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Development 104 Supplement, 67-73 (1988)
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Ectopic segmentation gene expression and metameric regulation in
Drosophila
D. ISH-HOROWICZ and FI. GYURKOVICS*
Developmental Genetics Laboratory, Imperial Cancer Research Fund Developmental Biology Unit, Zoology Department,
(Jniversity of Oxford, South Parks Road, Oxford OXl 3PS, UK
* Permanent address: Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences,H6701. Szeged, PO Box
Hungary
lntroduction
Embryonic pattern in Drosophila is organized by a
hierarchy of maternal and zygotic segmentation genes
that interact to define increasingly precise spatial
domains (reviewed by Scott & O'Farrell , 1986;
, 1987). A metameric body plan is already
established by the onset of gastrulation (3 h postfertilization) such that each segment expresses the segment-polarity genes engrailed (en) and wingless'(wg)
in single-cell-wide stripes (Kornberg, Sidon, O'Farrell & Simon, 1985; Fjose, McGinnis & Gehring,
1985; DiNardo, Kuner, Theis & O'Farrell, 1985;
Baker , 1987). The current challenge is to define
specific segmentation gene interactions, to determine
how they achieve this spatial refinement and how
their spatial relationships give rise to the structural
complexities of the mature larva.
Akam
The pattern of en expression defines two metameric
registers: the en domain lies at the posterior of each
segment and at the anterior of each parasegment
(Martinez-Arias & Lawrence, 1985; Fig. 1C). It has
been suggested that the parasegment is the primary
metamere of the early embryo and that establishing
en domains is crucial in defining metameric units (see
Howard & Ingham,1986; Ingham & Martinez-Arias,
1986; Lawrence, this volume). Moreover, initial
selector gene expression appears parasegmental
(Akam & Martinez-Arias, L985; CarroII et al. 1988).
However, a second feature of establishing metamerism is ensuring the stability of en domains. Various
mutant conditions cause the establishment of unstable en patterns that underlie subsequent loss of
pattern elements (DiNardo & O'Farrell , I9B7; IshHorowicz & Pinchin, L987; Martinez-Arias, Baker &
Ingham, 1988; DiNardo et al. 1988; Ish-Horowicz,
Gyurkovics, Pinchin & Ingham, L9B8).
The hierarchy of segmentation genes can be ranked
according to their interactions. Gap genes act before
521.,
the pair-rule genes; pair-rule genes act to establish
the patterns of segment-polarity genes (Howard &
Ingham, 1986; Caroll & Scott , 1986; Inghoffi, IshHorowicz & Howard, 1986; Ingham & MartinezArias, 1986; Harding et al. 1986; DiNardo & O'Farrell , 1987; Martinez-Arias & White, 1988; Inghoffi,
Baker & Martinez-Arias, 1988). The hierarchy is
further subdivided by distinctions between the pairrule genes . hairy (h), even-skipped and runt are
required for the patterning of fushi tarazu (ftz)
expression, indicating that they act earlier in establishing pair-rule spatial domains (Howard & Ingham,
1986; Carroll & Scott , L986; Ingham & Gergen, this
volume).
Genetic analysis of epistatic relationships favour
simple hierarchical models. The large number of
segmentation genes and the rapidity with which they
organize themselves make it clear that pattern cannot
be the result of strictly sequential gene action. As
gene interactions become more complicated, in number and temporal complexity, it becomes increasingly
difficult to distinguish between direct interactions and
indirect consequences.
An alternative approach to conventional genetic
analysis is the deliberate mis-regulation of segmentation gene expression. Struhl (1985) has constructed
a hybrid gene (11SF) in which the coding region of the
pair-rule gen e ftz is under the control of the (hsp70)
heat-shock promoter. The latter is inducible in essentially all tissues, overcoming the spatial specificity of
the endogenous ftz promoter. Indeed, heat shock of
HSF blastoderm embryos causes specific pair-rule
pattern defects, indicating that the striped pattern of
ftz is critical in establishing proper metameric pattern. We have shown that similar ectopic expression
of the pair-rule gen e (h) also causes pair-rule defects
(Ish-Horowicz & Pinchin, 1987).
Struhl (1985) has interpreted the HSF phenotype in
terms of a combinatorial role for ftz, suggesting that it
68
D. Ish-Horowicz and H. Gyurkovics
is required for the development of the ftz-expressing
domains and prohibited in the alternate non-expressing metameres (see also Gergen & Wieschaus, 1986;
Gergen, Coulter & Wieschaus, 1986). However,
Duncan (1986) has proposed an alternative interpretation of the H S F pair-rule defects whereby ectopi c ftz
interferes with the establishment of parasegmental
metamerism. Such a model demands that the metameres affected in pair-rule HSF embryos be parasegmental, not segmental as described by Struhl. A
further expectation is that the loss of pattern elements
would be due to size regulation within abnormal
metameres and that the apparent deletion need not
derive exclusively from cells expressing ectopi c ftz.
We have therefore re-examined the cuticular
phenotypes of heat-shocked HSF embryos in order to
define more precisely the affected structures. In this
paper, we present evidence that the pair-rule HSF
phenotype is indeed reciprocal to that of ftz mutant
GL.2.3
TI
T2
AI
T3
A2
A3
embryos. We also show that the odd-numbered
parasegments are still represented within such HSF
embryos. Thus, the ultimate developmental fates of
many cells appears to depend on patterning within a
metamere, rather than their precise blastoderm identities.
Results and discussion
Cuticular deletions in HSF embryos are
parasegmental
Struhl (1985) showed that heat shock of blastoderm
HSF. embryos causes specific pair-rule pattern
defects, describing the HSF cuticular phenotype as
deletions of alternate segments. He suggested that
such embryos retain segments T2, A1, ,A3 etc. and
that they are not reciprocal to the (roughly) parasegmental deletions in ftz mutant embryos (Fig. 1C).
A4
A5
A6
A7
A8
segments
parasegments
|lF
IF IF
t
ftz
W
ffi
l$i:-+.::::::liii{:?i::1r.: ji::ifl
ljlf
ilit $lr1*S.:r-.!19!n
3.i:e..li}}$:.:f +)sii:.::4
ffi
EflE&6[&d
Flf.EF.F.ffi.t
.
fri#.its.r$.Yi*tii.FC
!A:rt:ii?..iil:.:.ii{'tii:r}l
1'i+iufill+ir{dt<un:d{|
ffi-ffi
ffi.ffi
ffi
ffi
HSF(or
HS4sr
Fig. l. Ventral cuticle preparations of (A) ftz and (B) HSF embryos. Anterior is to the left. (C) Map of the apparent
ventral deletions in ftz and pair-rule HSF embryos. The different HSF interpretations [A and B] are according to Struhl
(1985) and this paper, respectively.
Gene expression and metameric regulation in Drosophila
69
()
'lr
d'1..
.l1' I\.
t)
\-\
"' ir
\\-r{
!
..>.
i-';:iilT:'\-l:-
$
l;'&
Fig. 2. Cuticular morphology of wild-type and heat-shocked HSF embryos. (A) Dorsal pattern of + l* embryos. The
band of anterior hairs in T3 is broader than that in T2, extending posterior to the Black dot sensory organs (triangular
arrowheads). The row of triangular hairs in pA2 (arrowed) is lacking in more anterior segments. (B) Dorsal pA^z
(arrowed) in a pair-rule HSFembryo. The ventral dentical band is from A1, demonstrating a fusion of characters within
the segment.(C) VeutralpAZ in pair-rule HSF;Ubx embryos showing the ventral pits (hollow arrowheads) and the
partial Keilin's Organs (solid arrowheads).(D) Dorsal pattern in heat-shocked HSF;Ubx embryo showingp{z row
(arrowed). (E) Pair-rule HSF embryo showing the anterior band of dorsal hairs extending into pT3. Anterior is
uppermost.
70
D. Ish-Horowicz and H. Gyurkovics
This would also argue against the parasegment as the
primary embryonic metamere. For example, retaining a complete ,4.L segment would require the fusion
of two adjacent parasegments, posterior PS6 and
anterior PS7.
It is difficult to distinguish whether the metameres
deleted in pair-rule HSF embryos are segmental or
parasegmental. Both phasings predict similar ventral
cuticular phenotypes as the losses of, pT2 (posterior
second thoracic segment lost if the deletions are
parasegmental) and pT3 (lost if the deletions are
segmental) are indistinguishable (Fig. 1C).
However, we have been able to show that the HSF
pair-rule phenotype affects parasegmental metameres by analysing the dorsal cuticular patterns. In
wild-type embryos, each segment includes a wide
anterior band of fine posterior-orientated hairs (for a
detailed description of the dorsal pattern, see LohsSchardin, Cremer & Ni.isslein-Volhard, L983). The
bands are separated by a naked region, except for
pAz to pA7 which develop a row of broader anteriorpointing triangular hairs (Figs 2A and 3). Such a row
distinguishes pAtz (PS8) from pA1 (PS7) which occasionally shows individual hairs but never a row.
The dorsal pattern of pair-rule HSF embryos
demonstrates that the apparent segments display
mixed segmental character. The 'Al-like' segment
includes the posterior row of hairs characteristic of
pA2 (Fig.2B). Analysis of heat-shocked HSF;Ubx
exact reciprocal. However, we have analysed the
patterns of segmentation gene expression in heatshocked HSF embryos and shown that the mechanisms leading to the two phenotypes are very different.
The pair-rule ftz phenotype can be attributed to the
absence of the even-numbered en stripes, i.e. misestablishment of the initial embryonic metameres
(Ingham & Martinez-Arias, 1986). fn heat-shocked
HSF embryos, all en stripes are initiated but alternate
domains become unstable (Ish-Horowicz et al. 1988).
This appears to be due to the repression of alternate
stripes of wg,leading to decay of the even-numbered
en stripes at the end of germ-band extension. The
same bands are lost in ftz mutant embryos; thus the
pair-fule metameric organizations of HSF and ftz
embryos are the same, not reciprocal. The different
cuticular phenotypes are due to differential selector
gene expression (Ish-Horowicz et a/. submitted). The
T1
n
T3
AI
A2
34s67PS
+l+
embryos, in which PS6 is transformed to PS5, confirm
that the affected metameres are parasegmental. In
such embryos, aA1 (PS6) becomes thoracic (PS5) and
.
includes an extra pair of partial Keilin's organs at the
parasegmental boundary (see Struhl, 1984) (Fig . 2C).
As expect€d, the p[z posterior row is unaffected
PS6
(Fig.2D). Both experiments indicate that pair-rule
HSF embryos retain PS6 and PS8 while lacking PS7.
The patterns of dorsal thoracic hairs in pair-rule
HSF embryos are also consistent with apparent loss
of the odd-numbered parasegments. In the 'T2-like'
segment, the anterior band of hairs extends past the
dorsal sense organs as is usually found in pT3 (PS6),
but not pT2 (PS5) (Fig.zA,E).
In some heat-shocked HSF embryos, the pattern
deletions extend over slightly more than a metamere.
In such embryos, the posterior row is often lackitg,
not only in pAZ and but also in more posterior
segments. We do not understand the basis for these
more extensive deletions, but such embryos may
explain the discrepancy between Struhl's and our
interpretations of the HSF phenotype.
ftz mutant embryos display apparent deletion of
the even-numbered parasegments (Ni.isslein-Volhard
& Wieschaus, 1,980; Wakimoto, Turner & Kaufmotr,
I98/; Ingham & Martinez-Arias, 1986). Thus, the
pair-rule HSF cuticular phenotype represents their
HSF
-
PS5
HSF;Ubx
Fig. 3. Diagram of the dorsal cuticular pattern in
HSF and HSF;Ubx embryos.
+
I+
Gene expression and metameric regulation in
final HSF cuticular phenotype is the result of meta-
parasegments
meric instability and homoeotic transformations.
embryos.
Odd-numbered parasegments are not deleted in pairrule HSF embryos
Clearly, the integrity and stability of parasegmental
domains are critical to the establishment of metamerism. Loss of alternate en stripes leads to loss of
alternate metameres. Nevertheless, the double-metameric primordia must regulate to give approximately
normal-sized cuticular segments. It has not yet
proved possible to follow the fates of individual cells
during this process. If size regulation is due to an
intrinsic response of cells to mutant patterns of gene
expression, their fates will depend on their individual
blastoderm identities. In pair-rule HSF embryos, the
deleted structures would derive from the non ftzexpressing, odd-numbered parasegment, &S envisaged in the combinatorial model. Alternatively, if the
cells are responding to external cues consequent on
inappropriate metameric size, regulation will involve
all regions of the metamere. In this latter case, both
Y
V
?
c
Fig.
4.
Drosophila 7I
will be represented in the
pair-rule
We used the ftz-lacZ fusion gene of Hiromi,
Kuroiwa & Gehring (1985) to mark the even-numbered parasegments in heat-shocked HSF embryos.
This fusion gene directs the synthesis of E. coli
ftgalactosidase (lacZ) under the control of the ftz
promoter. lacZ persists through germ-band shortening and retraction, so that it is still present when
segmental organization is overt (9 h).
In wild-type embryos that have completed germband shortening, the lacZ staining boundary extends
to the parasegmental boundary, anterior to the segmental groove (Lawrence, Johnston, MacDonald &
Struhl ,'L987; Fig. 38). The stained domain appears
narrower than a metamere due to weaker staining in
the posterior of the parasegment.
Odd-numbered parasegments are never stained
In pair-rule HSF embryos, there is a single stained
domain in each segment by the end of germ-band
retraction, reflecting the loss of alternate metameres.
The centre of each segment is stained, but not the
Y
D
ftgalactosidase staining
in HSF;ftz-lacZ
embryos. (A) Heat-shocked embryo showing stained domain within
each segmental metamere. (B) Non-heat-shocked embryo after germ-band retraction in which alternate metameres are
stained. The segmental grooves (arrowheads) extend deep into the embryo (see Martinez-Arias & Lawrence, L985).
lacZ staining in 5 h heat-shocked (C) and non-heat-shocked (D) embryos, showing broader staining in former. Anterior
is to the left.
72
+l+
PS
!B!!!B!!!B
ftz
HSF
PSI
BB!!!B!!BB
Fig. 5. Altered parasegmental primordia in (A) + I + and
(B) heat-shocked HSF embryos. The diagram represents
the putative cell states at blastoderm/early gastrulation
(from Ish-Horowicz et al. 1988).
anterior or posterior edges (Fig. 4,A.). The staining is
neither segmental nor parasegmental, and both parasegments contribute to the fused metamere.
Thus, the parasegmental deletions in heat-shocked
HSF embryos are misleading. They do not reflect the
loss of a parasegmental metamere, but rather its
inappropriate development. That the final cuticular
defects appear to be deletions of precise metameres
illustrates the importance of the parasegment as a
regulative unit in early development.
Although we have not followed the detailed time
course of lacZ staining, size regulation in pair-rule
HSF embryos must occur during germ-band shortening. The pattern is roughly norm aI at 5-6 h (Fig . 3C;
see below) and we have been unable to visualize any
cell death in 5 h embryos (not shown). The pattern of
lacZ staining suggests that the two parasegments do
not contribute equally to the final metamere. The
lacZ domain occupies considerably more than half
the final metamere (Fig.3C), indicating that the
majority of each segment derives from the evennumbered parasegments.
We have found that the heat-induced ftz expression
acts at blastoderm to advance the anterior margin of
ftz expression by one cell (Ish-Horowicz et al. 1988).
This leads to the broader IacZ staining in HSF
embryos than in wild-type (Fi g. 3C,D). The anterior
margins of the even-numbered en stripes advances
coincident with the change in ftz expression, causing
narrowing and widening of the odd- and even-num-
bered parasegmental anlagen respectively (Fig. 5).
Despite recruitment of extra cells into the evennumbered parasegments, they do not contribute exclusively to the final pair-rule HSF pattern.
Concluding remarks
The major advances
in studying embryonic pattern
formation have resulted from realization of the importance of the early analysis of developmental
perturbations. Lewis (1978) and Ni,isslein-Volhard &
Wieschaus (1980) pioneered the combination of genetic analysis and an emphasis on the study of larval
cuticular phenotypes. The current use of segmentation gene probes as molecular markers is a natural
extension of this philosophy and reflects the need to
overcome the inherent ambiguities of cuticle pattern
phenotypes as markers for very early events.
The ftz and HSF cuticular phenotypes are reciprocal; the same is true for the ectopic h (HSH) and h
mutant phenotypes (Ish-Horowicz & Pinchin, 1987).
However, the underlying causes of the complementary phttern defects differ completely. Ectopi c h acts
to repress the pair-rule gene ftz, thereby affecting the
establishment of en pattern. Ectopic ftz represses h/g,
a segment polarity gene, giving rise to instability of en
expression (Ish-Horowicz et al. 1988). Cuticular
phenotypes alone provided no distinction between
these different mechanisms. Only the analysis of
initial stages of embryogenesis, when pattern is being
established, allows us to define the direct interactions
that lead to the establishment of spatial domains.
We should like to thank Dr Gary Struhl for the HSF flies,
which he generously made available for our analysis. We
should also like to thank Phil Ingham for discussions in the
course of this work and our colleagues in the laboratory for
criticisms of the manuscript.
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