numb1 allele. Dysgenic males of the genotype
numb1 /CyO; Dr ∆ 2-3/+ were crossed to numb2 pr cn Elp/CyO
females. The F1 Cy+ progeny were collected and examined for
abnormal sensory bristle phenotypes on the fly nota. Each candidate
with abnormal bristles was retested for reproducible bristle defects by
crossing to the other numb alleles. One mutation (numbSW allele) was
isolated from screening 3600 F1 Cy+ progeny. This numbSW allele
displayed a double-socket bristle phenotype when trans-heterozygous
to other numb mutations.
All four alleles of numb were balanced over the second chromosome balancer CyO. To examine the bristle phenotypes, numbSW /CyO
flies were mated to numbX /CyO flies and the Cy+ flies were scored
for sensory bristle defects (X represents 1, 2, 3, or the Df(2L)A-C). To
study the inner cell fates of the numbSW mutant nota, we crossed
homozygous numbSW flies to numb2 pr cn Bc/CyO flies. Third instar
larvae that displayed the marker Black cell (Bc) were selected and
maintained at room temperature or 25°C till the formation of white
prepupae. After staging, the prepupae were dissected and stained with
antibodies that recognize the inner cells of each sensory organ.
For studying the bristle phenotypes caused by overexpression of
numb, the homozygous UAS-numb#34 flies were crossed to the GAL4
enhancer-trap line Gal4109-68/CyO, and Cy+ flies were analyzed.
Prepupae were collected from the same cross for pupal notum
antibody staining of the inner cells of each sensory organ.
The genetic interaction analysis between numbSW and Su(H)SF8 was
carried out as follows. The numb2 allele and Su(H)SF8 allele were first
recombined onto the same chromosome, generating flies of the
genotype yw; numb2Su(H)SF8 /CyO p[y+, ry+]. Homozygous yw;
numbSW flies were crossed to yw; numb2Su(H)SF8 /CyO p[y+, ry+] flies
and Cy+ flies were examined for phenotypes. For inner sense organ
cell analysis, third instar larvae with yellow mouth hooks (y−) were
selected and staged to prepupae for pupal notum staining.
Each of the four alleles of H was double balanced to numbSW using
CyO and TM6 p[y+, ry+], generating flies of the genotype yw; numbSW
/CyO; H/TM6 p[y+, ry+]. Females homozygous for yw; numbSW were
then crossed to yw; numbSW /CyO; H/TM6 p[y+, ry+] males. Bristle
phenotypes were scored in Cy+ and y flies (yw; numbSW ; H/+) as
compared to Cy+ and y+ flies (yw; numbSW ; +/TM6 p[y+, ry+]).
Double mutant mosaic analysis
For the loss-of-function mitotic recombinant clones in adults and
pupae, two different GAL4 enhancer trap lines, Gal4109-68 (Frise et
al., 1996; Guo et al., 1996) and sca-Gal4 (Nakao and Campos-Ortega,
1996) were used to drive UAS-FLP recombinase expression in the
sensory organ lineage. For adult notum yellow (y) and crinkled (ck)
marked clones, we mated yw UAS-FLP; p[y+, ry+]VC1 ck FRT40A
Gal4109-68/CyO females to yw; numb2 Su(H) SF8 FRT40A /CyO and yw;
A1-2-29 Su(H) SF8 FRT40A /CyO males. Marked numb null mutant
clones were generated by using two independent UAS-FLP transgenes: (1) p[UAS-FLP, w+] (a gift from J. Duffy, N. Perrimon and D.
Harrison; Zeng et al. personal communication) was used by mating
yw UAS-FLP; p[π M, w+] FRT40A to yw; p[y+, ry+] numb2 ck FRT40A
/CyO; 2) p[UAS-FLP, ry+] (a gift from K. Bunier and K. Golic; Frise
et al., 1996) was used by mating yw; p[π M, w+] FRT40A; UASFLP/TM6, p[y+, ry+] to yw; p[y+, ry+]VC1 ck numb2 FRT40A
Gal4109-68/CyO. For clones in the pupae, we mated yw UAS-FLP;
FRT40A sca-Gal4 (or Gal4109-68) females to the following males: yw;
numb2 Su(H) SF8 FRT40A/ CyO, p[y+, ry+], yw ; numb2 ck FRT40A/CyO,
p[y+, ry+] and yw; A1-2-29 Su(H)SF8 FRT40A /CyO, p[y+, ry+]. The fly
stock, A1-2-29 Su(H) SF8 FRT40A (Gho et al., 1996) was used along
with p[y+, ry+]VC1 numb2 ck FRT40A (Frise et al., 1996) to make the
recombinant double and single mutant chromosomes: numb2 Su(H)SF8
FRT40A and numb2 ck FRT40A. To identify y− larvae that were FRT40A
sca-Gal4 (or Gal4109-68) /numb2 Su(H)SF8 FRT40A, the p[y+, ry+]VC1
transgene (a gift from V. Corces) was jumped onto the CyO balancer
(CyO, p[y+, ry+]) to serve as a marker for larval mouth hooks.
Dissection of adult and pupal nota
Adult nota were dissected in PBS, soaked in 80% isopropanol, and
mounted in Hoeyer’s medium (Ashburner, 1989). White prepupae
(time zero) were collected and maintained at 25°C for 24-30 hours
(i.e. 24-30 hours after puparium formation (APF)) for cell fate transformation analysis. To examine the Numb protein localization,
prepupae were maintained at 25°C for 14-16 hours APF, 16-18 hours
APF, and 18-20 hours APF. Staged pupae were then dissected in PBS
and fixed for 10 minutes in 5% formaldehyde in PEM solution (100
mM PIPES pH6.9, 1 mM MgCl2, 1 mM EGTA).
Antibody staining and confocal microscopy
The fixed pupal nota were washed in PBT (PBS containing 0.3%
Triton X-100). The nota were incubated with primary antibodies for
2 hours at room temperature. After washing several times with PBT,
the nota were incubated with the secondary antibodies conjugated
with either fluorescein or rhodamine. The nota were washed first with
PBT and then with PBS and subsequently mounted in 90% glycerol
with 2% n-propyl-gallate. The antibodies used in this study have been
described previously: the rabbit anti-Prospero antibody (Vaessin et al.,
1991), the monoclonal anti-Elav antibody (mAb44C11) (Bier et al.,
1988), the rabbit anti-Numb antibody (Rhyu et al., 1994), the rat antiCut antibody (Blochlinger et al., 1990), and the rat anti-Su(H)
antibody (Gho et al., 1996), which were used at 1:1000, 1:5, 1:1000,
1:5000, and 1:1000 dilutions, respectively. The anti-Su(H) antibody
was kindly provided by F. Schweisguth. A Zeiss microscope and a
Bio-Rad MRC-600 confocal microscope were used to analyze the
images.
Generation of UAS-numb transformants
The full length numb cDNA was derived from the KpnI fragment of
hs-numb#2 (Rhyu et al., 1994) and subcloned into the KpnI site of the
pUAST vector. The pUAST-numb DNA was introduced into w−
embryos to generate transgenic flies by standard injection methods
(Rubin and Spradling, 1982). The transgenic flies were crossed to scaGal4 (Hinz et al., 1994) or Gal4109-68 flies (Frise et al., 1996) for phenotypic characterization. Fifteen out of 18 independent transformants
displayed similar phenotypes, i.e. balding and twinned hairs, although
the strength of the phenotypes varied from one tranformant to another.
#34 of pUAST-numb (on the third chromosome) was used for the
studies presented in this report.
In vitro binding assays
The Notch intracellular domain and various Numb fragments were
4438 S. Wang and others
cloned in-frame into the pGEX vectors (Pharmacia) for expression in
DH5α as GST-fusion proteins. The expression of fusion proteins was
induced for 3 hours with 1 mM isopropyl-D-thiogalactopyranoside
added to log-phase bacterial cultures. Following sonication of the
bacteria in PBS, Triton X-100 was added to a final concentration of
1%. The bacterial lysate was then centrifuged at 7,000 rpm (9000 g)
for 10 minutes in an HS-4 rotor in a Sorvall centrifuge. The supernatant was then mixed with glutathione Sepharose-4B beads
(Pharmacia) at 4°C for 30 minutes. The beads were then washed three
times in PBS/1% Triton X-100 and three times in PBS/0.1% NP-40.
The washed beads were kept at 4°C as a 50% suspension until use.
The plasmid pNB40-H, which contains the full length H cDNA,
was kindly provided by J. Posakony. [35S]Met-labeled H protein was
expressed from pNB40-H using the TNT-coupled in vitro transcription/translation rabbit reticulocyte system (Promega). For in vitro
binding analyses, 20 µl of the 50% suspension of beads (with bound
GST fusion protein) were mixed with 5 µl lysate of the [35S]Metlabeled in vitro translated protein. The protein mixture was
incubated in 150 µl PBS/0.1% NP-40 at 4°C for 30 minutes and the
beads were then washed 4-5 times with PBS/0.1% NP40. After
washing, SDS-PAGE loading buffer was added, the samples were
boiled, and the supernatant was analyzed by 10% SDS-PAGE
(Sambrook et al., 1989). The gels were dried and subjected to
autoradiography.
RESULTS
Reduction of numb function causes both hair to
socket and neuron to sheath transformations
The three previously isolated numb alleles (numb1, numb2 and
numb3) cause recessive embryonic lethality, rendering these
mutants unsuitable for the study of interactions of numb with
other genes during adult sensory organ formation. We have
therefore carried out a P-element revertant screen in order to
isolate more numb mutants that are viable and show visible
phenotypes in adult sensory bristles. We started with the allele
numb1, which was generated by insertion of the transposon
pUChsneo (see Materials and Methods for details), and
isolated a viable hypomorphic numb allele (numbSW) which
exhibits a double-socket phenotype (Fig. 1).
The sensory organs on the fly notum (dorsal thorax) include
both macrochaetae and microchaetae, large and small bristles,
positioned in a fixed pattern. Each bristle has an external
sensory structure that is composed of a hair and a socket (Fig.
1A). In the numbSW mutant flies, both macrochaetae and
microchaetae exhibit abnormal external sensory structures
(Fig. 1C,F). Double-socket bristles are produced at the expense
of a hair in these partial loss of numb function mutants. The
severity of the double-socket bristle phenotype varies with the
genotype (Table 1). The homozygous numbSW flies show a
small number of double-socket bristles (3 out of 26
macrochaetae on the notum), while transheterozygous
numbSW/numb2 flies display a very strong double-socket
phenotype (12 out of 26 macrochaetae). These abnormal
bristles with double sockets are observed on almost every part
of the fly, including notum, head, wing (Fig. 1E), abdomen,
legs and sex combs. This double-socket phenotype can be
rescued by overexpression of numb driven by the heat-shock
hsp70 promoter (data not shown), suggesting that this new
mutation is indeed a numb allele. The double-socket phenotype
generated in numbSW flies is similar to the double-socket
phenotype generated in some of the clones that have lost numb
Table 1. Numbers of double-socket macrochaetae in flies
that are trans-heterozygous for numbSW and various numb
alleles, as compared to those of the wild-type (+/+) flies
Average number
of double sockets
Genotypes
numbSW/numb1
numbSW/numb2
numbSW/numb3
numbSW/numbSW
numbSW/Df(2L)A-C
Notum
Head
11.2
12.4
11.8
3.0
15.4
7.9
10.2
9.2
1.9
11.3
Wild-type macrochaetae number: notum, 26; head, 14.
function (Rhyu et al., 1994) and confirms the function of numb
in the hair/socket lineage.
Besides a hair and a socket, a normal sensory bristle also
contains two inner cells, a neuron and a sheath cell (Fig. 1A).
To examine the inner cells of sensory bristles in the numbSW
mutant we have used a neuronal nuclear marker, the monoclonal antibody mAb44C11 (an anti-Elav antibody), and a
sheath cell nuclear marker, the anti-Prospero antibody, and
doubly stained numbSW mutant nota at 24-30 hours APF when
all four cells of a sensory organ are fully differentiated. In wildtype nota, each sensory bristle has one mAb44C11-positive
neuron and one Prospero-expressing sheath cell (Fig. 2A-C).
Fig. 2. Partial loss of function of numb transforms the neuron into a
sheath in an adult sensory organ. Pupal nota at 24-30 hour APF were
dissected and doubly stained with the neuronal marker mAb44C11
(red) and the sheath cell marker anti-Prospero antibody (green).
(A-C) Wild-type pupal notum. Each sensory organ contains one
sheath cell (A, green) and one neuron (B, red) that are positioned
next to each other (C). (D-F) A numbSW/numb2 mutant pupal notum.
Note the double-sheath cells (marked by *), the two green cells that
are positioned next to each other (D), are sensory organs in which the
neuron is transformed into a sheath cell.
Interaction of Su(H) with numb 4439
Table 2. Numbers of double-sheath cells in numbSW/numb2
mutant nota compared to those in numbSW + /numb2
Su(H)SF8, from pupal nota stained with anti-Prospero
antibody and mAb44C11 antibody
Pupa
no.
1 Pros+ cell
A
1
2
3
4
numbSW/numb2
B
1
2
3
numbSW+/numb2Su(H)SF8
9
8
10
9
8
4
7
2 Pros+ cells
Total
macrochaetae
counted
9
10
9
11
18
18
13
18
10
13
10
19
21
20
cells with distinct fates. Similar crescent localization of Numb
was also observed during the SOP development on the pupal
nota of numbSW mutants (data not shown). Therefore, the
numbSW mutation most likely affects the Numb signaling efficiency rather than Numb localization.
Overexpression of numb can cause cell fate
transformations opposite to those of the numb
hypomorphic allele
Previously, it has been shown that overexpression of numb
using the heat-shock hsp70 promoter (hs-numb) results in two
major phenotypes, balding (loss of external sensory organ
structures) and twinned hairs with no socket. These two phenotypes presumably are due to the transformation of the IIa cell
into the IIb cell and the socket cell into the hair cell in the IIa
Average neuron to sheath transformation: numbSW/numb2, 58%;
numbSW+/numb2Su(H)SF8, 55%.
In contrast, in the numbSW mutant nota, some sensory bristles
contain two Prospero-positive sheath cells but no neurons (Fig.
2D-F), indicating that the neuron is transformed into a sheath
cell. The total number of cells within each sensory bristle does
not seem to change, as revealed by staining with an anti-Cut
antibody which recognizes all four cells of the sensory organ
(data not shown). About 50%-60% of the sensory bristles have
a neuron to sheath cell transformation (Table 2A), showing that
numb also functions in the IIb cell lineage for the formation of
the neuron and the sheath cell. Therefore, partial loss of numb
activity in the numbSW allele causes both IIa and IIb cells to
divide symmetrically, resulting in the formation of two socket
cells and two sheath cells (Fig. 5B).
Previously it was shown that the Numb protein is asymmetrically localized in the dividing SOP cell, forming a crescent
in the cell cortex of SOP (Rhyu et al., 1994). We were therefore
interested in knowing whether Numb protein localization is
affected in the numbSW mutants. Since the Numb protein localization in later stages (i.e. the IIa and IIB divisions) during the
SOP development has not been studied, we have first examined
the Numb protein localization on the pupal nota of wild type
during each of the divisions (SOP, IIa, and IIb) of a
microchaete bristle (Fig. 3). In this experiment, we used the
anti-Cut antibody which stains the nuclei of all the cells within
each sensory organs, and the anti-Numb antibody, and doubly
stained pupal nota at 14-16 hours APF (Fig. 3A-C), 16-18
hours APF (Fig. 3D-F), and 18-20 hours APF (Fig. 3G-I). On
a notum at 14-16 hours APF when dividing SOPs are frequently present as revealed by the non-nuclear staining of the
anti-Cut antibody, a clear crescent staining of Numb is associated with each dividing SOP cell (Fig. 3A-C). At 16-18 hours
APF, each adult sensory organ is at the two-cell stage, with the
IIa cell being ready to divide. We observed a Numb crescent
in each dividing IIa cell (Fig. 3D-F). A couple of hours after
the division of the IIa cell, the IIb cell starts to divide (18-20
hours APF). Fig. 3G-I shows that a Numb crescent also forms
in the IIb cell before it divides during a three-cell stage. The
observation of asymmetric localization of Numb protein in the
dividing SOP, IIa cell and IIb cell suggests that Numb is preferentially segregated into one daughter cell during each of the
three divisions in the SOP lineage, thus generating daughter
Fig. 3. Numb is asymmetrically localized in all three divisions during
SOP development. Pupal nota at 14-16 hours APF, 16-18 hours APF,
and 18-20 hours AFP were dissected and doubly stained with the
anti-Cut (red) and the anti-Numb (green) antibodies. Similar results
were obtained with both wild-type and numbSW mutant pupal nota.
(A-C) A region of pupal notum at 14-16 hours APF. Note that a
Numb crescent (arrow) is formed during mitosis of a SOP cell (as
revealed by the cytoplasmic staining of anti-Cut antibody). The
arrowhead shows the two Cut-positive cells with nuclear staining,
corresponding to a IIa cell and a IIb cell resulted from a SOP
division. (D-F) A region of pupal notum at 16-18 hours APF. Arrow
indicates the Numb crescent. Note that the Numb crescent is
associated with the dividing cell, the IIa cell, whereas the
neighboring cell with nuclear staining of Cut is presumably the IIb
cell. The arrowhead shows a two-cell cluster positive for nuclear
staining of Cut, corresponding to a stage before the IIa cell enters
mitosis. (G-I) A region of pupal notum at 18-20 hours APF. Arrow
indicates the Numb crescent, which is associated with the IIb cell
during mitosis. The arrowhead shows the two cells next to the IIb
cell which are positive for nuclear staining of Cut. These two
neighboring cells are presumably the hair cell and the socket cell.
Brackets show two clusters of three cells with nuclear staining of
Cut, suggesting that the IIb cells in these three-cell clusters are at a
stage before entering mitosis.
4440 S. Wang and others
ments confirm the finding that numb functions in all three
divisions of the SOP lineage. Fig. 5 summarizes the cell fate
transformations in the IIa and IIb lineage within an adult
sensory organ in numbSW and UAS-numb, as compared to the
wild type.
Fig. 4. Overexpression of numb can generate phenotypes that are
opposite to those from reduction of numb function. The homozygous
UAS-numb (#34A) flies were crossed to Gal4109-68 and the
Gal4109-68/+; UAS-numb/+ flies were examined. (A,C) Twinned hairs
caused by the socket to hair transformation. (B,D) Balding
phenotype due to the transformation from IIa into IIb. (E-G) A pupal
notum of Gal4109-68/+; UAS-numb/+ mutant at 24-30 hour APF
doubly labeled with the anti-Prospero antibody (E, green) and the
mAb44C11 (F, red). The three neurons and one sheath cell
phenotype is marked by arrowhead, two neurons and two sheath cells
by *s, and two neuron with no sheath cells by arrows.
A Su(H) mutation acts as a dominant suppressor of
the numb hypomorph in determining the hair/socket
lineage but not the neuron/sheath lineage
Partial reduction of Su(H) activity produces a twinned hair
phenotype, resulting from transformation of the socket cell into
a hair cell (Schweisguth and Posakony, 1994), opposite to the
phenotype caused by decreased numb function. Conversely,
overexpression of Su(H) causes a double-socket phenotype,
presumably resulting from transformation of the hair cell into
a socket cell (Schweisguth and Posakony, 1994). To test
whether these genes act in the same pathway, we assessed the
influence of the Su(H) null allele, Su(H)SF8, on the hypomorphic allele numbSW. As described above, numbSW /numb2 flies
showed the double-socket phenotype in about 50% of the adult
sensory bristles (Table 1) and a neuron to sheath transformation in 50-60% of pupal sensory bristles (Table 2A). Removing
one copy of the Su(H) gene in numbSW /numb2 flies (i.e.
numbSW + /numb2 Su(H)SF8) strongly suppressed the doublesocket phenotype (Fig. 6A-C). In contrast, reduction of Su(H)
function had no effect on the numbSW double-sheath
phenotype. As shown in Fig. 6D-F and summarized in Table
2B, double-labeling of the sensory bristle cells with
mAb44C11 and anti-Prospero clearly showed the doublesheath cell phenotype characteristic of numbSW /numb2 mutant.
These results establish that Su(H)SF8 behaves as a dominant
suppressor of the hair cell to socket cell transformation but not
of the neuron to sheath cell transformation in the
numbSW/numb2 mutant.
neuron
neuron
hair
hair
sheath
sheath
socket
socket
sheath
neuron
socket
hair
Numb determines the hair cell identity by negatively
regulating Su(H) protein expression
cell lineage, respectively (Rhyu et al., 1994). We examined the
effects of numb overexpression on the cell fate of the inner
The strong genetic interaction betwen numbSW and Su(H)SF8
cells, the neuron and sheath cell (daughters of the IIb cell).
made us wonder how numb and Su(H) act antagonistically.
The UAS-GAL4 system (Brand and Perrimon, 1993) was
Shortly after division of the IIa cell, the Su(H) protein is
used to study the effect of numb overexpression.
Transgenic flies carrying one copy of UAS-numb
A
B
C
(#34A) were crossed to flies of a GAL4 enhancer-trap
line, Gal4109-68, which drives the transgene expression
SW
Wild type
numb
UAS-numb
in the SOP cell and its progeny. Two phenotypes
resulted from such overexpression of numb, twinned
hairs (Fig. 4A,C) and balding (Fig. 4B,D), the same
SOP
SOP
SOP
as the phenotypes caused by hs-numb (Rhyu et al.,
1994). Sensory bristles in the dissected pupal nota of
flies carrying both UAS-numb and Gal4109-68 were
IIa
IIb
IIa
IIb
IIa
IIb
doubly stained using the neuronal marker mAb44C11
and the sheath cell marker anti-Prospero antibody.
Four types of phenotypes were observed for the inner
cells: four neurons, three neurons and one sheath cell,
two neurons and two sheath cells, and two neurons
with no sheath cells (Fig. 4E-G). Most likely, transformation of both the IIa cell to a IIb cell and the
sheath to a neuron caused these phenotypes.
Therefore, overexpression of numb can cause phenoFig. 5. Schematic diagrams of the SOP cell lineage for (A) the wild type,
types opposite to the loss or reduction of numb
(B) the hypomorph numbSW, and (C) the overexpression of numb (UASnumb).
function phenotypes. These overexpression experi-
Interaction of Su(H) with numb 4441
activity. Numb may normally be present primarily in the hair
cell but not in the socket cell, thus resulting in low Su(H)
protein level in the hair cell and high Su(H) protein level in the
socket cell. When numb function is reduced, the level of Su(H)
protein is increased in the hair cell, leading to the transformation of the hair cell into a socket cell. Conversely, when numb
function is increased, the level of Su(H) protein is suppressed
in the socket cell, resulting in the transformation of the socket
cell into a hair cell.
Fig. 6. Reducing one copy of Su(H) suppresses the double-socket
phenotype but not the double-sheath phenotype of numbSW/numb2
mutants. (A) An adult notum from a numbSW + /numb2 Su(H)SF8 fly
showing the normal looking macrochaete and microchaete bristles.
This indicates that the double-socket phenotype generated in the
numb hypomorph is suppressed by removing one copy of the wildtype Su(H) gene. (B) The dorsal central region of the notum in (A) at
higher magnification. (C) The wing margin bristles from a numbSW +
/numb2Su(H)SF8 fly showing a similar suppression of the double
socket bristle phenotype. (D-F) Staining of a pupal notum from
numbSW + /numb2Su(H)SF8 mutants with the anti-Prospero antibody
(D, green) and mAb44C11 antibody (E, red). Note that the doublesheath cells (marked by *) are still present, indicating that the
suppression of the numbSW/numb2 phenotype by Su(H)SF8 is
restricted to the socket/hair lineage.
present in the nucleus of the socket cell but not the hair cell
(Gho et al., 1996), and it is required for socket cell formation
(Schweisguth and Posakony, 1994). We have examined Su(H)
protein expression in flies with partial reduction of numb
activity (numbSW mutant flies) and in flies with increased
numb activity (UAS-numb/Gal4109-68). Within each sensory
bristle of the wild-type pupal nota, a single sheath cell is
labeled with the anti-Prospero antibody and a single socket
cell is labeled with the anti-Su(H) antibody (Fig. 7A-C).
Similar immunofluorescence staining in the nota of the
numbSW/numb2 mutants showed a number of sensory organs
with two cells expressing Su(H) (Fig. 7D-F), consistent with
the double-socket phenotype (Fig. 1C,F). Moreover, in pupae
where Numb is overexpressed (UAS-numb/Gal4109-68), some
sensory organ cells have greatly reduced Su(H) expression
(Fig. 7G-I), consistent with the twinned hair phenotype (Fig.
4A,C).
These results suggest that numb may determine the fate of
the hair cell and the socket cell by controlling the level of Su(H)
Epistatic relationship between Su(H) and numb
The finding that loss-of-function mutations of numb and Su(H)
have opposite effects on the hair/socket cell fate specification
and the strong genetic interaction between these two genes
raise the question of their epistatic relationship. To investigate
this issue, we used UAS-FLP and specific GAL4 enhancer trap
lines to target FRT mediated clones of both numb and Su(H)
to the SOP lineage (Frise et al., 1996) (Zeng et al., personal
communication). We took advantage of two GAL4 enhancertrap lines, Gal4109-68 and sca-Gal4, which express GAL4
specifically in the SOP and its daughter cells. Both Gal4109-68
and sca-Gal4 show similar expression of a reporter gene during
SOP divisions, although the expression in sca-Gal4 appears to
be stronger. The reporter gene expression in sca-Gal4 also
starts earlier in proneural clusters before the SOP is singled
out. We expected that the FLP recombinase would be expressed
in a similar pattern from the UAS-FLP transgene. The SOP
lineage specific FLP expression induced mitotic recombination
between a FRT chromosome carrying mutations of numb,
Su(H), or numb Su(H) and a ‘wild-type’ FRT chromosome,
producing one daughter cell homozygous for the null
mutations and the other daughter cell homozygous for the wildtype genes.
Loss of numb function in the IIa cell due to UAS-FLP and
Gal4109-68 caused a double-socket phenotype (Fig. 8A-C),
which is indistinguishable from the double sockets observed in
the numbSW allele (Fig. 1C,F) or in heat-shock FLP-numb FRT
mutant clones generated during third instar larval and pupal
stage (Rhyu et al., 1994). Interestingly, sensory organs with
three sockets and one hair (Fig. 8D) or four sockets (data not
shown) were observed with a moderate frequency when clones
for the null mutation of numb were generated using the GAL4
line sca-Gal4. These phenotypes are most likely due to an early
mitotic recombination event induced by the early expression of
sca-Gal4 in the proneural cluster before the division that gives
rise to the SOP cells. In this case, the SOP itself would be
homozygous for the numb null mutation, causing transformation of the IIb cell into the IIa cell. Subsequently, loss of numb
function in the two IIa cells may cause one IIa cell to divide
symmetrically to form two socket cells, whereas the other IIa
cell may either give rise to a hair cell and a socket cell due to
an incomplete transformation or two socket cells due to a
complete transformation.
Twinned hairs as well as several other bristle phenotypes
were observed when either Gal4109-68 or sca-Gal4 was used
to remove Su(H) function in the SOP cell and its daughter
cells (Fig. 8E-I). The twinned hair phenotype suggests that
Su(H) functions in the IIa division to determine the socket
cell fate (Schweisguth and Posakony, 1994). Loss of Su(H)
function, like that of numb, also yields a broad spectrum of
transformations. The phenotypes are always in the direction
4442 S. Wang and others
Fig. 7. Su(H) protein expression in various numb
mutant background. Pupal nota at 24-30 hour
APF were dissected and doubly stained with the
anti-Su(H) antibody (B,E,H, red) and the antiProspero antibody (A,D,G, green). (A-C) A
notum from a wild-type pupa. Note that a
Su(H)-expressing cell (the socket cell, red) is
associated with a Prospero-expressing cell (the
sheath cell, green). (D-F) A notum from a
numbSW/numb2 mutant. Note that three
phenotypes are generated: two Prosperoexpressing cells and two Su(H)-expressing cells
(i.e. 2 sheath cells and 2 socket cells, *); two
Prospero-expressing cells and one Su(H)expressing cell (i.e. 2 sheath cells and 1 socket
cell, arrows); and one Prospero-expressing cell
and two Su(H)-expressing cells (i.e. 1 sheath cell
and 2 socket cells, not shown in the figure).
(G-I) A notum from a Gal4109-68/+; UASnumb/+ pupa. Note the disappearance of the
Su(H)-expressing cell in some sensory organs.
Two phenotypes are associated with this absence
of Su(H) protein expression: the presence (small
white arrowheads) or the absence (large white
arrowheads) of a Prospero positive cell. These
two phenotypes are presumably due to the
transformation from socket to hair and from
sheath to neuron, respectively.
Fig. 8. The double mutants of Su(H) and
numb showed a Su(H) mutant phenotype,
indicating that Su(H) acts downstream of
numb. (A-C) Adult nota (A,B) with a
scutellum (C) from the numb2 null mutant
clones generated by Gal4109-68. (D) A
notum from the numb2 null mutant clones
generated by sca-Gal4. Note that a three
socket and one hair phenotype
(arrowhead) is also generated in addition
to the double-socket phenotype.
(E-I) Su(H)SF8 mutant clones showing the
twinned hairs in nota (H,I) and scutellum
(G) as well as the intermediate phenotypes
caused by partial transformation of socket
into hair (E,F). (J-N) The Su(H) and numb
double mutant clones showing twinned
hairs in both nota (M,N) and scutellum (L)
as well as the intermediate phenotypes
caused by partial transformation of socket
into hair (J,K). Note that the various
phenotypes generated in the double
mutants are similar to those generated in
the Su(H) null mutants.
of forming two hair cells and no socket cell, and they range
from a remnant of a hair cell associated with an abnormal
socket to the formation of twinned hairs at the expense of
the socket cell. These phenotypes (Fig. 8E,F) may be
explained by partial transformation of the socket cell to a
hair cell and are therefore intermediate phenotypes toward
the formation of twinned hairs. We then examined the
phenotype in the double mutants where both numb and Su(H)
function were removed from the SOP and/or its daughter
cells. We found that a range of phenotypes was produced in
the numb2 Su(H)SF8 mosaic flies when either Gal4109-68 or
sca-Gal4 was used (Fig. 8J-N). Phenotypes of the double
mutants were indistinguishable from those due to loss of
function of Su(H) alone (Fig. 8E-I). Heat-shock FLP induced
mitotic recombination, generated in second instar larvae,
also produced double null mutant clones with phenotypes
Interaction of Su(H) with numb 4443
Fig. 9. The epistatic
relationship between numb and
Su(H) does not apply to the
neuron/sheath cell lineage.
Mutants of numb or Su(H) and
double mutants of numb and
Su(H) were generated by scaGal4. Pupal notum from numb
mutant (A-C) or Su(H) mutant
(D-F) or double mutant of
numb and Su(H) (G-I) were
doubly stained with antiProspero antibody (left panels,
green) and mAb44C11 (middle
panels, red). Note the presence
of the double-sheath
phenotypes (*s) in the numb
mutant and double mutants but
not in the Su(H) mutant.
indistinguishable from those of Su(H) null mutant clones
(data not shown). Therefore, the Su(H) gene appears to act
downstream of numb in the same genetic pathway in determining the fates of the IIa daughter cells, the hair cell and
the socket cell.
We have further examined the inner cells on the pupal nota
of these mutants at 24-30 hours APF by double staining with
the anti-Prospero antibody and the monoclonal antibody
mAb44C11 which specifically stain the sheath cells and the
neurons, respectively (Fig. 9). As expected, the loss-offunction mutation of numb caused a double-sheath phenotype
(Fig. 9A-C). In loss-of-function clones of Su(H), the
sheath/neuron cell lineage is completely normal (Fig. 9D-F).
In the double mutants where null mutations of both numb and
Su(H) were generated in the SOP lineage, the double-sheath
phenotype due to loss of numb function did not seem to be
affected by the removal of the Su(H) function (Fig. 9G-I).
Similar phenotypes of the inner cells in these double mutants
were generated when either Gal4109-68 or sca-Gal4 was used.
These results provide further evidence for Su(H) acting as a
downstream target of numb only in determining the cell fates
in the IIa lineage for the formation of the hair cell and the
socket cell. Consistent with this scenario, overexpression of
Su(H) via the heat-shock promoter (hs-Su(H)) did not affect
the neuron/sheath cell lineage, as revealed by double staining
with the anti-Prospero antibody and the mAb44C11 antibody
(data not shown). Our results strongly indicate that Su(H) and
numb function in the same genetic pathway, with Su(H)
acting downstream of numb and negatively regulated by
numb. However, this interaction is restricted to the hair/socket
lineage and there is no such epistatic relationship in the
Table 3. Numbers of the double-socket cells counted in
numbSW nota compared to those in numbSW; H/+ nota
Notum macrochaetae (number of double sockets)
Genotype
HB55
HDEB1
HB21
HD9
numbSW/numbSW;
12.1
13.0
11.7
7.1
H/+ numbSW/numbSW; +/+ numbSW/+; H/+
2.0
2.8
2.5
2.7
4.2
2.1
2.7
0.7
The wild-type notum contains 26 macrochaetae.
neuron/sheath cell lineage where apparently Su(H) is not
required.
H acts as a dominant enhancer of numb in
determining the hair/socket lineage
Previously, it has been shown that Su(H) and H act antagonistically to control the cell fates in the adult sensory organs
(Schweisguth and Posakony, 1994). The Su(H) mutation acts
as a dominant suppressor of the various haplo-insufficient
alleles of H to suppress the double-socket phenotype of H
mutants. The double-socket phenotype exhibited by H lossof-function mutants is similar to that of the numb loss-offunction phenotype. Given that numb and Su(H) act in the
same genetic pathway, we have further examined the genetic
interaction of H with numb. Homozygous numbSW flies
display a very weak double-socket phenotype (middle
column in Table 3; Fig. 10A,D). Removing one copy of H
greatly enhances the double-socket phenotype of the numbSW
flies (Fig. 10C,F). Table 3 summarizes the effect of four
different H alleles on the numbSW flies. We found that the
4444 S. Wang and others
Fig. 10. Partial loss of function of H
enhances the double-socket phenotype in
homozygous numbSW flies. (A,D) A notum
and a wing margin from numbSW flies,
showing the weak (low frequency) doublesocket phenotype. (B,E) A notum and a wing
margin from a HB21/+ fly showing the fairly
normal bristles on both the notum and the
wing margin. (C,F) A notum and a wing
margin from a numbSW; HB21/+ fly, showing
the greatly enhanced double-socket
phenotype.
number of double-sockets in numbSW mutants carrying one
copy of the H mutation is two- to three-fold greater than the
sum of those in H and numbSW single mutants (Table 3). Thus
the hair/socket cell lineage is very sensitive to the dosage of
both H and numb. H mutations function as dominant
enhancers of numbSW in the specification of cell fates of the
hair/socket cell lineage of adult sensory bristles.
In a genetic screen for modifiers of numb using the numbSW
allele, we isolated a dominant enhancer of numbSW (data not
shown). This enhancer mutant was subsequently found to be
allelic to H, further substantiating the genetic interaction we
observed between numb and H (Fig. 10 and Table 3).
H and Numb physically interact with each other
Given that numb, Notch, Su(H), and H all participate in the same
genetic pathway in determining the IIa cell lineage and that
Su(H) binds to H (Bailey and Posakony, 1995), Notch binds to
Su(H) (Fortini and Artavanis-Tsakonas, 1994), and Numb binds
to Notch (Guo et al., 1996), we wondered whether the interactions of numb with Su(H) and H are due to physical associations
of their gene products. We performed an in vitro binding assay
to determine whether Numb binds to Su(H) or H. As shown in
Fig. 11, a bacterial fusion protein, GST-Numb, and in vitro translated H protein were coprecipitated by glutathione agarose beads
(Fig. 11, lane 2). Similar coprecipitation of GST-Notch and the
in vitro translated H protein was also observed (Fig. 11, lane 1).
The in vitro translated H protein binds the Numb-N fragment
which encompasses the PTB domain of Numb (Fig. 11, lane 3),
whereas the GST-Numb-C (Fig. 11, lane 4) or GST alone (Fig.
11, lane 5) failed to bind to H. Similar binding analysis with the
in vitro translated Su(H) protein showed no detectable binding
of Su(H) to Numb (data not shown).
Fig. 11. The in vitro binding of
Numb and Notch to H. In vitro
translated 35S-labeled full length
H binds to GST-Numb (lane 2)
and GST-Numb-N (lanes 3), but
not GST-Numb C (lane 4) or GST
alone (lane 5). In addition, the in
vitro translated 35S-labeled H also
binds to GST-Notch intra (lane 1).
DISCUSSION
By isolating and characterizing a hypomorphic numb mutant,
numbSW, we have confirmed and extended previous studies to
show that numb functions in all three asymmetric divisions that
give rise to a simple external sensory organ. Moreover, we
found that the IIa lineage which gives rise to the hair and the
socket, but not the IIb lineage which gives rise to the neuron
and the sheath cell, requires the functions of Su(H) and presumably H. These findings, and the genetic interactions among
these genes, are discussed below.
numb functions in all three asymmetric cell
divisions during the formation of adult sensory
organs
Studies of numb in embryos indicate that numb functions during
the SOP division to produce the two distinct secondary precursors, the IIa cell and the IIb cell (Uemura et al., 1989; Rhyu et
al., 1994). Moreover, results from overexpression and mosaic
analysis in adult flies suggest that numb functions during both
SOP division and the IIa division in the development of adult
sensory organs (Rhyu et al., 1994). Therefore, numb plays an
important role in specifying the IIb cell fate and the hair cell
fate. Although it was proposed previously that numb also plays
a role in specifying the progeny cell fates of IIb, it has not been
demonstrated directly owing to the transformation of the IIb cell
into the IIa cell in numb null mutants. The characterization of
the newly isolated hypomorphic allele of numb (numbSW)
clearly indicates that numb is also involved in the sheath/neuron
lineage (the IIb cell lineage), as revealed by the double-sheath
cell phenotype on fly pupal nota as revealed by staining with
anti-Prospero antibody (Fig. 2). Similar double-sheath phenotypes were also observed in the external sensory organs in fly
embryos (data not shown). This involvement of numb in the
choice between the neuron and the sheath cell fates was also
confirmed by mosaic analysis using the UAS-FLP/FRT with
GAL4 and a numb null allele (numb2) (Fig. 9). In addition, we
have performed immunofluorescence analysis and found that
the Numb protein is asymmetrically localized in the dividing
SOP, the dividing IIa cell, and the dividing IIb cell (Fig. 3).
Thus, Numb functions to promote the IIb cell fate in the SOP
division, the hair cell fate in the IIa lineage, as well as the
neuron fate in the IIb lineage presumably by being preferentially segregated into the IIb cell, the hair cell, and the neuron,
respectively. The normal presence of asymmetric Numb localization in numbSW mutants suggests that the hypomorphic
mutation of numb may affect the Numb signaling efficiency.
Interaction of Su(H) with numb 4445
Su(H) and H are required for determining the
hair/socket cell lineage but not the neuron/sheath
cell lineage
It has previously been shown that Su(H) and H are required for
the determination of the socket cell and the hair cell within an
adult sensory organ, respectively (Schweisguth and Posakony,
1994). Loss of function alleles of Su(H) are potent dominant
suppressors of the double-socket phenotype in H mutants
(Schweisguth and Posakony, 1992), suggesting that reduction
of Su(H) activity favors the hair cell fate. Consistent with this
hypothesis, a twinned hair phenotype without a socket was
produced by either partial loss of function or the null mutant
of Su(H) in mosaic analysis (Schweisguth and Posakony,
1994). Overexpression of Su(H) produces the opposite transformation. When Su(H) activity is increased during the IIa cell
division, the hair cell is transformed into a socket cell, resulting
in a double-socket phenotype (Schweisguth and Posakony,
1994). In contrast, reduction of function or overexpression of
H during the later stage of the SOP lineage yields a doublesocket or twinned hair phenotype, respectively (Bang et al.,
1991; Bang and Posakony, 1992). Therefore, unlike Su(H),
which is required for socket cell formation, H activity seems
to be required for hair cell formation.
Although it was proposed that the Su(H) and H activities are
required for all three binary cell fate decisions in sense organ
development (Posakony, 1994), Schweisguth and Posakony
(1994) observed that flies which carry extra copies of Su(H)
contain a single neuron within each sensory organ, as revealed
by staining with the anti-HRP antibody. This observation raised
the possibility but did not prove that Su(H) is not involved in
the neuron/sheath cell fate decision because it was not known
whether the extra copies of Su(H) caused overexpression of
Su(H) at the right place and right time to produce
sheath/neuron
transformation.
Importantly,
whether
neuron/sheath decision is affected by Su(H) loss-of-function
mutant was not tested. Here, we have used anti-Prospero
antibody, which labels the nuclei of sheath cells, and the monoclonal antibody mAb44C11, which labels the nuclei of
neurons, to analyze sensory bristle cells of pupal nota of both
the null mutant (Fig. 9A-C) and mis-expression (hs-Su(H), data
not shown) of Su(H). These experiments show that the
neuron/sheath cell lineage is not affected by the loss or increase
of Su(H) activity.
numb interacts with Su(H) and H in determining
hair/socket lineage
As described above, numb is involved in all three asymmetric
divisions during the formation of a sensory organ in both
embryos and adults. The twinned hair phenotype due to loss of
function of Su(H) is opposite to the double-socket phenotype
due to loss of function of H or numb during the IIa cell division.
It therefore raises the possibility that numb, Su(H), and H may
determine the IIa cell lineage by acting in the same genetic
pathway. Consistent with this, we found that loss of activity of
one copy of Su(H) almost completely prevented the doublesocket formation in numb mutants, indicating that Su(H)SF8
behaves as a dominant suppressor of numb mutants. Conversely, we showed that various haplo-insufficient alleles of H
act as dominant enhancers of the numbSW allele, since partial
reduction of H function greatly increased the double-socket
phenotype in flies homozygous for numbSW.
In contrast to the suppression of the double-socket
phenotype of numbSW/numb2 mutants (Fig. 6; Table 2), we
failed to detect any effect on the double-sheath phenotype of
the numbSW/numb2 mutants when one copy of the wild-type
Su(H) gene was removed, suggesting that this genetic interaction between numb and Su(H) is restricted to the IIa lineage.
Su(H) acts downstream of numb in the hair/socket
lineage
Having found that partially reduced Su(H) activity prevents the
double-socket phenotype in the partial loss of numb function
mutant, we asked whether Su(H) acts downstream of numb by
examining mitotic recombination induced clones that lack the
functions of both Su(H) and numb in the SOP lineage.
Adult flies carrying clones of null mutations of both numb
and Su(H) showed a twinned hair phenotype, resembling the
loss of Su(H) function phenotype but opposite to the loss of
numb function phenotype (Fig. 8). This result indicates that
Su(H) is epistatic to numb in the same genetic pathway that
leads to the determination of daughter cell fates of IIa. Given
that complete removal of Su(H) functions had no effect on the
neuron/sheath cell lineage nor did it alter the double-sheath cell
phenotype generated by null mutations of numb (Fig. 9), it
appears that Su(H) has no essential role in the neuron/sheath
cell specification.
How might numb, Su(H), and H regulate one another to
specify the hair or the socket cell fate? Our immunostaining
results indicate that the Numb protein is asymmetrically
localized in the dividing IIa cell (Fig. 3D-F). Therefore, this
intrinsic factor Numb is probably preferentially segregated into
the future hair cell upon the asymmetric division of the IIa cell.
High level of Numb leads to down regulation of Su(H) and the
hair fate. Conversely, low level of Numb leads to high level of
Su(H) and the socket fate. How does Numb (and possibly H)
level affect Su(H) level is unknown. An intriguing possibility
is that the regulation is at the level of protein stability as has
been shown in the case of Tramtrack in photoreceptor specification (Li et al., 1997; Tang et al., 1997).
Numb/Notch signaling mediated by Su(H)-dependent
versus Su(H)-independent pathways
Notch and numb are involved in an antagonistic manner in all
three asymmetric divisions of the SOP lineage during embryogenesis and adult sensory organ formation (Hartenstein and
Posakony, 1990; Rhyu et al., 1994; Guo et al., 1996). It has
been proposed that Notch is a downstream target gene that is
negatively regulated by numb in the determination of daughter
cell fate during asymmetric division of the SOP and its
daughter cells in the PNS (Guo et al., 1996) as well as the
asymmetric division of MP2 in the CNS (Spana et al., 1995;
Spana and Doe, 1996) during embryogenesis. Most likely, this
epistatic relationship of Notch and numb also applies to adult
sensory organ development.
Su(H) is known to function in the Notch signaling pathway.
Drosophila sense organ development provides a striking
example for Su(H) action in only a subset of the binary cell
fate decisions controlled by Numb/Notch signaling. Existence
of Su(H)-independent Notch signaling is not limited to
Drosophila sense organs. Lecourtois and Schweisguth (1995)
found that Su(H)-independent Notch signaling is involved in
mesectodermal cell fate specification. In a vertebrate muscle
4446 S. Wang and others
cell culture system, Shawber et al. (1996) have demonstrated
that Notch can inhibit muscle cell differentiation by a
mechanism that is independent of CBF1 (a human homolog of
Su(H)). An important task for the future is to identify the
molecular components used in Su(H)-independent Notch
signaling pathways. The Drosophila sense organ should prove
to be a useful system for this purpose.
We wish to thank C-P. Shen for various GST-fusion proteins of the
Numb fragments, M. Guo for the GST-Notch construct, F.
Schweisguth for the Su(H) antibody and the fly stock pLacWA1-2-29
Su(H) FRT40A, J. Posakony for the H cDNA. We thank S. Ralls for
help with the revertant screen for numbSW isolation. We thank Ming
Chan, Ming Guo, Zack Ma, Chaoyang Zeng, Salim Abdelilah, and
Manuel Utset for critically reading the manuscript. S. Wang is a postdoctoral research associate, S. Younger-Shepherd is a research
associate, and L. Y. Jan and Y. N. Jan are investigators of the Howard
Hughes Medical Institute.
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(Accepted 12 September 1997)