Download Origin and specification of type II sensory neurons in

Development 121, 2923-2936 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
2923
Origin and specification of type II sensory neurons in Drosophila
Rachel Brewster and Rolf Bodmer*
Department of Biology, University of Michigan, 830 N University, Ann Arbor, MI 48109-1048, USA
*Author for correspondence: E-mail [email protected]
SUMMARY
The peripheral nervous system (PNS) of Drosophila is a
preferred model for studying the genetic basis of neurogenesis because its simple and stereotyped pattern makes
it ideal for mutant analysis. Type I sensory organs, the
external (bristle-type) sensory organs (es) and the internal
(stretch-receptive) chordotonal organs (ch), have been postulated to derive from individual ectodermal precursor
cells that undergo a stereotyped pattern of cell division.
Little is known about the origin and specification of type II
sensory neurons, the multiple dendritic (md) neurons.
Using the flp/FRT recombinase system from yeast, we have
determined that a subset of md neurons derives from es
organ lineages, another subset derives from ch organ
lineages and a third subset is unrelated to sensory organs.
We also provide evidence that the genes, numb and cut, are
both required for the proper differentiation of md neurons.
INTRODUCTION
Type I sensory neurons in Drosophila innervate the sensory
organs to which they are related by lineage (Bodmer et al.,
1989; Hartenstein and Posakony, 1989). Each of these sensory
organs is thought to derive from a single ectodermal precursor
(SOP) which gives rise to one or several monodendritic
neurons and several support cells. Type I sensory organs have
been classified into two major classes: mechano- or chemosensory organs that have external sensory structures in the cuticle
such as bristles, campaniform and basiconical sensilla (es
organs) and chordotonal organs that are internally located
stretch receptors (ch organs). In addition, the larval PNS also
contains numerous type II neurons that have multiple dendrites
(md neurons, Ghysen et al., 1986; Bodmer and Jan, 1987). In
contrast to es and ch neurons, md neurons (with one exception)
do not seem to be associated with support cells. Md neurons
are thought to function as stretch or touch receptors. Three
different subclasses of md neurons have been distinguished
based on their morphology (Bodmer and Jan, 1987): md-da
neurons are the most abundant subclass and have extensive
subepidermal dendritic arborisations, md-bd neurons have
bipolar dendrites and md-td neurons extend their dendrites
along tracheal branches. The origin and lineage relations of
these cells with other PNS cells has not been established.
Moreover, the genetic basis for md neuron differentiation is
poorly understood.
Mutations in some genes involved in sensory organ (es
and/or ch) development have also been shown to affect the
formation of md neurons. It is not clear, however, if md
neurons are affected independently of sensory organs or as a
consequence of alterations in sensory organ development. ch
and es organs are absent in atonal and AS-C mutants, respectively. Many md neurons are also missing in these mutants
The peripheral nervous system (PNS) of Drosophila embryos
has provided many insights into the genetic mechanisms of how
neural precursor cells are determined and assume a particular
developmental pathway of differentiation (for reviews see
Campuzano and Modellel, 1992; Ghysen and DamblyChaudiere, 1993; Jan and Jan, 1993). The model that has
emerged suggests that a given area of the early ectoderm where
sensory organs will develop becomes competent for producing
neural precursors due to the action of ‘proneural’ genes, such as
atonal and genes of the achaete-scute-Complex (AS-C) (e.g.
Cabrera et al., 1987; Dambly-Chaudiere and Ghysen, 1987;
Romani et al., 1989; Ruiz-Gomez and Ghysen, 1993; Jarman et
al., 1993). Some of these genes are expressed in a limited
number of ectodermal cells endowing each of these cells with
the potential to become a neural precursor. Another set of genes,
the ‘neurogenic’ genes, such as Notch and Delta, are then
required to limit the number of cells that will become neural precursors to one per cluster (Hartenstein and Campos-Ortega,
1986; Goriely et al., 1991; Bodmer et al., 1993; for review on
neurogenic genes see Artavanis-Tsakonas and Simpson, 1991;
Campos-Ortega, 1993). Once a neural precursor has been
singled out, selector-type genes, such as cut and poxneuro
(poxn), are required to initiate the correct developmental
program of a particular type of sensory organ (Bodmer et al.,
1987; Blochlinger et al., 1991; Dambly-Chaudiere et al., 1992).
Other genes are then responsible for specifying cellular identities of sublineages of neural precursor cells. The gene numb, for
example, is required for the correct specification of second order
precursor cells (Uemura et al., 1989; Rhyu et al., 1994; for other
genes affecting PNS development, see Salzberg et al., 1994).
Key words: Drosophila, peripheral nervous system, neurogenesis,
sensory neuron, numb, cut
2924 R. Brewster and R. Bodmer
(Dambly-Chaudiere and Ghysen, 1987; Jarman et al., 1993). It
is possible that md neurons have their own atonal- or AS-Cdependent precursor cells or, alternatively, md neurons may
derive from es or ch organ lineages. In the latter case, a failure
to recruit es and ch SOPs will automatically result in the loss
of md neurons.
In numb mutants, a normal number of es or ch SOPs are
formed, but the second order precursor cells give rise mainly
to support cells instead of neurons and their glial-like sibling
cells. Many md neurons are also missing in numb mutants
(Uemura et al., 1989; Rhyu et al., 1994). The neural selector
gene, cut, is required for specifying es organ identity: in cut
mutant embryos, the number of sensory organs and md neurons
is unchanged but es precursor cells develop as ch organs
(Bodmer et al., 1987). In addition to the es organs, about two
thirds of the md neurons also express cut (Blochlinger et al.,
1990), many of them are in close physical association with es
organs. Interestingly, in deficiencies of AS-C all Cut-positive
md neurons are absent, whereas, in atonal mutants, a complementary subset of (Cut-negative) md neurons is missing
(Dambly-Chaudiere and Ghysen, 1987; Jarman et al., 1993; R.
B. unpublished). In order to better understand the role of these
(and other) genes in the specification of md neurons, it is
necessary to know their origins and lineages.
Observation of cell division patterns of sensory organs in
other insects had suggested that the cells within an es or ch
sensory organ derive from a common precursor that divides
near the organ’s final location (e.g., Wigglesworth, 1953;
Lawrence, 1966; Jagers-Rohr, 1968; for review see Bate,
1978). Due to the relatively small size of Drosophila cells, it
has not been possible to confirm these lineages by simple visualization of SOP divisions. BrdU labelling of Drosophila
embryos provided further insights concerning the division
pattern of the putative SOPs (Bodmer et al., 1989). Exposure
to BrdU at progressively later times resulted in fewer and fewer
labelled cells within the postulated sensory organ lineages,
since many precursor cells had already completed their last Sphase (i.e. last possibility for BrdU incorporation) at the time
of exposure. A likely pattern of SOP division was inferred from
the patterns of BrdU-labelled cells (Bodmer et al., 1989). Md
neurons were shown to be generated around the same time as
type I SOPs, and it was speculated that they derive from type
II ectodermal precursors that divide close to their final position.
The main drawback of BrdU incorporation studies with respect
to SOP lineages stems from the inability to mark individual
SOPs. Since all dividing PNS cells get labeled, the interpretation of sensory organ lineages heavily relied on the underlying
assumption that the cells within a sensory organ lineage derive
from a common precursor.
In order to reassess the lineages of type I sensory organs and
determine the lineages of type II (md) neurons in the PNS of
Drosophila, we have used the flp/FRT site-specific recombination system of yeast (Golic and Lindquist, 1989; Struhl and
Basler, 1993). Using this method, we show that each type I
sensory organ derives indeed from a single ectodermal
precursor cell. In addition, we have determined that many of
the md neurons are part of the type I sensory organ lineages.
Our results indicate that one subset of the md neurons is related
to es organs, a second subset is related to ch organs and a third
set does not appear to be related to either es or ch organs. Consistent with these proposed lineage relationships, we find that,
in numb mutants, the sensory organ related md neurons are
usually absent or transformed into support cells. We also show
that, in cut mutant embryos, the identity of AS-C-dependent md
neurons (many of which are related to es organs) has changed
to what seems to be characteristic of a subset of atonaldependent md neurons. This suggests that AS-C and cut are not
only required for es organ formation, but also for specifying a
subset of md neurons.
MATERIALS AND METHODS
Fly stocks
The fly stocks used to generate lacZ-positive clones were: hsp 70-flp
(flipase construct inserted on the X chromosome, Struhl and Basler,
1993) and Act-Draf-nuclacZ (construct inserted on the third chromosome; Struhl and Basler, 1993). The Act-Draf-lacZ stock has a constitutive actin promoter separated from a lacZ reporter gene by a
segment of DNA that contains a transcriptional stop codon and is
flanked by two FRT repeats. The presence of Draf in this construct is
incidental. The transformant flies were provided by G. Struhl.
In order to determine whether md neurons are affected in numb
mutants, numbn7/CyO males (Uemura et al., 1989) were crossed to
females from the E7-3-36 P-element enhancer trap line (second chromosome insertion), which marks all md neurons (Bier et al., 1989).
Since the numb mutation and the E7-2-36 P-element insertion are both
on the second chromosome, recombinants from this cross were
selected (numb, E7-2-36/CyO).
Transformations of md neurons in cut mutants were assessed using
the enhancer trap line, E7-3-49 (third chromosome insertion), which
marks a subset of md neurons (Bier et al., 1989). This line was crossed
into a cut mutant background of the genotype: yw,ctdb7/FM7c. ctdb7
is a small deletion in the cut gene of ~1 kb that is homozygous lethal
and considered a null mutation (Blochlinger et al., 1988).
Generation of lacZ-expressing clones in the embryo
lacZ-expressing clones were generated using the yeast flp/FRT sitespecific recombination method according to Struhl and Basler (1993).
Homozygous hsp 70-flp virgin females were crossed to Act-DrafnuclacZ males. F1 embryos, hemizygous for hsp70-flp and heterozygous for Act-Draf-nuclacZ, were collected on grape plates with a dab
of yeast and submitted to various levels of heat shock (28°, 30°, 32°
or 34°C) for 30-40 minutes, at one of the following developmental
stages: 2-4 hours, 4-6 hours or 6-8 hours of development after egg
laying. Heat shocks were administered either in a water bath or in an
incubator in which water had been prewarmed to the desired temperature. The embryos were then allowed to develop at 18°C until they
reached 14-16 hours of development. They were then fixed and
stained with antibodies. In order to avoid scoring the same clones
twice, the coordinates of each PNS clone were taken. Embryos
submitted to a mild heat-shock treatment (30°C) had less than 20
clones and on average less than one clone per embryo that included
PNS cells embryos heat-shocked at 32°C had on average two or three
PNS-containing clones. These two treatments were used in the vast
majority of the collected data. An excess of 5000 embryos was scored.
To test the efficacy of the flipase-mediated recombination, embryos
were heat shocked at 37°C for 30-40 minutes or given two consecutive 20-30 minute heat shocks at 37°C with an interval of 30 minutes
at room temperature. With this regime, embryos expressed lacZ in
virtually all cells. Another control consisted of raising the embryos at
18°C until they reached 14-16 hours of development without heat
shock. These embryos had very few lacZ-expressing clones (data not
shown). Embryos bearing only the Act-Draf-nuclacZ construct were
also tested for lacZ expression (after heat shock for 30 minutes at
32°C). These embryos did not express the reporter gene.
PNS lineages in Drosophila 2925
Immunocytochemical staining
hsp70-flp;Act-Draf-nuclacZ embryos or mutant embryos (for cut and
numb) marked with enhancer trap lines E7-2-36 or E7-3-49 were
double labeled with a rabbit antibody against β-galactosidase (β-gal,
Cappel, 1:2000) to monitor the lacZ-positive clones or md neurons,
and one of the following antibodies: monoclonal antibody 22C10
(1:100; all PNS neurons marked), monoclonal antibody 21A6 (1:10,
labels scolopales and dendritic caps; Zipursky et al., 1984), antiProspero antibody (1:4, stains all thecogen and scolopale cells; Spana
and Doe, personal communication) or the antibody RK2 (1:500, stains
glial cells and ligament cells; Campbell et al., 1994). Embryos were
then incubated with the appropriate HRP-coupled secondary antibody
(Biorad). The lacZ construct that we have used confers nuclear localization of this β-gal reporter gene product. Since the intensity of the
anti-β-gal staining tended to mask that of 22C10, the two antibodies
were used sequentially. The staining procedure is essentially the same
as described in Bodmer and Jan (1987). In some cases, the DAB
(Sigma) color reaction for one antibody was carried out in the
presence of 8% nickel chloride, which results in a black product that
can then be distinguished from the brown DAB product. numb
homozygous mutants were recognized using the following criteria:
absence of most peripheral neurons (determined with 22C10 antibodies) or absence and disorganization of md neurons (determined
with anti-β-gal staining). cut mutant embryos were recognized by the
transformation of dendritic caps into scolopales (determined with
21A6 antibodies, see Bodmer et al., 1987).
RESULTS
Generation of small clones in the early embryo
using the yeast flp/FRT method
The PNS of Drosophila embryos is composed of numerous es
and ch organs as well as md neurons that are arranged in a
segmental, highly stereotyped fashion, allowing reliable identification of all cellular components (Fig. 1A,B; Ghysen et al.,
1986; Bodmer and Jan, 1987; Hartenstein, 1988). We have
studied cell lineages in the PNS by inducing random clones in
the early embryo using a site-specific recombination method
from yeast, the flp/FRT system (Golic and Lindquist, 1989;
Struhl and Basler, 1993). Small clones of lacZ-positive cells
were generated randomly in embryos, before or during the
division of PNS precursors (Fig. 1C, see also Materials and
methods). These clones were induced by activation of a site-
Fig. 1. The embryonic PNS of Drosophila. (A) Diagram of
all the PNS cells in an abdominal hemisegment of a wildtype embryo. In blue: chordotonal (ch) organs, in yellow:
external sensory (es) organs, in red: multiple dendritic neurons (md). Nomenclature according to Ghysen and Dambly-Chaudiere (1986),
Bodmer and Jan (1987) and Bodmer et al. (1989). Abbreviations: da, neuron with large dendritic arbors (diamond shape); td, tracheae
innervating neuron (drop shape); bd, bipolar dendrite neuron (triangle shape); n, neuron; g, glial cell; th, thecogen cell; tr, trichogen cell; to,
tormogen cell; l, ligament cell; s, scolopale cell, c, cap cell; a, attachment cell; v,v′,l and d refer to the two ventral, lateral and dorsal clusters
(see B) respectively. Anterior is always to the left and dorsal is up. (B) Lateral view of a stage 16 wild-type embryo (14-16 hours of
development), stained with the monoclonal antibody 22C10 (cytoplasmic staining) and a marker for multiple dendritic neurons (the lacZexpressing E7-2-36 enhancer trap line, nuclear staining). T, thoracic segments; A, abdominal segments. (C) Same stage embryo containing
hsp70-flp;Act-Draf-lacZ constructs (see Materials and Methods) that was heat shocked for 30 minutes at 32°C at blastoderm stage. Nuclei of
clonally derived cells are revealed by staining with anti-β-gal antibodies. This embryo contains two clones: a purely ectodermal clone (wavy
bracket) and a mixed ectodermal and sensory clone (round bracket indicates labelled cells of a single ch organ in lch5 and arrowhead points to
two ectodermal cells).
2926 R. Brewster and R. Bodmer
specific recombinase, flipase (flp), which fuses a constitutive
promoter (of actin) to the coding region of a lacZ reporter gene
(constructs and transgenic flies of this system are described in
Struhl and Basler, 1993). Prior to flipase expression, these
sequences are separated by two flipase recombination target sites
(FRTs) in between which a stop codon is located. The lacZ gene
product can therefore only be detected in the cells in which the
intervening FRT/stop/FRT sequence has been recombined away.
A heat-shock promoter-flp construct was used to control the
timing and the level of flp expression. A flp-mediated recombination event can occur at any time after flp induction, thus
marking all or a subset of the cells that belong to a given lineage.
We reasoned that comparison of larger with smaller clones
should enable us to determine the sequence of cell divisions of
a given SOP. Since the SOPs of the embryonic PNS start
dividing between 5 and 7 hours of development, we heatshocked embryos at blastoderm (2-4 hours of development) to
maximize the labelling frequency of SOPs, or after gastrulation (4-6 or 6-8 hours of development) expecting to label a
higher proportion of SOP sub-lineages (Fig. 2). Embryos were
aged to 14-16 hours of development (stage 16, Campos-Ortega
and Hartenstein, 1985) and stained for lacZ expression. All
cells in clones that included PNS cells had approximately the
same level of staining of lacZ expression, which indicates that
Fig. 2. Variations of expected flp-induced clone
sizes in a typical sensory organ lineage. The number
of labeled cells in a particular sensory organ lineage
depends on when during the lineage a recombination
event took place. An early event at the precursor
level or before is expected to label all cells that are
related to a particular sensory organ. Later events
should produce progressively smaller clones, in
which only the cells that are generated later in the
lineage are labelled. Single cells can be labelled as
well.
Fig. 3. lacZ-expressing clones in type I es and ch organs. Types of clones observed in es organs (A-D) and ch organs (E-H). β-gal staining is
nuclear and 22C10 staining is cytoplasmic. (A,B) Two examples in which all cells of an individual es organ are labelled (neuron, thecogen,
tormogen and trichogen) as well as a number of ectodermal cells (indicated by asterisk or line). In A, lesC is labelled; in B desD is labelled
(note the neuronal dendrite between n/th and to/tr). (C) Only the thecogen and neuron and (D) only the tormogen and trichogen of desD are
labelled. Arrowheads point to the position of unlabelled es organ cells. (E,F) All cells of a single scolopidium of lch5 are labelled (cap,
scolopale, neuron and ligament cell). (E) An ectodermal cell (ec) is also labelled that corresponds to the position of an ectodermal attachment
cell, described by Matthews et al. (1990) as an anchor point for ch organs. (G) Clone composed of three cells: ligament, neuron and scolopale
cell. (H) Clone composed of two cells: ch neuron and scolopale cell. Arrowheads point to unlabelled ch organ cells. Abbreviations as in Fig. 1.
PNS lineages in Drosophila 2927
the flipase was not expressed preferentially in any given cell
type (a representative embryo is shown in Fig. 1C). To help
identify PNS clones, the embryos in some experiments were
double labelled with the monoclonal antibodies 22C10 (a
neuronal membrane marker) or 21A6 (which stains the
dendritic caps of es organs and the scolopales of chordotonal
organs; Zipursky et al., 1984; see Bodmer et al., 1987).
External sensory (es) lineages
First we examined clones containing only es or ch organ cells,
because the outcome of these studies could be compared with
the findings of previous lineage studies of the embryonic PNS
(Bodmer et al., 1989). The cells of es organs tend to be tightly
grouped together. We chose to concentrate on the lineages of
two dorsal es organs (desC/D) and one lateral es organs (lesC)
since individual cells in these organs are spaced further apart
(see Fig. 1A for exact location within a segment). The neuron
was easily recognized with the use of the 22C10 marker and
the neuron-associated glial cell, the thecogen, was localized on
the basis of its vicinity to the neuron along its dendrite. The
support cells, the tormogen and trichogen, were distinguished
based on their position adjacent to the 21A6-positive dendritic
cap, which is located at the tip of the dendrite at the base of
the future cuticular structure associated with the sensory organ
(Hartenstein, 1988).
Three types of clones were observed in desC/D and lesC
(Fig. 3A-D; Table 1). 42 clones were composed of all es organ
cells: neuron, thecogen, tormogen and trichogen (Fig. 3A,B).
This indicates that the cells within es organs in Drosophila
embryos are indeed clonally related. Many of these clones,
however, also included a few labelled ectodermal cells
(asterisks in Fig. 3) indicating that the recombination event
took place in an ectodermal cell that underwent one or more
rounds of cell divisions before the SOP was formed. 13 clones
were composed of neuron/thecogen (Fig. 3C) and 10 clones
were composed of tormogen/trichogen (Fig. 3D; Table 1).
These findings are consistent with the previously reported
BrdU data (Bodmer et al., 1989) indicating that es organs are
generated through two sets of cell divisions: neuron/thecogen
derive from one SOP daughter cell, tormogen/trichogen from
the other (Fig. 5A). Individually labeled es organ cells were
also observed, consistent with a late recombination event (see
Fig. 2).
Chordotonal (ch) lineages
An abdominal segment is composed of three single ch organs
(v′ch1, vchB and vchA) and a cluster of five ch organs (lch5)
(Fig. 1A). Unlike most es organs, the cells of ch organs are
neatly arranged in a row. Whereas the ligament cells of the ch
organs (scolopidia) in the lch5 cluster are clearly visible, this
is not always the case for the ligament cells in v′ch1, vchB and
vchA. We therefore restricted our analysis of ch lineages to the
lch5 cluster.
The majority of the clones that we observed in the lch5
cluster (82 cases) were composed of all four cells within one
scolopidium (ligament, neuron, scolopale and cap cell). These
clones frequently included a few ectodermal cells, as was also
observed for es organ clones (Figs 1C, 3E,F; Table 1). This
argues in favor of a clonal relationship between the cells of
individual ch organs. 34 clones consisted of the neuron and
Table 1. Number of lacZ + PNS clones
es organs
des C + D
les C
lesA + ldaA
lesB + ldaB
v′esB + v′ada
v′es2 + v′pda*
md (da) neuron ± es organs
n, th, to, tr
n, the
to, tr
n(s)
md + es (all) md + es (n + th)
29
13
1
0
0
1
10
3
2
1
0
1
10
0
0
0
0
5
0
0
0
0
0
17
0
0
27
21
33
68b
0
0
0
2a
0
12
n, s
c
md + ch (n, s, c)
md + ch (n, s)
md alone
34
26c
0
0
0
ch organs
l, n, s, c
lch5
82c
l, n, s
25
md + es (n)
md alone
0
0
4a
0
0
78b
0
0
2
4
1
27
md (td) neuron + ch organs
ch organs
n, s, c**
n, s
s, c
c
vchA + v′td(v)***
1
0
0
2
vchB + v′td(d)***
1
12e
3
9
13 + vpda
12 − vpda
1 + vpda
56 − vpda
6
6
*v′es2 is a poly-innervated es organ (2 neurons).
**The ligament cell was not identifiable in the ventral ch organs.
***In rare cases (2% of the clones) the td neurons might have been confused due to variability in their position.
aIn 1 or more cases other es organ cells might have been faintly stained.
bIn 17 cases a group of cells in a ventro-lateral position to v′pda were also labeled.
cIn approx. 40% of the cases, an ectodermal cell dorsal to the cap cell was also labeled.
dOnly the neuron was labeled.
eColabelling of n ans s, 3 cases; n alone, 4 cases; s alone, 5 cases.
fIn 24 cases the neuron was colabeled with v′td(d).
4 + vpda
1d − vpda
4 + vpda
29f − vpda
6
6



14 v′td
2928 R. Brewster and R. Bodmer
scolopale cell (Fig. 3H; Table 1) indicating that they are sibling
cells. 25 cases clones were composed of three cells that consistently included neuron, scolopale and ligament cell (Fig. 3G;
Table 1), which is indicative of a serial mode of ch SOP
division: the cap cell is produced first then the ligament cell
and finally neuron and scolopale cell (Fig. 5D). In this model,
the ligament/neuron/scolopale derive from a common second
order precursor. BrdU-labelling studies also suggested a serial
division pattern for ch SOPs; however, the ligament cell was
thought to be produced first. This sequence of cell divisions
implies that cap/neuron/scolopale are generated from a
common second order precursor (Bodmer et al., 1989). These
two contradictory observations can be reconciled in a model
where the first ch SOP division produces two second order precursors, one that gives rise to the ligament/neuron/scolopale
cell lineage, and the other that generates the cap and another
(ectodermal) cell. This other, cap/ectodermal cell precursor,
then replicates somewhat later than the first division of the
secondary precursor for the ligament, neuron and scolopale cell
(see Fig. 5D). Consistent with this model is the observation
that an ectodermal cell, dorsal to the cap cells, was often colabelled in clones that expressed lacZ in all cells of a scolopidium (ec-labelled cell in Fig. 3E). In 40% of the cases, where
only the cap cell was labelled to the exclusion of other ch cells,
this dorsal ectodermal cell was also labelled (Table 1). Indeed,
two ectodermal cells have been identified in the position of
these cap-related ectodermal cells as attachment cells for lch5
(Matthews et al., 1990; see also Fig. 1A). In many cases,
however, it seems the cap-related ectodermal cells fail to differentiate in this ectodermal position (perhaps because they
degenerate), thus escaping detection at the stage of analysis. In
addition, we have observed one or two labelled ectodermal
cells in embryos that have incorporated BrdU at the time of
PNS neurogenesis (R. B. and R. B., unpublished). These cells
were in the same position as the ch attachment cells and only
labelled when cap cells were also labelled. These observations
further support the proposed model of ch lineages (Fig. 5D).
A considerable number of clones (29) observed in the lch5
cluster consisted of two or more fully labelled scolopidia (individual ch organs) (data not shown). Since the frequency of
clones observed in the PNS is relatively low, most if not all of
these larger clones are probably not due to independent recombination events. This raises the possibility that two or more
scolopidia within the lch5 cluster derive from a common
precursor. Alternatively, the labelled scolopidia may have originated from a recombination event in an early ectodermal cell
(at blastoderm stage), which, after further cell divisions, gave
rise to independent ch SOPs (within the lch5 proneural region).
Clones of less than four cells per ch organ were never observed
when more than one scolopidium of lch5 was labelled.
Therefore, it is unlikely that swapping of equivalent cells
amongst neighboring scolopidia occurs with appreciable
frequency.
Md cell lineages
Md neurons constitute the third class of sensory neurons in the
Drosophila PNS. The majority of these neurons have extensive
dendritic arbors below the epidermis. Md neurons are easily
identifiable by location and shape, using the 22C10 marker.
Some md neurons are in close association with es or ch organs
(see Fig. 1A). We explored the possibility that these md
neurons could be related by lineage to their neighboring type
I sensory organs. A few PNS cells have been shown to migrate
away from their point of origin. For example, lch5 originates
in the dorsal region and ends up more laterally (Bodmer et al.,
1989; Salzberg et al., 1994), and v′ch1 originates in a ventral
cluster and migrates dorsally (Ghysen and O’Kane, 1989).
Therefore, we scored all subectodermal cells that were labelled
close to and at a distance from a labelled md neuron. Our
results suggest that md neurons derive from three types of
lineages (summarized in Fig. 5). One type of lineage probably
gives rise to md neurons exclusively (termed ‘solo-md’
lineage), whiles the others produce md neurons and es organs
(‘md/es’ lineage) or md neurons and ch organs (‘md/ch’
lineage).
Md/es lineages
Several md neurons of the da subclass are located adjacent to
es organs (Fig. 1A). In practically every case (129 out of 131
cases), when all cells of one of these es organs were labelled,
the neighboring md neuron was also labelled (Table 1):
lesA/ldaA (27 cases, Fig. 4A), lesB/ldaB (21 cases, Fig. 4B),
v′esB/v′ada (33 cases, Fig. 4C), v′es2/v′pda (68 cases, Fig.
4D). This strongly suggests that a subset of md neurons are
descendants of es SOPs. Since lesA, lesB and v′esB are
monoinnervated es organs and v′es2 is a polyinnervated es
organs, we will refer to these lineages as md/mono-es and
md/poly-es, respectively.
In order to determine the pattern of cell division of the cells
belonging to the md/mono-es lineages, we scored clones in
which only part of the md/es cells were labelled. For
v′esA/v′ada, 2 cases consisted of the es neuron, thecogen and
md neuron; and for ldaA/lesA, 4 cases consisted of the es
neuron and md neuron (Table 1). This suggests that the md
neuron derives from a second order precursor, which also
generates the es neuron and thecogen, probably by an additional division of the neuronal precursor. Due to the small
number of such clones, the order in which the md neuron, es
Fig. 4. lacZ-expressing clones that include type II md neurons.
(A-F) md/es clones. (G-J) md/ch clones. (K-M) Solo-md clones.
(A-D) Two-segment-wide micrographs with anti-β-gal-labelled
clones including all cells of an individual es organ (brackets indicate
position of labeled es organ cells and unlabeled es organ in adjacent
segment) as well as one md neuron (indicated by arrowhead, see Fig.
1A for PNS map). (A) ldaA/lesA, (B) ldaB/lesB, (C) v′ada/v′esB and
(D) v′pda/v′es2. (E,F) Two-segment-wide micrograghs consisting of
clones composed of v′es2 neurons with (E) and without (F) colabelling of v′pda (arrowhead). This suggests that this md neuron
derives from the same sublineage as the two v′es neurons. (G-I)
Clones that include all cells of a ch organ. (G) Labelling of vchA,
vchB (brackets), v′td2 (arrowheads point to dorsal (d) and ventral (v)
cell and vpda (small arrow) due to an early recombination event).
(H) Clone composed of all vchA cells, v′td(v) and vpda. (I) Clone
composed of all vchB cells and v′td(d). (J) Clone composed of only
the vchB neuron and v′td(d). (K) Clone composed of dbd neuron
(black arrowhead) and associated glial cell (curved arrow). An
unstained dbd neuron in an adjacent segment is indicated by an open
triangle. (L) Two clones that include vmd5 neurons in two adjacent
segments. In the clone on the left three vmd5 cells are labelled, on
the right only one cell of vmd5 is labelled (position of the labelled
md neurons is indicated by arrowheads). (M) vpda is the only
labelled cell in the right-hand segment (arrowhead). Open triangle
indicates position of an unlabelled vpda in adjacent segment.
PNS lineages in Drosophila 2929
K
2930 R. Brewster and R. Bodmer
neuron and thecogen cell are produced could not be determined
unequivocally.
The situation is similar for poly-es neurons (Table 1): in 12
cases, we observed that the md neuron (v′pda) was co-labelled
with the thecogen cell and the two es neurons of v′es2. This
suggests that, in md/poly-es lineages, the md neuron is also
generated from a second order precursor cell derived from the
first division of the v′es2/v′pda precursors. In 78 clones, the
md neuron and both es neurons co-labelled exclusively (Fig.
4E). Co-labelling of the es neurons and the thecogen to the
exclusion of the md neuron was almost never observed (Table
1). This means that, in the case of md/v′es2, the md and es
neurons are generated by additional divisions of the neuronal
precursor. The two v′es2 neurons were stained alone in 17
cases (Fig. 4F) and the md neuron was labeled alone in 27 cases
(Table 1), suggesting that the es neurons are siblings. In 5
cases, we observed that only the tormogen and trichogen were
co-labelled suggesting that they are also siblings (Table 1). We
conclude that the first SOP division gives rise to the
tormogen/trichogen precursor and the precursor for the
thecogen cell and the md/es neurons. The latter secondary
precursor then generates the thecogen cell and the precursor
for the md and es neurons. These lineages are consistent with
the BrdU-labelling studies, which show that the
tormogen/trichogen precursor replicates first, followed by the
precursor of the thecogen and the neurons. In the case of
md/v′es2, the precursor of the two es neurons replicates last. It
is likely that the division pattern for an md/poly-es SOP is
essentially the same as the division pattern that generates
md/mono-es, except that the precursor for the neurons of v′es2
undergoes another division to generate two es neurons. The
proposed lineages for md/mono-es and md/poly-es are summarized in Fig. 5B,C.
Md/ch lineages
Two types of clones that contained co-labeled md neurons and
ch organs were observed: one type of clone was composed of
the ventral v′td2 (an md neuron belonging to the td subclass)
and vchA; the other type of clone included the dorsal v′td2
neuron and vchB (see Fig. 1A). The v′td2 neurons are located
at some distance from vchA or vchB in the v′ cluster. Table 1
(bottom half) shows the number and types of chordotonal
organ-associated md clones that we have observed. Since the
ligament cells of vchA and vchB were not easily visible in most
clones (too close to the ch neurons), we excluded them from
the analysis. Clones composed of an md neuron and all cells
of a vch organ were observed in 82 cases (Fig. 4H,I; Table 1).
These data suggest that a subset of md neurons derives from
ch organ lineages. The other abdominal ch organs, lch5 and
v′ch, do not seem to have lineage relations to md neurons. In
some embryos, we observed vpda, an md neuron belonging to
the da subclass, co-labelled with v′td/vchA clones or with
clones including both v′td/vchA and v′td/vchB organs (Fig.
4G,H). As discussed below, this probably means that an early
recombination event generated several neighboring PNS precursors, similar to the situation with lch5.
In order to determine more precisely the relation between
vpda and v′td2 to vch organs, we scored smaller clones. 29
md/vchB clones were composed of the dorsal v′td2 and a
partially labelled ch organ (i.e. the neuron and scolopale were
labelled to the exclusion of the cap cell). In most of these cases,
Fig. 5. PNS lineages. (A-F) Proposed PNS lineages. Open symbols:
precursor cells. Filled symbols: postmitotic cells. List of cells
belonging to lineages A through F is given below lineage diagrams.
Question marks indicate uncertainty about lineage classification.
(A) Lineage of ‘pure’ es organs. (B) Lineage of md neurons related
to mono-es organs. (C) Lineage of md neurons related poly-es
organs. (D) Lineage of ‘pure’ ch organs. (E) Lineage of md neurons
related to ch organs. vpda*: part of v′td(v)/vchA lineage only (Fig.
4M) or together with vchA (Fig. 4H). Thus, vpda is likely to be a
solo-md neuron that may be generated independently but in close
vicinity of the vchA precursor. (F) Lineages of solo-md neurons.
the ch neuron alone was co-labelled with the dorsal v′td2 (Fig.
4J). In contrast, only 3 cases showed the vchB neuron and
scolopale cell not co-labelled with the dorsal v′td2. These
observations suggest that the md/vchB lineage is likely to be
similar to other ch organ lineages, the only difference being
that the ch neuron undergoes another cell division to generate
an md neuron (Fig. 5E). The md/vchA lineage is most likely
identical to the md/vchB lineage with respect to the td neuron
(Table 1), but it is unclear if the other vchA-associated md
neuron, vpda, is indeed part of the md/vchA lineage. Only 13
out of 25 cases show co-labelling of vpda with the other
md/vchA cells. This means that vpda is either generated first
in the md/vchA lineage or that it arises from an independent
ectodermal precursor in close proximity to the md/vchA
precursor (Fig. 5E). Consistent with the latter interpretation is
the observation that vpda is occasionally co-labelled with a
vchB clone (5 cases) and often by itself (40 cases) (Fig. 4M).
We classify vpda tentatively as a solo-md neuron (see below).
Solo md lineages
A number of md neurons were not usually or consistently colabelled with other sensory organ cells. These md neurons are
the dorsal bd neuron (dbd), the da neurons in the ventral vmd5
cluster and possibly a few da neurons in the dorsal cluster (see
Fig. 1A). 48 clones were observed that labelled the dbd neuron
and/or its neighboring glial cell. 20 of these clones label both
the dbd neuron and the glial cell, excluding other PNS cells
(Fig. 4K), which suggests that they are sibling cells and that
PNS lineages in Drosophila 2931
they derive from their own ectodermal precursor (Fig. 5F, top).
This is further supported by BrdU-labelling studies, which
showed that these cells divide around the same time and are
thus likely to be siblings.
The clones that include vmd5 neurons were quite heterogeneous, varying between 1 and 4 labelled vmd5 neurons (Fig.
4L), often in conjunction with ves organs. In 19 cases, vmd5
neurons were labelled to the exclusion of other PNS cells and,
in 19 other cases, vmd5 neurons were co-labelled with vesA,
vesB or vesC. There did not seem to be a consistent pattern in
which es organs were most frequently co-labelled with vmd5
neurons. Although we can not rule out a complex lineage relationship of ves organs and vmd5 neurons, it seems most likely
that the vmd5 neurons are generated from md-specific precursors, in the vicinity of ves SOPs (Fig. 5F). Some or all of the
dorsal md neurons are probably generated by a similar
mechanism although clones including these cells have not been
quantified (since they are difficult to identify individually).
Specification of md neurons is altered in numb and
cut mutants
md neurons in numb mutants
It has been suggested that in numb mutants the first SOP
division generates two identical second order precursors,
which results in the overproduction of sensory organ support
cells (tormogen/trichogen) at the expense of neuron/thecogen
cells (Uemura et al., 1989; Rhyu et al., 1994). Our lineage
analysis suggests that md neurons related to es or ch organs
are descendants of the neuronal second order precursors (Fig.
5B,C,E). In numb mutants, we would thus expect that these
SO-related md neurons are absent. To test this hypothesis, we
have crossed an enhancer trap line, which expresses the lacZ
reporter gene exclusively in all md neurons (E7-2-36; Bier et
al., 1989), into a numb mutant background. Embryos doubly
labelled for all PNS neurons and for lacZ expression show that
the sensory organ-associated md neurons are usually missing
in numb mutants (Fig. 6A,B). Although this observation is consistent with a lineage relationship between a subset of md
neurons and sensory organs, one can not rule out that the md
neurons are affected independently by numb as well. This is
supported by the observation that the solo-md neurons, vpda
and some vmd5 (and several dmds) were often missing in
numb (Fig. 6B).
The transformation of the neuron and thecogen cells into
tormogen/trichogen-like cells is frequently incomplete (see
Uemura et al., 1989). We reasoned that, if the es-related md
neuron (md/es) and the es neuron derive from the same second
order precursor lineage, both es and md/es neuron should either
be present (incomplete transformation) or absent (complete
transformation). Embryos, doubly labelled for all PNS neurons
and for lacZ reporter gene expression in md neurons, show that
this prediction was correct: both the es and md/es neurons
either did or did not express neural markers simultaneously
(Fig. 6B). Similar observations were made in numb mutant
embryos that were doubly labelled for md neurons and
thecogen cells: every time an md/es neuron was present the
neighboring thecogen cell was also labelled (Fig. 6C,D). Interestingly, two or three cells positive for the thecogen marker
were frequently present in the location of md neurons/mdrelated es organs (indicated by brackets in Fig. 6D). This
suggests that the first SOP division appears to be normal in
some cases, but the secondary precursor for the thecogen and
neurons now generates only thecogens (Fig. 6G, top panel).
These results also indicate that numb not only acts in generating asymmetry during the first SOP division but also during
both secondary SOP divisions (see also Rhyu et al., 1994).
Since our md marker is only weakly expressed in md-td
neurons associated with ch organs, we have not examined their
fate in numb mutants. Taken together, our analysis of numb
mutants strongly supports the conclusions from the lineage
studies.
To determine if numb also affects other md lineages, we
examined the fate of solo-md neurons in these mutants. We
observed only 2-3 neurons in vmd5 or the dorsal cluster. Due
to the lack of other markers, we do not know if a hypothetical
solo-md precursor divides less or if the fate of its progeny is
altered. The md-bd neuron of the dorsal cluster is also absent
in numb mutants (Fig. 6E,F). Our lineage studies suggest that
dbd is generated from a dbd-specific ectodermal precursor,
which divides once to give rise to a glial cell and dbd (Fig. 6E).
In numb mutants, instead of one dbd neuron and one glial cell,
two glial cells are formed at the expense of the dbd neuron
(Fig. 6F,G, lower panel). Therefore, numb not only affects the
es and ch lineages but also other components of the PNS.
It had previously been shown that in numb mutants an excess
of cap cells are produced but the fate of the ligament cell was
not clear. Since lch5 neurons are rarely formed in numb
mutants (Uemura et al., 1989), we expected that the second
order precursor is often transformed into a cap-like precursor.
Consistent with our proposed ch lineages, we find that in lch5
not only neurons and scolopales are missing but also the
ligament cells (Fig. 6E,F).
cut mutant phenotype
The da subclass of md neurons appears to be a morphologically homogeneous population of cells (Bodmer and Jan,
1987). The observation that these cells are generated by
different types of lineages (this study) and that the axonal projections to the CNS are not identical for all da-md neurons
(Merritt and Whitington, 1995), raises the possibility that there
are distinct subpopulations of da neurons. To address this
question and identify genes that may play a role in the specification of md neurons, we sought for cell markers that differentially label subsets of da neurons. One such marker is the cut
gene product. cut is first expressed in es SOPs and later in their
progeny including the es-related md-da neurons (see Figs 5B,C,
7C; Blochlinger et al., 1990). Since cut functions as a developmental switch to specify the correct identity of es organs
(Bodmer et al., 1987; Blochlinger et al., 1991), we wondered
if cut may also be involved in the specification of da neurons.
In cut mutants, the morphology and position of md neurons is
not affected (data not shown). To test for more subtle changes,
we used an enhancer trap line, E7-3-49 (Bier et al., 1989),
which marks a subset of md neurons that is non-overlapping
with the Cut-positive da neurons: vpda, dbd and 3-4 dorsal da
neurons (Fig. 7A,D). At least one of the cells marked with E73-49 (vpda) is atonal-dependent (Jarman et al., 1993).
The lacZ expression of the E7-3-49 enhancer trap line is dramatically altered in cut mutant embryos. In addition to the cells
in which it is normally expressed, β-gal staining is now
observed in the da neurons that are normally Cut positive: all
dorsal cluster da neurons, ldaA&B, v′ada, v′pda and 3-4
2932 R. Brewster and R. Bodmer
Fig. 6. Cell fate changes of md neurons in numb mutants. (A-F) Two segments of wild-type (A,C,E) and numb mutant (B,D,F) embryos at
stage 16 are shown. (A,B) Embryos double-labelled for nuclear md-specific lacZ expression of the enhancer trap line, E7-2-36 (Bier et al.,
1989), and 22C10 (cytoplasmic labelling of PNS neurons). Arrowheads indicate md neurons. Dotted lines indicate vmd5. Open triangles
indicate lesA and v′esB neurons which are also occasionally present in numb mutant embryo (B). In B, left arrowhead points to v′ada and right
arrowhead to ldaA (in an adjacent segment); asterisks indicate missing md neurons. (C,D) Embryos double-labelled with thecogen/scolopalespecific anti-Prospero antibodies (black, Vaessin et al., 1991) and for md-specific lacZ expression of the E7-2-36 line (brown). Arrowheads
indicate md neurons. Open diamond indicates the Prospero-positive thecogen cell of v′esB. Asterisks indicate missing md/es neurons. Brackets
show md/es organs with 2-3 Prospero-positive cells indicating transformation of the md and/or es neuron into thecogen cells. (E,F) Embryos
double-stained with 22C10 antibodies (brown) and glia-specific anti-RK-2 antibodies (Campbell et al., 1994) (black). Arrowhead indicates a
dbd-md neuron and the curved arrow the dbd-related glial cell (black). RK-2 antibodies also label the ligament cells of lch5 (outlined by dotted
lines in E), which are absent in numb (F). Note the transformation of the dbd neuron into an RK-2-positive glial cell in numb (F).
(G) Diagrammatic representation of the lineage changes associated with the numb mutation. Upper panel: either the first SOP division produces
two equal secondary SOPs (a,a′) giving rise to only support cells (no neuron or thecogen cells are formed), or the secondary SOP (b) is formed
but gives rise to another thecogen precursor (b′) in addition to the normal thecogen cell. Lower panel: dbd/glia lineage in wild-type and numb
mutants.
neurons of vmd5 (Fig. 7A-E). We conclude from these observations that there are at least two subclasses of da neurons, Cutnegative and Cut-positive da neurons, and that cut is likely to
be involved in specifying the identity not only of es organs but
also of a subpopulation of da neurons (Fig. 7F).
DISCUSSION
Lineage relationships in the PNS
The analysis of patterns of cell division in a number of insects
and BrdU-labelling studies in Drosophila embryos and wing
imaginal discs have provided evidence that the cells within
individual es and ch organs are derived from single precursor
cells (reviewed in Bate 1978; Bodmer et al., 1989; Hartenstein
and Posakony, 1989). The pattern of BrdU incorporation in
embryos was suggestive of a cell division pattern that was
different for es organs and for ch organs (Bodmer et al., 1989).
Using the yeast flipase method to generate small clones in the
embryo (Golic and Lindquist, 1989; Struhl and Basler, 1993),
we have reassessed the lineages of type I sensory organs (es
and ch) and examined the lineages of type II sensory neurons
(md neurons) and their relationship to type I sensory organs,
by scoring an excess of 5000 embryos.
es and ch organ lineages
We scored clones that included cells of the dorsal most es
organs (desC/D) and the lateral ch organs (lch5). We observed
that a majority of clones were composed of all cells belonging
to an es or ch organ, which indicates that es and ch organs
derive from individual SOPs. The cellular compositions of
clones that included only a fraction (but more than one) of
labelled cells within an individual es or ch organ allowed us to
PNS lineages in Drosophila 2933
Fig. 7. Identity changes of md neurons in cut mutants. (A,B) Two abdominal segments of stage 16 embryos stained for lacZ expression of the
enhancer trap line, E7-3-49, which labels most md neurons that are Cut-negative (see C,D), and with 21A6 (Zipursky et al., 1984), which is
specific for es-associated dendritic caps and ch-associated scolopales. Segment boundary cells are also labeled in this enhancer trap line (arrows
indicate the position of the segment boundary). The wild-type pattern of β-gal-positive md neurons is indicated by arrowheads in one of the
segments of A and B. md neurons that ectopically express lacZ in cut mutants (pointed out by asterisks in B) are in the identical position of md
neurons that normally express the Cut protein (C, symbols in blue). A diagrammatic representation of the wild-type expression pattern of the
E7-3-49 line is given in D (symbols in red indicate position of lacZ-positive mds). (E) The lacZ expression pattern of the E7-3-49 line is
expanded in cut mutants, to encompass virtually all md neurons, suggestive of a change in identity of the md neurons that are normally Cutpositive. The level of ectopic lacZ expression is variable. (F) cut mutant phenotype in the PNS: not only are the es organs transformed into ch
organs (see Bodmer et al., 1987), but the subset of md neurons that is normally Cut-positive (mdes and mdsolo) is also transformed into E7-3-49positive md neurons (mdE7) (see text).
infer the most likely lineage relationships. For es organs, we
observed co-labelling of either the neuron/thecogen or the
tormogen/trichogen, confirming the previously proposed
division pattern for es SOPs (Fig. 5A; Bodmer et al., 1989).
For ch organs, we observed the following combinations of
labelled cells: ligament/scolopale/neuron, cap/attachment cell
or scolopale/neuron. This suggests a division pattern for ch
organs that is a modification of what had been previously
proposed, but which is also consistent with the BrdU studies
(illustrated in Fig. 5D). In this model, the two second order
precursors replicate at different times: the ligament/
scolopale/neuron precursor does so before the cap and attachment cell precursor. In agreement with the proposed ch lineage
pattern is the finding of several clones in which the cap and
attachment cell are co-labelled (Table 1), indicating that the
recombination event took place in the immediate precursor of
these two cells.
A relatively large number of multiple ch organs was labelled
in lch5 (the lateral chordotonal cluster), suggesting that ‘super
SOPs’ could give rise to more than one ch organ. It has previously been suggested that there are at least two ch organ precursors for lch5 that emerge during early stage 10 (Ghysen and
O’Kane, 1989). Since BrdU-labelling studies have shown that
the precursors that give rise to lch5, divide in a graded fashion,
always proceeding from anterior to posterior in each segment
(Bodmer et al., 1989), we expected that clonally related ch
organs would be adjacent to one another. However, the number
of adjacent scolopidia that were co-labelled was far lower than
the number of non-adjacent clones (R. B. and R. B., unpublished). Moreover, the clones observed were composed of
random combinations of scolopidia (identified by their position
along the anterior-posterior axis). This makes it unlikely that
multiple scolopidia in lch5 are generated by a fixed lineage
pattern but rather argues in favor of the existence of multiple
precursors (possibly five) for the lch5 cluster. We speculate
that these precursors emerge independently, in close vicinity
to one another, in the posterior lateral region of each segment
(Ghysen and O’Kane, 1989). One cannot rule out, however,
that clonally related precursor cells rearrange themselves
randomly after they have been generated.
Multiple md lineages
Three types of clones were observed that included md neurons:
a subset of md neurons that were almost always co-labelled with
closely juxtaposed es organs (md/es), another subset of md
neurons that co-labelled with ch organs (md/ch) and a third
2934 R. Brewster and R. Bodmer
group of md neurons that did not co-label with other sensory
organs in a consistent and reproducible pattern (solo mds). We
conclude from this clonal analysis that es- and ch-associated md
neurons are related to these sensory organs by lineage and that
they share a common SOP (lineages are summarized in Fig. 5).
Clones that labeled all cells of the es organs, v′es2, v′esB,
lesA and lesB, always included the associated md-da neuron,
whereas the chordotonal organs vchA and vchB co-labelled
with one of the v′td2 md neurons. We inferred the patterns of
cell division from smaller clones where only a fraction of a
sensory organ was labelled (see Fig. 4; Table 1): SO-related
md neurons derive from secondary SOPs that also give rise to
the SO neuron(s).
The vpda md neuron often co-labelled with vchA and vmd5
neurons often co-labelled with one of the ves organs. These md
neurons may also have lineage relations to sensory organs,
possibly generated by an early ectodermal division (see Fig.
5E). As we argued for co-labelling of multiple scolopidia of
lch5, it is likely that the high frequency of co-labelled
vpda/vchA or vmd5/ves simply reflects the close proximity of
independently emerging SOPs, and is not due to a necessary
lineage relation. This possibility is supported by the finding
that in mutations of the rhomboid gene, vchA is usually absent
without the concomitant deletion of vpda (Bier et al., 1990).
Therefore, we classify vpda and vmd5 as solo-md neurons. The
dorsal bd neuron and its sibling glial cell were never consistently co-labelled with SO cells and was therefore also classified as a solo md neuron.
The putative es-related md neurons also happen to be among
the md neurons that are missing in AS-C mutants (DamblyChaudiere and Ghysen, 1987). This suggests that genes of ASC are required for the formation of precursors common to es
organs and a subset of md neurons. Similarly, the atonal gene
seems to be required for the precursors common to vchA,
vchB, the v′td2-md neurons and vpda (Jarman et al., 1993).
Fate of md neurons in lineage mutants
In mutants of the numb gene, the second order precursor of es
organs which gives rise to the neuron and thecogen cell is
usually transformed into its sibling, the tormogen/trichogen
second order precursor (Uemura et al., 1989). Our proposed
model of md/mono-es lineages predicts that the fate of the md
neurons related to es organs may also be altered in numb
mutants since these md neurons appear to have the same
second order precursor as the neuron and thecogen cell. The
finding that most md neurons are absent in numb mutants
supports this finding, but it could be argued that md neurons
and sensory organs are affected independently. Close examination of partially transformed md/es organs (see also Uemura
et al., 1989) indicate that, whenever a thecogen cell and an es
neuron is formed, the associated md neuron is formed as well
(Fig. 6B). This strongly supports that es-related md neurons
derive from the same second order precursor as neuron and
thecogen cell.
numb not only affects the lineage of es and ch organs in the
PNS but also that of solo-md neurons, since most of them are
absent (Uemura et al., 1989). Although the fate of most solo-md
neurons in numb mutants could not be determined due to lack
of specific markers, the dorsal bd neuron (dbd), is a notable
exception. In numb mutants, dbd is transformed into its associ-
ated glial cell, consistent with them being siblings and requiring
numb for distinguishing between a neuronal and glial cell fate.
In poxn mutants, the poly-es organs are transformed into
mono-es organs (Dambly-Chaudiere et al., 1992). At least for
v′es2, the formation of the associated md neuron is not affected
in these mutants (C. Dambly-Chaudiere and A. Ghysen,
unpublished data). This means that, although the identity of the
whole v′es2 has changed in poxn mutants, the only lineage
defect concerns the immediate precursor of the es neurons.
Md neurons have previously been classified into three broad
categories: da neurons characterized by large dendritic arrays,
td neurons whose dendrites extend along trachea and bd
neurons which have bipolar dendrites (Bodmer and Jan, 1987).
Our clonal analysis of the PNS suggests that md neurons can
be further classified into different subtypes defined by lineage.
Two markers, Cut and E7-3-49 label non-overlapping subsets
of md neurons. In addition to es organs, cut is expressed in the
es-related da neurons and some of the potential solo da neurons
(e.g. in vmd5). E7-3-49 expresses the lacZ reporter in a set of
da neurons that is essentially complementary to the cutexpressing da neurons (Fig. 7C,D). In cut mutant embryos, the
E7-3-49 driven lacZ expression in md neurons is expanded to
encompass virtually all da-md neurons including all es-related
da neurons (see Fig. 7B,E). In contrast, when the Cut protein
is overexpressed via a heat-shock promoter (Blochlinger et al.,
1991) during the time of PNS neurogenesis in embryos that are
wild-type for cut, E7-3-49 driven lacZ expression is greatly
reduced or absent in those md neurons that normally express
lacZ (i.e. vpda and the dorsal cluster da neurons)(R. B. and R.
B., unpublished observations). Therefore, cut seems to be a
necessary and sufficient component for the specification of the
correct identity of es-related da neurons, similar to its role in
es organ development (Bodmer et al., 1987; Blochlinger et al.,
1991). Since the cut gene product contains a homeodomain
(Blochlinger et al., 1988), cut could act directly on downstream
genes as a transcriptional activator and/or as a repressor. A vertebrate Cut-related protein, CDP/Clox/Cux1 (Neufeld et al.,
1992; Andres et al., 1992; Valarche et al., 1993), has been
shown to function as a transcriptional repressor in co-transfection experiments (Andres et al., 1992; Valarche et al., 1993;
Dufort and Nepveu, 1994; see also Skalnik et al., 1991). A
possible repressor function by Drosophila Cut is supported by
our finding that E7-3-49 driven lacZ expression is apparently
suppressed in md neurons that normally or ectopically express
Cut. Thus, cut function seems not only required for the specification of es organs but also for a subset of md neurons. Other
selector-type genes, which may function in parallel or downstream of cut, are likely to be required to further specify the
fate of different cell types, including es-related md neurons and
es neurons.
How do axonal projections of md neurons correlate
with their lineage identity?
Morphologically, md-da neurons look quite similar (Bodmer
et al., 1987). However, the fact that md-da neurons derive from
distinct lineages, raises the possibility that these cells form
functionally distinct subpopulations, and the axonal projections of different types of md neurons in the CNS may reflect
these differences. Merritt et al. (1993) have previously shown
that the central projections of es neurons are distinct from those
of ch neurons and that cut is required for the correct projec-
PNS lineages in Drosophila 2935
tions of es organs. Md neurons also have distinct central projections (Merritt and Whitington, 1995). (1) Most md-da
neurons form one class and project into a discrete longitudinal
CNS fascicle. They are dependent on AS-C and virtually all
express cut. (2) In contrast, the atonal-dependent v′td2 and
vpda md neurons have a projection pattern that is different
from the majority of the md-da neurons. (3) The dbd-md
neuron and one of the dorsal md-da neurons do not depend on
a known proneural gene and their projections seem to be
distinct from all others. This suggests that the central projections of md neurons reflects their requirement for proneural
genes (Merritt and Whitington, 1995).
How do the lineage relations of md neurons correlate with
a particular projection pattern? The es-related da neurons and
most solo-da neurons seem to have an undistinguishable projection pattern, but the ch-related v′td2 neurons and the dbdmd neuron have distinct projections. Although lineage relationships may not be an absolutely reliable indicator of md
neuronal identity, md neurons of similar lineages also have
similar projections in the CNS (e.g. all es-related md neurons
project into the same fascicle). Thus, it seems that proneural
genes, selector genes (cut) and lineage relationships, as well as
other unknown factors, have influential roles in determining
the identity of md neurons.
We are much indebted to Gary Struhl for generously providing us
with the flipase and FRT-lacZ transformant flies. We thank Greg
Gibson, Alain Ghysen and David Merrit for critical reading of the manuscript. We also thank Corey Goodman for the 22C10 antibody,
Andrew Tomlinson for the RK2 antibody, Chris Doe for the antiprospero antibody and Seymour Benzer for the 21A6 antibody. We
also thank the Dr Yuh-Nung Jan and the Bloomington Stock Center
for sending fly stocks. This work was supported by a grant from NIH
to R. Bodmer.
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(Accepted 13 May 1995)