623
Development 107, 623-636 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Complex spatio-temporal accumulation of alternative transcripts from the
neurogenic gene Delta during Drosophila embryogenesis
CASEY C. KOPCZYNSKI and MARC A. T. MUSKAVTTCH
Programs in Genetics and Molecular, Cellular and Developmental Biology, Department of Biology, Indiana University,
Indiana 47405, USA
Bloomington,
Summary
Delta (Dl) function is required for proper specification of
epidermal and neural lineages within the neurogenic
ectoderm of Drosophila melanogaster. We have determined the spatial accumulation offiveDl transcripts that
arise as the result of alternative RNA processing during
embryogenesis. We find that these transcripts accumulate in all cells of the neurogenic ectoderm immediately
preceding neuroblast segregation, indicating that transcription of Dl does not differ between presumptive
neuroblasts and presumptive dermoblasts. Dl transcripts also accumulate transiently in mesodermal and
endodermal cells, suggesting that Dl may function in
developmental processes in addition to differentiation of
the neurogenic ectoderm. We find that three of the Dl
transcripts are localized to the base of the nucleus during
cellularization. The apparent association of these three
transcripts with polysomes suggests that they accumulate within the cytoplasm at the nuclear periphery and is
consistent with the hypothesis that Dl encodes multiple
translation^ products.
Introduction
vealed that the Notch product is a transmembrane
protein with an extracellular domain that contains 36
cysteine-rich, epidermal growth factor-like repeats
(Wharton etal. 1985; Kidd etal. 1986). The structure of
this polypeptide led to the hypothesis that TV and other
neurogenic genes may function in a system of cell-cell
communication required for the proper differentiation
of neurogenic ectoderm into neural and epidermal
lineages (Wharton et al. 1985; Kidd et al. 1986). This
hypothesis has received direct experimental support
from embryonic cell transplantation experiments
(Technau and Campos-Ortega, 1987). These experiments revealed that the neurogenic mutant phenotype
of a cell lacking the function of TV, Dl, mam, neu or bib
can be rescued when the cell is transplanted among wild
type ectodermal cells. Somatic mosaic analyses have
further revealed that while single TV mutant cells can be
rescued by surrounding wild type cells, 'patches' of two
or more cells are rarely rescued (P. Hoppe and R.
Greenspan, in preparation), indicating that the nonautonomous function of TV is locaHy restricted.
The subsequent molecular characterization of Dl and
E(spl) has provided further support for the hypothesis
that the neurogenic genes function in intercellular
communication (Vassin et al. 1987; Kopczynski et al.
1988; Hartley et al. 1988). The predominant embryonic
and maternal Dl transcripts encode a putative transmembrane protein (D1ZM) with an extracellular
domain that contains nine cysteine-rich repeats hom-
The central nervous system of Drosophila is derived
from a subset of ectodermal cells located within the
neurogenic regions of the embryo (Campos-Ortega and
Hartenstein, 1985). Neurogenesis begins within these
regions shortly after gastrulation when presumptive
neuroblasts, progenitors of the nervous system, delaminate from the ectodermal epithelium and segregate into
the interior of the embryo. The cells that remain within
the epithelium after neuroblast segregation are dermoblasts, progenitors of the ventral and cephalic epidermis of the larva. Thus, the neurogenic ectoderm of
Drosophila gives rise to both neural and epidermal
lineages.
The zygotic function of six genes - Notch (TV), Delta
{Dl), Enhancer of split [E(spl)], neuralized (neu),
mastermind (mam) and big brain (bib) - is known to be
required to establish epidermal identity within neurogenic regions of the embryo (Poulson, 1937; Lehmann
et al. 1983). Homozygosity for loss-of-function mutations in any one of these genes leads to hypertrophy of
the central nervous system and reduction of the ventral
and cephalic epidermis. Histological studies indicate
that this phenotype results directly from the misrecruitment into the neural lineage of ectodermal cells that
otherwise would enter the epidermal lineage (Lehmann
etal. 1983).
Molecular and biochemical analyses of TV have re-
Key words: Drosophila, neurogenic gene, ectodermal
differentiation, in situ hybridization, alternative transcripts.
624
C. C. Kopczynski and M. A. T. Muskavitch
ologous to the 36 EGF-like repeats present in the Notch
polypeptide (Vassin etal. 1987; Kopczynski etal. 1988).
Additional polypeptides distinct from D1ZM may be
encoded by less abundant (minor) embryonic and
maternal Dl transcripts (Kopczynski et al. 1988). A
neurogenic gene within the E(spl) region has been
identified recently (Hartley et al. 1988) that encodes a
putative protein with homology to /3-transducin, a
protein that is involved in vertebrate phototransduction
(reviewed in Gilman, 1987). These results reveal a
potential for functional interactions among these products and the Notch product that is particularly interesting given the array of genetic interactions that has been
described among N, Dl and E(spl) (Campos-Ortega et
al. 1984; Dietrich and Campos-Ortega, 1984; Vassin et
al. 1985; Alton etal. 1989; Shepard etal. 1989).
The accumulation of N and E(spl) transcripts in cell
types other than epidermal and neural precursors has
led to the suggestion that these genes may affect
developmental processes in addition to differentiation
of the neurogenic ectoderm (Hartley et al. 1987; Artavanis-Tsakonas, 1988; Hartley et al. 1988). We have
determined the spatial accumulation patterns of the
predominant and minor Dl transcripts during embryogenesis in order to address the question of whether Dl
function is required solely for delineating epidermal
and neural identities within the embryonic ectoderm. In
contrast to a previous report (Vassin et al. 1987), we
find that Dl transcripts are present in derivatives of all
three germ layers of the embryo and are not restricted
to neurogenic regions of the ectoderm. Thus, the spatial
and temporal accumulation patterns that we observe
suggest that Dl may act pleiotropically during embryogenesis. We also present evidence suggesting that the
minor Dl transcripts are subcellularly localized within
the cytoplasm to the nuclear periphery.
Materials and methods
In situ hybridization
Embryo collection, fixation and paraffin embedding were
performed as described in Ingham et al. (1985). Paraffin
blocks containing embryos were cut into 5/OTI sections that
were collected on glass microscope slides coated with poly-Dlysine (SO^gmF1 in 10mM-Tris-HCl pH8.0). Sections were
dewaxed by two 10 min incubations in xylene, then rehydrated
by incubation for 2 min each in 100 % EtOH, 95 % EtOH (v/v
in H 2 O), 70% EtOH, 50% EtOH, 30% EtOH and PBS
(130mM-NaCl, 7mM-Na2HPO4, 3mM-NaH2PO4). The slides
were then treated with 0.25 % (v/v) acetic anhydride in 0.1 Mtriethanolamine pH 8.0 for 10 min (Hayashi et al. 1978), rinsed
in PBS, dehydrated through the above EtOH series and air
dried. The subsequent hybridization, washing and autoradiography of the slides were performed as described in Ingham
et al. (1985). After autoradiography, embryo sections were
stained with Mayer's hematoxylin (Clayden, 1971) for
1-4min and with Giemsa stain (Fisher) for 2 min, dehydrated
through the above EtOH series, then incubated twice for
2 min in xylene before mounting in Permount (Fisher).
Morphological features present in individual sections were
identified, when necessary, on the basis of comparison with
alternate Giemsa-stained sections.
Probes
Antisense RNA probes were generated using T3 or T7 RNA
polymerase and S-UTP as described in Hartley et al. (1988).
Each probe was labelled to a specific activity of
2xl0 8 ctsmin~ 1 ^g~ 1 and used at a concentration of
Q3ngiA~lkb~l. Templates employed for the synthesis of
probes (Fig. 1) included: a 1.5 kb Pstl/EcoRl DNA fragment
corresponding to the 3' end of cDNA insert D12 (Kopczynski
et al. 1988) for 5.4Z and 4.5M, a 2.2 kb Dl genomic fragment
(fragment F, ibid.) for 3.5z, a 0.4kb Dl genomic fragment
(fragment H, ibid.) for 2.8z and a 2.5 kb Dl genomic fragment
(fragment B, ibid.) for 5.4z. A 3 kb Ngenomic Sg/II fragment
(ibid.) was used to generate a probe for N transcripts and a
118 bp Taql fragment from the 3'-untranslated region of the
beta3-tubulin gene was used to generate a beroi-tubulinspecific probe (Kimble et al. 1989). A sense-strand RNA
probe, synthesized using a 2.2 kb Dl genomic fragment
(fragment L, Kopczynski et al. 1988) as a template, was used
to determine levels of non-specific ('background') hybridization to sections (data not shown).
Polysomal RNA preparation
Fertilized eggs were collected for a 3h interval at 25°C, aged
an additional 2h at 25°C, then harvested and dechorionated
as described in Alton et al. (1988). The dechorionated
embryos were blotted dry, then 2g of embryos were hom5.4z
3.5z
2.8z
5.4Z/4.5M
sense
(126)
(96)
R
B
Sc
RR
RSP
PP
R
I
I
I
I I
II I
II
I
5.4 kb (Z)/5.4 kb(z)
4.5 kb (M)
3.6 kb (m)
3.5 kb (z)
2.8 kb (z)
Fig. 1. Transcript-specific probes used for in situ
hybridization. Thefilledarrows above the restriction map
represent the templates used to generate antisense-strand
RNA probes that were employed to detect 5.4z, 3.5z, 2.8z
and 5.4Z/4.5M transcripts, respectively (see MATERIALS
AND METHODS). The hatched arrow represents the
template for a sense-strand RNA probe used to assess
background levels of hybridization. The open arrows below
the restriction map represent a composite of the restriction
fragments that hybridize to the specified Dl transcripts
(Kopczynski et al. 1988). This diagram indicates that the
restriction fragment used to generate the 2.8z-specific probe
hybridizes to 5.4Z and 4.5M as well. However, RNA blot
analysis with this fragment reveals that the 2.8z signal
intensity obtained is approximately 20 times those observed
for 5.4Z and 4.5M (data not shown). In addition, the in situ
hybridization patterns obtained with this probe are identical
to those obtained with probes specific for 5.4z or 3.5z (data
not shown). We therefore conclude that this fragment can
be employed to specifically detect the 2.8z transcript in in
situ hybridization analyses. Numbers in parentheses are
coordinates from a chromosomal walk that encompasses Dl
(ibid.) given in kilobasepairs of DNA. Only selected
restriction sites are presented. (B), BamHl; (P), Pstl;
(R), EcoRI; (S), Sail; (Sc), Sad.
In situ analysis of Delta
ogenized in 15 ml ice-cold polysome buffer [40 mM-Tris-HCl
pH8.5, 5mM-KCl, 10mM-MgCl2, 0.5% (v/v) diethylpyrocarbonate] using a Dounce homogenizer. The homogenate was
centrifuged at 5000 revs min"1 for 2min at 4°C in a Beckman
JS13 rotor, and 2 ml of the supernatant were layered over a
20-50% (w/v) sucrose gradient in Mg2+-sucrose buffer
(20 mM-Tris-HCl pH8.5, 50mM-KCl, 10mM-MgCl2,
lmgmP 1 heparin) and centrifuged at 25 000 revs min for
2h at 4°C in a Beckman SW27 rotor. The gradient was
collected as 1.5 ml fractions through an ISCO UA-5 spectrophotometer. These fractions were subsequently pooled to
give the five larger fractions indicated in Fig. 9. Total RNA
was prepared from these fractions as described in Anderson
and Lengyel (1981). The purified RNA pellets were each
resuspended in 160 (A of deionized, distilled H2O and stored
at -70°C.
To achieve efficient EDTA-disruption of polysomes, 8 ml of
homogenate were layered onto a 3 ml sucrose pad (1.5 Msucrose in Mg2+-sucrose buffer) and centrifuged at
35 000 revs min"Ffor 90 min at 4°C in a Beckman SW41 rotor.
The polysome pellet was then resuspended in 8 ml of EDTA
gradient buffer (20 mM-Tris-HCl pH8.5, 50mM-KCl, 40 mMNa2EDTA, lmgml" 1 heparin) and 2 ml of resuspended
polysomes were layered onto a 20-50 % (v/v) sucrose gradient in EDTA gradient buffer. This gradient was run in parallel
with the Mg2^containing sucrose gradient and processed as
described above.
RNA blot analysis
RNA samples were denatured with glyoxal, fractionated on
agarose gels and transferred to nylon membranes as described
in Kopczynski et al. (1988). ^P-labeling of the DNA probes
and hybridization of the blots were performed as described in
Feinberg and Vogelstein (1983). The transcript-specific
probes used were Dl genomic fragments B (5.4z), F (3.5z), I
(2.8z) and L (5.4Z/4.5M) (Fig. 1, Kopczynski et al. 1988).
Results
The Dl transcripts
Dl encodes at least six distinct transcripts: 5.4Z, the
predominant embryonic transcript; 5.4z, 3.5z and 2.8z,
the minor embryonic transcripts; 4.5M, the predominant maternally-loaded transcript; and 3.6m, a minor
maternally-loaded transcript (Kopczynski et al. 1988).
The 4.5M transcript appears to encode the same polypeptide as 5.4Z and is probably expressed zygotically as
well as maternally (ibid.).
We have assessed the spatial accumulation patterns
of Dl transcripts using the probes that are depicted in
Fig. 1. The hybridization patterns we present for the
predominant transcripts, which were obtained with the
probe that recognizes 5.4Z and 4.5M, are identical to
those obtained with a probe specific for 5.4Z after the
zygotic accumulation of Dl transcripts begins (data not
shown). We present the spatial accumulation pattern of
only one minor transcript, 3.5z, because we observe no
difference between the patterns obtained with a 3.5zspecific probe and probes specific for 5.4z or2.8z (Fig.l,
data not shown). The accumulation of 3.6m transcripts
has not been assessed because we have not yet defined a
probe specific for this transcript. The stages of embryonic development referred to below have been defined
by Campos-Ortega and Hartenstein (1985).
625
The pattern through gastrulation
Maternally-loaded Dl transcripts are uniformly distributed throughout the embryo during the nine nuclear
division cycles that precede syncytial blastoderm
(Fig. 2A). The minor zygotic transcripts are not detectable above background [as assessed by hybridization
with a sense strand probe (data not shown)] during this
time (Fig. 2B). Zygotic accumulation of the predominant and minor transcripts begins at the start of
cellularization (early stage 5) and is localized to the
presumptive neurogenic regions of the embryo
(Figs 2C, D, 3A, B). The transcripts are initially distributed in a dorsoventral gradient within the ventral
neurogenic region (Fig. 3A, B), but become evenly
distributed within this region as cellularization proceeds
(Fig. 3C, D).
The predominant and minor transcripts accumulate
in all regions of the ectoderm during gastrulation with
the exception of the presumptive foregut (stages 6 and
7, Fig. 2E-H). Dl transcripts are also present in the
anterior and posterior midgut invaginations and at low
levels in the mesoderm (Fig. 2E-H). Thus, Dl transcripts are expressed in all three germ layers of the
embryo at this time.
Two significant differences between the predominant
and minor transcript accumulation patterns become
apparent during cellularization. First, the minor transcripts are localized to the base of each nucleus,
whereas the predominant transcripts are distributed
throughout the cortical cytoplasm (Fig. 3E, F). This
localization of minor transcripts could represent accumulation at a basal position within the nucleus or
accumulation in the cytoplasm that surrounds the basal
half of the nucleus. These possibilities cannot be
distinguished by in situ hybridization (however, see
below). Second, the accumulation of the minor transcripts appears to extend into the dorsal ectoderm and
presumptive mesoderm significantly earlier than the
zygotic accumulation of the predominant transcripts
(Fig. 3C, D). However, it is possible that the zygotic
accumulation of predominant transcripts begins at the
same time within these regions, but is more difficult to
detect due to the more diffuse cytoplasmic distribution
of the predominant transcripts. The presence of the
predominant maternally-loaded transcript throughout
the cortical cytoplasm of the precellular embryo
(Fig. 3A) may also obscure the initial accumulation of
predominant transcripts outside of the neurogenic regions.
The pattern during germ band elongation and extended
germ band
The spatial accumulation of the predominant and minor
Dl transcripts from approximately three-and-one-half
through seven hours of embryogenesis (stage 8 through
stage 11) is presented in Fig. 4. The accumulation of Dl
transcripts in endodermal derivatives (anterior and
posterior midgut) remains relatively constant throughout this interval. However, the accumulation of Dl
transcripts in the ectoderm and mesoderm changes
continuously as these germ layers give rise to their
626
C. C. Kopczynski and M. A. T. Muskavitch
vNR
Fig. 2. Distribution of £>/ transcripts from presyncytial blastoderm through gastrulation. Parasagittal and sagittal sections of
embryos hybridized with the 5.4Z/4.5M (left column) or 3.5z (right column) probes are shown. All sections are oriented
dorsal side up and anterior to the left. (A, B) Alternate parasagittal sections through a stage 3 embryo (darkfield
illumination). Maternally-loaded 4.5M transcripts are evenly distributed throughout the embryo; 3.5z transcripts are not
detectable above background. (C, D) Parasagittal sections through mid- (C) and early (D, darkfield) stage 5 embryos.
5.4Z/4.5M and 3.5z transcripts are most prevalent within the procephalic (pNR) and ventral (vNR) neurogenic regions of
the embryo. (E, F) Alternate sagittal sections through a stage 6 embryo. 5.4Z/4.5M and 3.5z transcripts are present within
most cells of the embryo, including germ band mesodermal cells (ms) and cells in a region of the embryo that will give rise
to the cephalic mesoderm and anterior midgut (arrowheads). Transcripts are not detectable in cells of the presumptive
foregut (fg). (G, H) Alternate parasagittal sections through a stage 7 embryo. 5.4Z/4.5M and 3.5z transcripts are present in
ectodermal, mesodermal and endodermal [anterior and posterior midgut (pm)] cells. Cells of the presumptive foregut remain
unlabeled at this stage. The arrowheads point to labeled cells of the cephalic mesoderm and anterior midgut primordium.
Exposure times: (A, B, D, G) 4 days; (C, E, F, H) 10 days.
respective differentiated tissues. We therefore present
the temporal changes in Dl transcript accumulation
patterns within the context of ectodermal and mesodermal differentiation during this interval.
Dl function is required during the period of neuroblast segregation to establish epidermal identity in the
neurogenic ectoderm (Lehmann et al. 1983). The pattern of Dl transcript accumulation within the neuro-
genic ectoderm during this period is presented in detail
in Fig. 5. The predominant and minor transcripts accumulate in all cells of the ventral and procephalic
neurogenic ectoderm during stage 8 immediately prior
to neuroblast segregation (Figs4A, B, 5A, B). Dl
transcripts are still present in the peripheral ectodermal
cells during stage 10 after the majority of the germ band
neuroblasts have segregated (Figs 4C, D, 5C, D). Dl
In situ analysis of Delta
dec
cyt
Fig. 3. Distribution of Dl transcripts during cellularization.
Transverse and parasagittal sections hybridized with the
5.4Z/4.5M (left column) or 3.5z (right column) probes are
shown. (A, B) Alternate transverse sections through an
early stage 5 embryo at approximately 50 % egg length
(100% egg length at anterior end). Arrowheads mark the
approximate boundaries of the presumptive ventral
neurogenic region (vNR). Labeled nuclei are most apparent
in the ventral neurogenic region, indicating that the 5.4Z/
4.5M and 3.5z embryonic transcripts are most prevalent
within this region of the embryo. However, maternallyloaded 4.5M transcripts are present throughout the cortical
cytoplasm at this stage. Note that the highest levels of
5.4Z/4.5M and 3.5z transcripts are present over the ventralmost nuclei of the ventral neurogenic region.
(C, D) Alternate transverse sections through a late stage 5
embryo at approximately 40% egg length. The zygotic
accumulation of 5.4Z/4.5M transcripts remains most
apparent within the presumptive ventral neurogenic regions
of the ectoderm, although some hybridization is evident
over nuclei in regions representing the presumptive
mesoderm (ms) and dorsal ectoderm (dec). The
accumulation of 3.5z transcripts in the presumptive
mesoderm and dorsal ectoderm is much more evident.
5.4Z/4.5M and 3.5z transcripts appear evenly distributed
within the ventral neurogenic region at this stage.
(E, F) Parasagittal sections through the anterior regions of
two late stage 5 embryos. 5.4Z/4.5M transcripts are
distributed over nuclei (nuc) and throughout the cortical
cytoplasm at this stage; 3.5z transcripts are localized to the
base of the blastoderm nuclei. Exposure times: (A, B) 4
days; (C-F) 10 days.
627
transcripts are also present in neuroblasts during this
stage, but the neuroblast layer does not appear as
heavily labeled in longitudinal sections as the mesodermal and peripheral ectodermal cell layers (Fig. 4C, D).
Transverse sections of stage 10 embryos hybridized with
the 5.4Z/4.5M probe reveal areas of lower grain
density within the neuroblast layer, suggesting that
these transcripts are not present at comparable levels in
all neuroblasts (Fig. 5C). This is clearly the case for 3.5z
since both labeled and unlabeled neuroblasts are distinguishable (Figs 5D, 6D). The difference between the
5.4Z/4.5M and 3.5z hybridization patterns within the
neuroblast layer is apparently due to persistence of the
nuclear localization of the minor transcripts during this
interval. The levels of the predominant and minor Dl
transcripts in the neuroblast layer decrease during stage
11 as neuroblast segregation is completed (Figs 4E-H,
5E, F).
The accumulation of Dl transcripts within other
regions of the ectoderm is the same for the predominant
and minor transcripts and can be followed in Figs 4, 5,
and 6. Dl transcripts accumulate in all cells of the
ectoderm during stage 8 with the exception of some
cells in specific regions of the proctodeum and presumptive foregut (Figs 4A, B, 5A, B). Transcript accumulation subsequently falls off in segmentally repeated
arrays of dorsolateral ectodermal cells during stage 10
(Fig. 6A). These cells are apparently incorporated into
the tracheal pits during stage 11 (Fig. 6B). Dl transcript
accumulation is similarly diminished within ectodermal
cells of the labial segment just prior to the incorporation
of these cells into the salivary gland imaginations at the
end of stage 11 (Fig. 6C, D). The accumulation of Dl
transcripts in the ventral ectoderm is restricted to
ventrolateral clusters of cells at late stage 11 (Fig. 4G,
H), and by stage 12 Dl transcript levels are reduced
throughout the ventral epidermis (Figs 6E, 8C).
The accumulation of the predominant and minor Dl
transcripts in the differentiating mesoderm can be
followed in Figs 4, 5 and 7. Low levels of Dl transcripts
are present in the mesoderm of stage 8 embryos during
early germ band elongation (Figs 4A, B, 5A, B). The
mesoderm becomes organized into a monolayer of cells
during stage 9 within which Dl transcripts accumulate to
relatively high levels by early stage 10 (Figs 4C, D, 5C,
D). A second, more internal layer of mesodermal cells
is established by mid-stage 11 that will subsequently
give rise to the visceral musculature (Campos-Ortega
and Hartenstein, 1985). Dl transcripts do not accumulate in these cells (Fig. 7C), though Dl transcripts are
present in other mesodermal cells located over the
developing ventral nerve cord and between the tracheal
pits (Figs 4E, F, 6B, 7C). These regions of the mesoderm no longer contain detectable levels of Dl transcripts by the end of stage 11 (Figs 4G, H, 7E).
The pattern of Dl transcript accumulation in the
mesoderm was analyzed further by comparing the
accumulation of Dl transcripts to the accumulation of
transcripts that encode beta3-lubu\in, a marker for
mesodermal differentiation (Gasch et al. 1988; Leiss et
al. 1988; Kimble et al. 1989). beta3-i\xbu\\n transcripts
628
C. C. Kopczynski and M. A. T. Muskavitch
Fig. 4. Distribution of D/ transcripts from germ band elongation through extended germ band. Parasagittal sections of
embryos hybridized with the 5.4Z/4.5M (left column) or 3.5z (right column) probes are shown. All sections are oriented as
in Fig. 1. (A, B) Alternate sections through a stage 8 embryo. 5.4Z/4.5M and 3.5z transcripts are present throughout the
ectoderm with the exception of the foregut (fg) and proctodeum primordia. 5.4Z/4.5M and 3.5z transcripts are also present
in the dorsal wall of the posterior midgut primordium (pm) and at low levels in the mesoderm (ms). (C, D) Alternate
sections through a stage 10 embryo. 5.4Z/4.5M and 3.5z transcripts are present at relatively high levels in both the
mesodermal and peripheral ectodermal cell layers. Lower levels of transcripts are present between these cell layers where
neuroblasts (nb) that will give rise to the ventral nerve cord have segregated. 5.4Z/4.5M and 3.5z transcripts are also
present in the primordia of the anterior (am) and posterior midgut. The arrowheads point to unlabeled cells of the
proctodeum. (E, F) Alternate sections of a mid-stage 11 embryo. This embryo was sectioned such that the bottom of the
section represents a ventral region of the embryo and the top of the section a more lateral region of the embryo. 5.4Z/4.5M
and 3.5z transcripts are less abundant in the neuroblast layer than in the peripheral ectoderm, anterior midgut and posterior
midgut. 5.4Z/4.5M transcripts are apparent ventrally in metameric clusters of mesodermal cells and laterally in mesodermal
cells located between the tracheal pits; 3.5z transcripts are observed in the same region of the mesoderm laterally, but do
not appear to be restricted ventrally to metameric clusters of mesodermal cells in this section. (G, H) Alternate sections of a
late stage 11 embryo (H, darkfield illumination). 5.4Z/4.5M and 3.5z transcript levels are reduced throughout the mesoderm
and ventral ectoderm. Three ventrolateral clusters of ectodermal cells consisting of neuroblasts and underlying dermoblasts
remain heavily labeled with the 5.4Z/4.5M probe. 5.4Z/4.5M and 3.5z transcripts are apparent in the anterior midgut,
posterior midgut and dorsal wall of the proctodeum at this stage. Abbreviations: (pNR) procephalic neurogenic region;
(st) stomodeum; (vNR) ventral neurogenic region. Exposure times: (A, B, E, F) 10 days; (C, D, G, H) 4 days.
first accumulate during stage 10 in cells of the cephalic
mesoderm (Gasch et al. 1988). Adjacent sections of a
stage 10 embryo reveal that the predominant Dl tran-
scripts are present at much lower levels in the cephalic
mesodermal cells that accumulate the beta3-lub\\\\n
transcript than in the surrounding mesodermal and
In situ analysis of Delta
629
Fig. 5. Distribution of Dl transcripts during neuroblast
segregation. Transverse and parasagittal sections of
embryos hybridized with the 5.4Z/4.5M (left column) or
3.5z (right column) probes are shown. (A, B) Alternate
transverse sections through a stage 8 embryo at
approximately 70% egg length. Arrowheads mark the
boundaries of the procephalic (pNR) and ventral (vNR)
neurogenic regions. All ectodermal cells within these
regions accumulate 5.4Z/4.5M and 3.5z transcripts.
Hybridization to these transcripts is only slightly above
background within the mesoderm (ms) at this stage.
(C, D) Alternate transverse sections through a stage 10
embryo at approximately 30% egg length. 5.4Z/4.5M and
3.5z transcripts are present at similar levels in the
mesodermal and peripheral ectodermal cell layers. Regions
of reduced hybridization within the neuroblast population
that separates these cell layers are observed with the 5.4Z/
4.5M probe. Individual labeled and unlabeled neuroblasts
are distinguishable with the 3.5z probe (D, small
arrowheads). Most cells within the peripheral ectoderm are
labeled at this stage, although unlabeled cells are
occasionally observed with the 3.5z probe. (E, F) Alternate
parasagittal sections through a mid-stage 11 embryo. 5.4Z/
4.5M and 3.5z transcripts are present at low levels in the
mesodermal and neuroblast (nb) cell layers relative to the
peripheral ectoderm. Neuroblast segregation is complete at
this stage. Abbreviations: (cf) cephalic furrow; (dec) dorsal
ectoderm; (pm) posterior midgut; (vec) ventral ectoderm.
Exposure times: (A, B) 10 days; (C, D) 20 days; (E, F) 4
days.
~y
nb
anterior midgut cells (Fig. 7A, B). We also find that as
6efa3-tubulin transcripts accumulate during stage 11,
first in the innermost mesodermal cell layer and then in
the remaining mesodermal cells, the accumulation of
predominant Dl transcripts decreases within these regions of the mesoderm in the same temporal order
(Fig. 7C-F). Similar results were obtained with the 3.5z
probe (data not shown). Thus, the reduction in Dl
transcript levels observed in specific cells of the mesoderm correlates with the onset of beteJ-tubulin transcript accumulation in those cells.
The pattern from germ band shortening to hatching
The number of embryonic cell types in which Dl
transcripts accumulate continues to decline until the
end of embryogenesis (Fig. 8). No differences between
the accumulation patterns of the predominant and
minor transcripts have been observed during this interval (data not shown).
Dl transcripts are present through the end of embryogenesis in the nerve cord, optic lobe primordia and
anterior regions of the embryo that could represent
sensory organs of the head (Fig. 8A, C, E, G, J, L). The
accumulation of Dl transcripts in the nerve cord becomes restricted during this interval to the peripheral
cells of the central nervous system, which include
neuroblasts, ganglion mother cells and newly born
neurons (Fig. 8C, G, J, L). We are unable to determine
within which of these cell types Dl transcripts accumulate since these cells are not morphologically distinguishable after stage 12. Dl transcripts may also
accumulate in the developing peripheral nervous system during this time (Fig. 8E, I).
Mesodermal accumulation of Dl transcripts is observed during stage 12 in regularly spaced clusters of
subepidermal cells (Fig. 8A). We do not observe hybridization of the £>ef&3-tubulin probe to transcripts
within these cells (data not shown), but we infer that
they are mesodermal based on their location and the
absence of a segregated peripheral nervous system
during this stage (Campos-Ortega and Hartenstein,
1985). Similarly spaced subepidermal cell clusters are
labeled during stage 14 (Fig. 8E, I), but these clusters
apparently contain fewer cells than those observed
during stage 12. These clusters could represent a subset
of the previously labeled mesodermal cells or cells of
the peripheral nervous system. Transcripts are also
present during stage 14 in regions that correspond to the
developing gonads (Fig. 8E). By stage 17, Dl transcripts are no longer detectable in mesodermal cells
(Fig. 8G).
Other changes in Dl transcript accumulation during
this interval include the disappearance of Dl transcripts
from the anterior midgut during stage 12 (Fig. 8C) and
from the posterior midgut and entire epidermis during
stage 17 (Fig. 8G). The only nonneural accumulation of
Dl transcripts that remains during stage 17 is in the
630
C. C. Kopczynski and M. A. T. Muskavitch
Fig. 6. Dl transcript levels are reduced in differentiating ectoderm. Parasagittal and transverse sections hybridized with the
5.4Z/4.5M (A, B, C, E) or 3.5z (D) probes are shown. (A) Parasagittal section through the dorsolateral ectoderm (dec) of a
stage 10 embryo showing alternate regions of high and low transcript levels. (B) Parasagittal section through the dorsolateral
ectoderm of a mid-stage 11 embryo showing relatively low levels of transcripts in ectodermal cells that have invaginated to
form the tracheal pits (arrowheads). (C) Parasagittal section through the anterior region of a mid-stage 11 embryo.
Transcripts are not detectable in ectoderm of the labial segment (lb). Labeled cells of the developing stomatogastric nervous
system are apparent in this section (arrowheads). (D) Transverse section through a late stage 11 embryo at approximately
70% egg length. Transcripts are absent from the labial epidermis which has begun to invaginate to form the salivary gland
placodes (arrowheads). Transcripts are present in the epidermis of the procephalic lobe (pi) just above the labial segment.
Only one of the two neuroblasts (nb) visible above the labial epidermis is labeled. (E) Transverse section through a late
stage 12 embryo at approximately 60 % egg length. Transcripts are not detectable within the ventral epidermis (vep) below
the ventral nerve cord (vnc), but are present in lateral and dorsal regions of the epidermis. Abbreviations: (am) anterior
midgut; (ms) mesoderm; (pr) proctodeum. Exposure times: (A-C) 4 days; (D, E) 20 days.
proventriculus and in portions of the pharynx and
hindgut (Fig. 8G, K).
The Dl pattern compared to N
The dynamic pattern of Dl transcript accumulation
prior to germ band shortening contrasts with the
ubiquitous accumulation of N transcripts during this
interval (Hartley et al. 1987). By the end of embryogenesis, however, the N and Dl transcript accumulation
patterns are almost identical (Fig. 8G, H; ibid., Vassin
et al. 1987). A comparison of the progressive loss of N
and Dl transcripts from specific regions of the embryo is
presented in Fig. 8. It is evident that the temporal order
of events that leads to their respective final accumulation patterns is not the same. For instance, N transcript levels remain high in the epidermis long after Dl
transcript levels drop (Fig. 8E, F). Conversely, Dl
transcript levels remain high in the posterior midgut
long after N transcript levels drop (Fig. 8E, F). These
data reveal that Dl transcript accumulation is regulated
independently of N transcript accumulation within
many regions of the embryo. In the developing central
nervous system, however, the accumulation of Dl and N
transcripts is indistinguishable throughout this period
(Fig. 8A-D, G, H, J). Thus, the expression of Dl and N
in the central nervous system during the later stages of
neurogenesis may be coordinately regulated.
Sedimentation of the minor Dl transcripts in polysome
gradients
The resolution of our in situ hybridization analysis does
not allow us to determine whether the minor Dl
transcripts are localized within nuclei or within the
cytoplasm at the nuclear periphery. We therefore fractionated 2 to 5h embryo homogenates on sucrose
gradients to compare the sedimentation rates of the
minor embryonic Dl transcripts to the sedimentation
rates of polysomes. Fig. 9A reveals that the major
fraction of each minor transcript cosediments with
polysomes in the gradient. Fig. 9B further demonstrates that the sedimentation of these transcripts in the
gradient is EDTA-sensitive, a characteristic of polysome-bound transcripts (Penman et al. 1968). These
results are consistent with the hypothesis that the minor
embryonic Dl transcripts are associated with polysomes
and therefore localized within the cytoplasm. It remains
possible, however, that the minor transcripts accumulate within the nucleus as EDTA-sensitive ribonucleo-
In situ analysis of Delta
B
am
vec* *
vec
vms
-fy<f?
sms
- .
•'
vms
p
sms
•S
pm
/<•
i.*j
pm
Fig. 7. D/ transcript levels are reduced in differentiating
mesoderm. Parasagittal sections of embryos hybridized with
the 5.4Z/4.5M probe (left column) or a £>efaJ?-tubulin probe
(right column). (A, B) Alternate parasagittal sections
through the anterior region of a stage 10 embryo. Dl
transcripts are not detectable in cells of the cephalic
mesoderm that accumulate £>eta3-tubulin transcripts
(arrowheads). (C, D) Parasagittal sections through
dorsolateral regions of the germ band of two stage 11
embryos. Dl transcripts are present in ectodermal and
mesodermal cells between the tracheal pits, but are absent
from the inner-most layer of mesodermal cells that
represents the presumptive visceral mesoderm (vms). beta3tubulin transcripts are present in the presumptive visceral
mesodermal cells, but not in the mesodermal cells between
the tracheal pits. (E, F) Alternate parasagittal sections
through an early stage 12 embryo. Dl transcripts are no
longer present between the tracheal pits in those cells of the
somatic mesoderm (sms) that accumulate ftetoJ-tubulin
transcripts. Abbreviations: (am) anterior midgut;
(vec) ventral ectoderm. Exposure times: (A-F) 4 days.
protein particles that cosediment with polysomes in
sucrose gradients.
Discussion
Our previous molecular analysis of Dl revealed that at
least six distinct transcripts are encoded by this gene
(Kopczynski et al. 1988). The two predominant transcripts, 5.4Z and 4.5M, appear to encode the same
polypeptide which we have designated D1ZM (ibid.).
The structure of the minor transcripts indicates that
these transcripts must encode polypeptides significantly
different from D1ZM if they are translated. It was
631
therefore important to characterize the accumulation of
the predominant and the minor transcripts in our
analysis of the spatial expression of Dl. Our results
reveal that the predominant and minor Dl transcripts
accumulate in the same tissues at approximately the
same times throughout embryogenesis. The only apparent difference in their timing is the accumulation of the
minor transcripts within the dorsal ectoderm and presumptive mesoderm approximately 20 min prior to the
detectable accumulation of the predominant transcripts
in these regions. This difference and the differences
observed between the respective hybridization patterns
in the neuroblast layer during stage 10 are probably due
to the different subcellular distributions of these transcripts.
The association of the minor embryonic transcripts
with the basal half of the nucleus represents a newly
described form of transcript localization in Drosophila.
The cosedimentation of these transcripts with polysomes suggests that they are localized within the cytoplasm. Interestingly, the transcripts of a number of
genes involved in early pattern formation are localized
within the cytoplasm above the nucleus during cellularization (Hafen etal. 1984; Ingham et al. 1985; Harding et
al. 1986; Kilcherr et al. 1986; MacDonald et al. 1986;
Gergen and Butler, 1988). It has recently been suggested that this apical localization may function to prevent
diffusion of these transcripts out of their specific
domains of accumulation (Edgar et al. 1987). This same
hypothesis could be invoked to explain the subcellular
localization of the minor Dl transcripts, although the
functional significance of restricting Dl transcripts to
neurogenic regions at the start of cellularization is not
obvious. Alternatively, the subcellular localization of
the minor transcripts could reflect their translation in
association with a specific subcompartment of the
endoplasmic reticulum. Further characterization of
these transcripts and their putative translation products
will be required to address these issues.
The apparent association of all four embryonic Dl
transcripts (5.4Z, 5.4z, 3.5z and 2.8z) with polysomes
supports the hypothesis that Dl encodes multiple polypeptides. The accumulation of these transcripts within
the same cells during embryogenesis further suggests
that the embryonic Dl product may function as a
multimeric complex. If this were the case, then genetic
interactions among Dl alleles would exhibit some degree of complexity. Indeed, interallelic complementation has been observed among various Dl mutations
(Vassin and Campos-Ortega, 1987; Alton et al. 1988;
M.A.T. Muskavitch, unpublished data).
Cell transplantation experiments have revealed that
Dl can act nonautonomously to promote the entry of a
cell that lacks Dl function into the epidermal lineage
(Technau and Campos-Ortega, 1987). We therefore
wanted to determine whether the expression of Dl
within the neurogenic region is restricted to a subset of
ectodermal cells. We find that this is not the case. Dl
transcripts accumulate in most if not all cells of the
neurogenic ectoderm immediately prior to and during
neuroblast segregation. This result is particularly inter-
632
C. C. Kopczynski and M. A. T. Muskavitch
«•'•
ol
pmg
pmg
»--Mfe.**>t>7*i*'' :
vnc
esting since the transcripts of two other neurogenic
genes, N (Hartley et al. 1987) and E(spl) (Hartley et al.
1988), also appear to accumulate ubiquitously within
the neurogenic ectoderm. Thus, there is no apparent
distinction between presumptive neuroblasts and presumptive dermoblasts with respect to their accumulation of Dl, N, or E(spl) transcripts.
It has been proposed that the neurogenic genes may
function in a system of intercellular communication by
which segregating neuroblasts prevent neighboring ec-
todermal cells from entering the neural lineage (de la
Concha et al. 1988). This model requires that a functional distinction exists between neuroblasts and adjacent cells within the ectoderm. The apparently uniform
distributions of Dl, N and E(spl) transcripts within
neurogenic regions do not provide an evident basis for
such a distinction, although post-transcriptional regulation of one or more of these genes cannot be
excluded. The asymmetric expression of another neurogenic gene or a gene not yet known to be involved in
In situ analysis of Delta
Fig. 8. Distribution of Dl and N transcripts from germ band
shortening to hatching. Horizontal and parasagittal sections
hybridized with the 5.4Z/4.5M probe (A, C, E, G, I-L) or
a N probe (B, D, F, H) are shown. (A, B) Alternate
horizontal sections through a mid-stage 12 embryo. Dl
transcripts are present in the optic lobes (ol) and posterior
midgut (pm) and in subsets of cells within the proctodeum
(pr) and somatic mesoderm (arrowheads). N transcripts are
present in many of the same cell types, but are relatively
more abundant in the epidermis and less abundant in the
posterior midgut than Dl transcripts. (C, D) Alternate
parasagittal sections through a late stage 12 embryo. Dl
transcripts are abundant in the brain (br), ventral nerve
cord (vnc), posterior midgut and anterior and posterior
regions of the foregut. Dl transcript levels are much
reduced in the ventral epidermis and anterior midgut (am).
N transcripts are abundant in the ventral epidermis, foregut
and developing nervous system and are present at lower
levels in the anterior and posterior midgut. (E, F) Alternate
horizontal sections through a stage 14 embryo. Dl
transcripts are present in the optic lobes, brain, posterior
midgut and cells at the anterior tip of the embryo that may
represent sensory organs of the head. Dl transcripts are also
present in the germ band in clusters of cells just below the
epidermis and in regions that correspond topologically to
the developing gonads (arrowheads). N transcripts are still
abundant in the epidermis and brain, but are present only
at very low levels in the posterior midgut.
(G, H) Parasagittal sections through two stage 17 embryos.
Dl transcripts are present in the hindgut (hg),
proventriculus (pv), peripheral cells of the central nervous
system and cells that may represent anterior sense organs
(arrowhead). N transcripts are present in the same regions
of the embryo as Dl transcripts, but are relatively more
abundant in the anterior regions of the embryo than Dl
transcripts. Dl and N transcripts are absent from the ventral
epidermis (vep) at this stage. (I) Horizontal section through
a stage 14 embryo showing the accumulation of Dl
transcripts in clusters of cells just below the epidermis.
(J) Parasagittal section through the anterior half of a stage
14 embryo. Dl transcripts are present in peripheral cells at
the base of the nerve cord, in the brain and at the anterior
tip of the embryo (arrowhead). High levels of Dl transcripts
are also present in the pharynx and in the posterior region
of the esophagus (es). (K) Horizontal section through a
stage 17 embryo. The labelled cells of the esophagus
present at stage 14 have become incorporated into the
proventriculus. (L) Glancing section through the ventral
nerve cord of a stage 17 embryo. The relatively heavy
labeling of the peripheral cells of the central nervous system
is apparent in this section. Exposure times: (A, C, E, G,
i-L) 20 days; (B, D, F, H) 4 days.
lineage specification within the ectoderm may provide
the basis for this distinction. Alternatively, it is possible
that the neurogenic genes function in an intercellular
communication system that represses the entry of all
neurogenic ectodermal cells into the neural lineage.
Assumption of the neural fate would then occur as
specific cells lose their susceptibility to this inhibitory
influence. In this model, the neurogenic genes would be
expressed in most or all cells of the neurogenic ectoderm, as we and others have observed. However, the
available data do not exclude either of these models
from further consideration.
633
Dl and N transcripts accumulate in the peripheral
cells of the developing central nervous system long after
neuroblast segregation is completed. The progressive
loss of Dl transcripts from the neuroblast layer prior to
germ band shortening suggests that the relatively dense
peripheral labeling of the developing central nervous
system may correspond to ganglion mother cells and/or
newly born neurons. This possibility is particularly
intriguing given that the differentiation of sibling neurons has been shown to involve cell-cell interactions in
grasshopper embryos (Kuwada and Goodman, 1985).
Accumulation in the peripheral nerve cord could also,
or alternatively, represent the reinitiation of Dl expression during late neurogenesis in neuroblasts that
will divide post-embryonically (Truman and Bate,
1988).
VSssin et al. (1987) inferred that the spatial accumulation pattern of the predominant Dl transcripts during
embryogenesis reflects the specific expression of Dl in
regions of the embryo within which precursors of the
central and peripheral nervous systems develop. However, these authors (ibid.) did not document the accumulation of Dl transcripts in the mesoderm. Our
results suggest that the dynamic pattern of Dl transcript
accumulation during embryogenesis reflects the initial
expression of Dl in many different cell types of the
embryo followed by the progressive reduction of Dl
expression in those cell types as they differentiate. This
hypothesis is supported by our observation that Dl
transcript levels decrease in mesodermal cells as they
begin to accumulate beta3-tubu\in transcripts, as well as
in ectodermal cells immediately prior to their incorporation into tracheal pits and salivary glands, respectively.
The peripheral labeling of the central nervous system is
also consistent with this hypothesis since the less
differentiated cells of the developing nervous system
are located at the periphery of the nerve cord. The late
expression of Dl transcripts in the foregut and hindgut
could represent an exception to this general hypothesis.
However, other genes essential for normal pattern
formation are expressed equally late in these regions
(Fjose et al. 1985; Ingham et al. 1985; Kornberg et al.
1985; Mlodzik etal. 1985; Krause etal. 1988), suggesting
that the differentiation of these tissues may not be
complete until very late in embryogenesis.
What role might Dl play in undifferentiated tissues?
The predominant Dl product, D1ZM, is a putative
transmembrane protein with an extracellular domain
that contains a cysteine-rich motif that has been implicated in protein-protein interactions in other systems
(Carpenter and Cohen, 1979; Gray et al. 1983; Scott et
al. 1983; Sudhof et al. 1985; Wharton etal. 1985; Kidd et
al. 1986; Furie and Furie, 1988; Jones et al. 1988;
Montell and Goodman, 1988; Rees et al. 1988). The
structure of D1ZM suggests that it is involved directly in
the cell-cell interactions that are required for proper
differentiation of the neurogenic ectoderm. It is therefore possible that Dl is involved throughout the embryo
in a number of developmental processes that require
intercellular communication for accurate differentiation. Indeed, the Dl loss-of-function phenotype in-
634
C. C. Kopczynski and M. A. T. Muskavitch
Mg+
l
+
260
A
5.4Z
4.5M
B
C
D
'
E
t
5.4z
3.5z
A '
B ' C ' D
'
E
5.4Z
4.5M
5.4z
P
2.8z
3.5z
2.8z
Fig. 9. £>/ transcripts cosediment with polysomes in sucrose gradients. (A) Fractionation of a 2-5 h embryo homogenate in a
20-50% sucrose gradient (in the presence of Mg2"1"). Fractions A-E correspond to large polysomes (A), median-sized
polysomes (B), small polysomes (C), monosomes (D) and free ribosomal subunits (E) as inferred from the A ^ profile.
Total RNA was purified from these fractions and equal volumes of each RNA sample were fractionated on denaturing
agarose gels, transferred to nylon membranes and hybridized with transcript-specific probes. The RNA blots are presented
below the A26o profile. The majority of each transcript is present in fractions A-C. (B) Fractionation of polysomes isolated
from a 2-5 h embryo homogenate after incubation in 40mM-Na2EDTA. The A260 profile shows that the majority of the
polysomal RNA sediments at a position in the 20-50% gradient expected for free ribosomal subunits (fraction E). Total
RNA preparations from fractions A-E were analyzed by RNA blot analysis as in (A). The RNA blots below the A ^
profile reveal that the majority of each transcript sediments in fractions D and E.
eludes defects in the development of muscles, gut,
gonads and other tissues (Lehmann et al. 1983), but
these defects have been assumed to result from indirect
effects of gross neural hypertrophy and reduction of the
epidermis. With the aid of stage- and tissue-specific
molecular markers, it should be possible to determine
whether Dl plays such a pleiotropic role during embryonic development.
The authors thank David Hartley and Spyros ArtavanisTsakonas for introducing us to in situ hydridization techniques, Mary Kimble for providing the fcefai-tubulin probe
and Drs Thomas Kaufman, Thomas Blumenthal and the
members of our group for careful reading of the manuscript.
C.C.K. was supported by a training grant in molecular biology
from the National Institutes of Health. M.A.T.M. was supported by a Junior Faculty Research Award from the Ameri-
can Cancer Society. This work was supported by a grant from
the National Institutes of Health.
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