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TIG -- March 1985
16 Snow, M. tt. L. (1981) Autonomous developments of 30 Gnmeberg, H. (1952)TheGeneticsoftheMouse, 2ndEdn.,
Martinns Nijhof, The Hague, The Netherlands
parts isolated from primitive-streak stage mouse
embryos../. Embryol. Exp. Morphol. 65, 269-287
31 Nusslein-Volhard, C. and Wieschaus E. (1980)
17 Goodrich, E. S. (1958) Studz'es on the structure and
Mutants affecting segment number and polarity in
development of vertebrates, Dover Publications
Drosophila. Nature 287, 795-801
18 Noden, D. M. (1983) The embryonic origins of avian 32 Flint, O. P. and Ede, D. A. (1978) Cell interactions in
cephalic and cervical muscles and associated connecthe developing somite: in v4vo comparisons between
tive tissues. Am. J. Anat. 168, 257-276
amputated (am~am) and normal mouse embryos. J. Cell
Sci. 31,275-291
19 Leivo, I., Vaheri, A., Timpl, R. and Wartiovaara, J.
(1980) Appearance and distribution of collagens and 33 Flint, O. P., Ede, D. A., Wilby, O. K. and Proctor, J.
laminin in the early mouse embryo. Dev. Biol. 76,
(1978) Control of somite number in normal and ampu100-114
tated mouse embryos. J. EmbryoL Exp. Morphol. 45,
20 Tuckett, F., Lira, L. and Morriss-Kay, G. M. The
189-202
ontogenesis of cranial neuromeres in the rat embryo. I. 34 Slack, J. M. W. Homeotic transformations in man:
implications for the mechanism of embryonic developA scanning electron microscope and kinetic study. J.
ment and for the organization of epithelia. J. Theoretic.
Embryol. Exp. Morphol. (in press)
Biol, (in press)
21 I)etwiler, S. R. (1934)Anexperimentalstudyofspinal
nerve segmentation in amblystoma with reference to 35 Varnum, D. S. and Stevens, L. C. (1974) Rachiterata: a
the plurisegmental contribution to the brachial plexus.
new skeletal mutation in chromosome 2 of the mouse.
J. Exp. ZooL 67, 395-441
.[ Hered. 65, 91-93
22 Keynes, R. J. and Stern, C. D. (1984) Segmentation in 36 Griineberg, H. (1961) Genetical studies on the skelethe vertebrate nervous system. Nature 310, 786-789
ton of the mouse. XXIX. Pudgy. Genet. Res. 2,384-393
23 Kieny, M., Mauger, A. and Sengel, P. (1972) Early 37 Danforth, C. H. (1930) Developmental anomalies in a
regionalization of the somitic mesoderm as studied by
special strain of mice. Am. ]. Anat. 45, 275-287
the development of the axial skeleton of the chick 38 Lutz, H. (1949) Sur la production experimentale de la
embryo. Dev. Biol. 28, 142-161
polyembryonic et de la monstruosite double chez les
24 Pinot, M. (1969) Etude experimentale de la moroiseaux. Arch. Anat. Micr. Morph. Exp. 38, 79-144
phogenese de la cage thoracique chez l'embryon de 39 Hyman, L. (1951) The Invertebrates, Vol. II., McGraw
poulet. J. Embryol. Exp. MorphoL 21,149-164
Hill
25 Cooke, J. (1981) The problem of periodic patterns in 40 Eakin, R. M. (1968) Evolution of photoreceptors. In
embryos. Phil. Trans. R. Soc. London, Ser. B. 295,
E~vlutionary Biology, Vol. 2, (Dobzhansky, T., Hecht,
509-524
M. K. and Steere, W. C., eds) pp. 166-173, Appleton26 Slack, j. M. W. (1983) From Egg to Embryo,
Century-Crofts
Cambridge University Press
41 Vagvolgyi, J. (1967) On the origin of molluscs, the
27 Gerhardt, J. D. and Mintz, D. (1972) Clonal origins of
coelom, and segmentation. Syst. Zool. 16, 153-168
somites and their muscle derivatives: evidence from 42 Clark, R. B. (1964) Dynamics in Metazoan Evolution,
allophenic mice. Dev. Biol. 29, 27-37
Clarendon Press
28 Gregg, B. C. and Snow, M. H. L. (1983) Axial
abnormalities following disturbed growth in
mitomycin C-treated mouse embryos../. Embryol. Exp.
Morphol. 73, 135-149
B. Hogan, P Holland and P Schofield are at the Laboratory
29 Genetic variants and strains of the laboratory mouse of Molecular Embryology, National l~titute for Medical
(1981) {Green, M. C., ed.), Gustav Fischer Verlag
Research, Mill Hill, London NW71AA, UK
Many forms offish, bird, and beast
Brought forth an Infant form
Where was a worm before
Blake
First Book of Urizen
Molecules and puzzles from
the antennapedia homoeotic
gene complex of Drosophila
The intimacy of evolution and genetics is beauMatthew P. Scott
tifully illustrated
by
homoeotic
mutants,
How are loci that control the number of body segments related to loci that regulate
mutants in which one
segmental identi(y?In the A ntennapedia complexof Drosophila, the two kinds of lociare
part of an organism is
close together, and molecularanalysis has revealed shared and distinguishing features.
transformed into a semblance of another part.
An example of a homoeotic mutant is the fruit fly synthesis of specialized products, is regulated. The
(Drosophila) shown in Fig. 1 in which the antennae are structural components of an arm are probably nearly
transformed into legs. Such mutations remind us that identical to those of a leg; it is the organization of the
the organization of pattern can be altered by relatively two that differs.
Drosophila have bodies with about five head segminor genetic changes, and that small changes in
regulatory functions may be more important in the ments, three thoracic segments (each with a pair of
evolution of multiceUular organisms than small legs) and eight abdominal segments. The segments
changes in structural components. Homoeotic muta- can be seen at the earliest stages of differentiation in
tions are thought to identify 'master switch' genes the embryo and also in the larvae and in adults. The
homoeotic genes affect the identifies of body segthat control the selection of developmental pathways.
An important point is that pattern, and not merely the ments; other classes of genes affect the number of seg-
TIG -- March 1985
Fig. 1. A fly of the genoOepeAntp~/CxD. The antennae are
transformed into legs.
ments or their polarity. One of the notable successes of
Drosophila developmental genetics has been the
identification of two clusters of homoeotic genes. The
pioneering work was done by E. B. Lewis, though
many groups have contributed. Lewis continues to
investigate the cluster of genes known as the Bithorax
Complex (BX-C), which contains homoeotic genes
that are active in the thoracic and abdominal body
segments 1. BX-C mutations can cause, for example,
the transformation of the third thoracic segment into a
second thoracic segment. Lewis has proposed that it is
the array of homoeotic genes active in a particular
body segment that determines the identity of the
segment. A second homoeotic gene cluster, the
Antennapedia Complex (ANT-C) contains genes that
are active in the head, thorax, and/or abdomen. The
ANT-C was first recognized as a homoeotic gene
cluster by T. C. Kaufrnan and his colleagues2, and has
been studied intensively using genetic and
developmental methods 3~. However, the ANT-C is
not as well understood yet as the BX-C, the latter
having had the benefit of many more years of
attention.
tend to be at the left end of the complex, whil~I~iVuic? w ~
as Scr (Sex combs reduced, in which mutations turn first
legs into second legs) and loss-of-functionmutations in
Antp that normally act in the thorax are at the
righthand end of the ANT-C as it is usually
represented. In the BX-C most of the genes are in the
order of the body segments they affect x, so the order of
the genes in both complexes may be important. The
ANT-C is located in two fairly heavily stained
polytene bands, 84A4,5 and 84B1,2, on the right arm
of chromosome 3 (Fig. 2); the proximity of the ANT-C
to the centromere results in low frequencies of
recombination. In the ANT-C, a rough estimate of
recombination frequency is 0.0001% per 1000 base
pairs of DNA 9. This is in contrast to, for example, the
white locus near the tip of the X chromosome, where
the frequency is about 0.002% per 1000 base pairs ~°. It
appears that the size of the ANT-C will be roughly 300
kilobase pairs (kb), which is similar to the length of the
sum of the thoracic and abdominal regions of the
BX-C.
The Antp locus: a c o m p l e x locus within the
complex
The A n ~ locus inhabits polytene band 84B1,2, and
spans about 103 kb of DNA (Fig. 3). A major unsolved
question is why Antp needs so much DNA - the
finished transcripts are only 3.5 and 5.0 kb longs. The
details of transcription have not yet been worked out
but at least five exons have been identified through
their homologies with cDNA clones9.11.The exons are
distributed as shown in Fig. 3. Since the exons at
+100, +112 and +200 all hybridize to both of the
major transcripts, it is likely that differential RNA
processing gives rise to the different RNA species.
However, the possibility that DNA rearrangements
also occur has not been ruled out. Two different types
of cDNA clones have been described; one type hybridizes to the exons at + 100, + 112, + 170 and +2009'H,
while the second type hybridizes to the same two
exons at +100 and +112 and to an exon at +130,
but not to the exons at + 170 and + 200 (Fig. 3) H. Since
the cDNA libraries used were prepared from total
embryonic RNA including nuclear RNA, it is possible
that some cDNA clones represent processing intermediates rather than finished RNA species.
The molecular structure of Antp suggests that
The A n t e n n a p e d i a h o m o e o t i c gene c o m p l e x
there is room for genetic complexity and the diverse
My purpose here is to summarize what has been array of different mutations confirms this expectation
learned about the molecular organization of the ANT- (Table 1). Different dominant mutations may turn
C. The genetic aspects of the ANT-C are reviewed in antennae into legs (Anlp), dorsal head into dorsal
Ref. 7 and the developmental effects of ANT-C muta- thorax (A ntpCtx), or second and third legs into first legs
tions upon embryos are described in Ref. 8. Of the (AntpS~). The dominant mutations cause abnormal
ANT-C loci, AnqJ (Antennapedia, a homoeotic locus in activity of the A ntp locus and are therefore referred to
which dominant mutations transform antennae into as 'neomorphic' or 'gain of function' alleles4,s.7,~6.~7.All
legs, Fig. 1, and loss-of-function mutations transform of the dominant mutations are related because they
legs into antennae) and ftz (fushi tarazu, 'segment are each recessive lethals as well and they fail to comdeficient', a segmentation locus active in the zygote, plement each other. Thus, the abnormal activity of the
recessive mutation of which results in embryos with dominant alleles is accompanied by the loss of their
half the normal number of body segments) have been ability (in most cases) to provide the normal Antp
studied most extensively by molecular techniques. functions. A single functionalA ntp÷ allele is sufficient
While these two loci will be the focus of this for normal development; Antp + normally functions in
discussion, a few points about the ANT-C as a whole the thorax and perhaps the abdomen, but not in the
are worth mentioning. Loci that are normally active in head. Therefore, at least some of the dominant alleles
the head (e.g. pb, proboscil:w_dia, in which recessive apparently cause Antp to be active in the head where
mutations turn the labial palps into legs or antennae) the locus would normally be silent.
TIG-
March 1 9 8 5
transcription unit. The dominant mutations are fairly
rare, perhaps because only breakpoints in precisely
defined places within A n t p can lead to a dominant
effect. Alternatively, perhaps certain kinds of DNA
sequences must be brought into juxtaposition with the
A n t p DNA for a dominant phenotype to result.
The dominant mutations that are associated with
chromosome rearrangements may offer clues to how
A n t p functions. Is it the 5' part of the split
transcription unit or the 3' ,,art, or the combination of
the two, that is responsible for the mutant phenotype?
This question has been answered for one allele. A ntp 8,
in an elegant genetic experiment2. The A n t p I} allele,
which causes the antennae to be transformed into
legs, is associated with a small cytologically visible
inversion with one breakpoint within A n t p at + 140
(Ref. 9). About three-fifths of A n t p is therefore displaced away from the remaining 3' 40 kb. Kau_fi~anet
al. 2 isolated Drosophila in which the two ends of the
A n t p B inversion (and therefore the 5' and 3' parts of
Antp) had segregated from each other following
recombination within the inverted region. The result
the alleles that are completely recessive are actually
the source of the genetic observations that lead to the
formal designation of A n ( p as 'complex'. Certain
recessive lethal alleles exhibit a complementation
pattern as follows: allele A fails to complement B or C,
while B complements C (Ref. 7). Therefore, it seems
that type B or C mutations affect only a subset of the
functions of A n t p , while a type A allele affects all of the
functions. A molecular analogue, which may or may
not apply to A ntp, is that a type A mutation would be in
an exon that is included in all A n t p products, while a
type B or C mutation would be in an exon that is incorporated into only a subset of the A n t P products.
The task of mapping the A n t p locus onto the
chromosome walk was made easier by the fact that
most of the dominant A n t p alleles are associated with
cytologically visible chromosome rearrangements.
The breakpoints are distributed over a region of about
60 kb, from + 199 near the 5' exon to about + 140
(Refs. 9, 11). Therefore, abnormal gene activity
results from mutations that split the locus within the
Clonld.~l~j.men t
/!~:
D
|; i
I
E
/'
!1: , , ; V l I~ ') ( | l , ;d |
,i ~J
I
;
FIA
B
C
D
E
83 84
A n t p Ns* Rf7
J
Scr
J
1
l,
I sc~w."x2
J
I scxW'RX4
,
I!
~'RXJ
_Hu
I
i%;;
-
;wr cel
,
lob [pb
Fig. 2. A d r a ~
mum zen
2;- . . . . . .
Ofd [Scrl ftz
.
.
.
.
.
i "1 ']
IScrl
Antp
""
elb a
elb b dco
Ipl
mab
ecl a ]
of the r~ht arm o/ polyt~,~ chro~oso~z threein the vi~'ni~ of theAnt~napedia Complex{ANT-C). The ~mple-
mentation groups in and near the complex are shown below and the open boxes indicate def'wiency mutations that were used in o r d ~ n g
the loci The genetic analyses are the work of Thomas Kaufman and his colleagues at Indiana Universi(yz'zlz~° and the figure was
generously provided by T. Kaufman. The part ~f the chron~mme that has been c~ned as a series ~f ~ver~ap~n.ngpieces in phage vect~rs is
indicated above the chromosome. Symbols for genetic lock Tpl = Triplo-lethal, a T = alpha tubulin gene, roe = roughened eye, rn =
rotund, dsx = doublesex, twr = twisted bristles roughened eyes, cel= cell lethal, pb = probosm'pedia,mum = multimorph, zen =
zerknullt, Dfd = Deformed, Scr = Sex combs r e d ~ d , ftz = fushi tarazu, Antp = Antennapedia, elba° = embryo-/arvalboundary
lethal, dco = discsovergrown,lpl = larval pupat lethal, mab = malformed abdomen, eel ° = eclosion defective. Scr lesions are located
on the physical map on both sides of ftzg; hence its placonent in two positions, pb, Dfd, Scr and Antp are homeotic loc;~ ftz is a pair rule
7~cgraentation loc~ and several of the other loci affect eorly development.
TIG-
review
March 1985
Table 1. Mutations affecting the Antennapedia and ftz loci of Drosophila
Mutation
A n/'pa
Antp ct~
Anti) s~
Deficiency(Df) in Anti,
Recessive Ant/)- alleles
Deficiencyfor ftz
f~pl
Phenotype
antennae--~ second thoracic segrnen: legs
dorsal head --~dorsal thorax (sometimes eye --*-wing)
second and third legs----first legs
Hemizygous: (Dff+) no phenotype
Homozygous: lethal, second and third thoracic segments--~ first thoracic segments
Hemizygous(Antp-/+): no phenotype
Homozygous: lethal, same as Antp deficiency. However, some alleles complement to
give viable wild-type flies
Hemizygous (Df/+ ): no phenotype
Homozygous: lethal, half the usual number of segments form
Dominanteffect: posterior balancer organs, the halteres (vestigialwings on the third
thoracic segment) transformed into posterior wing tissue
Hemizygous(/tzRPl/Df):lethal, some segmental boundaries missing but not as many as
with aft2 null allele(Ref. 12)
was that the dominant phenotyge of antenna to leg
conversion was associated with the 3' part of Antp".
Therefore, the 3' 40 kb of An!p, juxtaposed to novel
chromosomal sequences, is sufficient to transform the
antennae. No phenotype was associated with the 5'
portion of the disrupted gene.
A simple model for what could be happening is that
the dominant Ant/) alleles are gene fusions in which a
3' part of Antp capable of directing the formation of
thoracic structures comes under the control of a promoter or other regulatory element that normally
causes gene expression in the head (or everywhere).
Abnormal expression of the 3' part of Antp in the head
(apparently 40 kb is enough) would result in the
observed antenna-to-leg transformation. This model
predicts that novel RNA and perhaps protein species
are encoded by the dominant alleles, and that different
alleles would encode different products. It also predicts that Antp dominant alleles will be expressed in
Me head, where the wild-type gene is inactive.
The (extra sex combs) Sex alleles of Antp, which
transform second and third legs into first legs, are
diverse in molecular structure. The So: phenotype is
reminiscent of the embryonic phenotype of Antp
recessive lethal mutants. The homozygous Antpmutants die as embryos but exhibit thoracic segmental transformations (T2 -4" T1, occasionally also
T3 --~ T1) that are analogous to the Sex dominant
effects 3. Therefore, the abnormal activity of Sex
alleles~7results in a phenotype that mimics the loss of
Antp function. The dominant alleles may be antimorphic, that is, they may interfere in a non-lethalway
with the activity of the wild-type Antp allele that is
present on the other homologue, giving rise to a
phenotype resembling partial loss of Antp function.
One of the two Sex alleles, Scxw, is associated with a 50
kb inversion from +85 to + 135, so that the 3' part of
Antp is inverted9. The only change yet detected in the
DNA of the original Seta allele is an insertion of about
3 kb just downstream of the 5' exon9. How the
molecular structure of Soc~ and &x w relate to the
phenotype or to antimorphic effects is not yet clear,
although it is possible that in ScxWDrosophila the 50 kb
inversion results in the production of antisense Antp
RNA, which could interfere with some function of the
normal RNA.
Transcription of a 103 kb gene probably takes about
one hour ~s. One might predict that genes expressed
very early in development would require shorter
transcription time and have a shorter length. An
example consistent with this idea is the fushi tarazu
(ftz) gene ~2.19,an ANT-C gene (Fig. 3) which has a 1.95
kb transcription unit and a single 150 bp intron. The,
ftz gene functions only at the blastoderm stage, as has
been demonstrated by mosaic analysiss, by the
temperature-sensitive period of a temperature-sensitive alleles, by Northern blots offlz RNA ]2'19, and most
dramatically by in situ hybridization offt2 probes to
frozen sections of embryos at different stages of
development~4. It will be interesting to learn whether
other genes that need to be expressed at an early stage
also tend to have short transcription units.
ftz: a s e g m e n t a t i o n l o c u s a m o n g t h e
h o m o e o t i c loci i n t h e A N T - C
It is now common to refer to genes that govern the
number and polarity of segments as 'segmentation
genes' and to distinguish them from the homoeotic
genes that regulate segment identity. Neither class of
gene is 'tissue specific', since genes in each class
affect, for example, both neural tissue and epidermis.
The flz segmentation locus was discovered during
work designed to saturate the ANT-C with recessive
lethal mutations2°. The phenotype offtz is that half the
usual number of segments develop, a lethal event in
i
"
Mntp
-
H
"tZO
t'1~
10
"*0
"4O
"~0
•
9
Ubx
bxd
Fig. 3. C,ene structure of the Antennapedia (Anrp)am/fushi
tarazu (ftz) loci of the A ntennapedia Complex (Refs 9, 11) and of
the Ultrabithorax (Ubx) and bithoraxoid (bxd) /oci of the
bithorax complex (Refs 1, 15, 23 and (R. Saint, P Beachly, D.
Peattie and D. Hogness, personal communica~m). Coordinates
are in kilobase pairs. Known exons and their splm'ng patWrns are
shown; they are based on the analysis of cDNA clones. The ftz
locus contains a 150 bp intron (not shown). The transcription and
processing of RNA from these genes may of course be more complex than the initial results shown here. The locations of homoeoboxes are indicated by 'H'. The bxd/ocus,a horm~ttk"/ocus,dz~es
not contain a homoeobox.
TIG -- March 1985
~
ien:WySgous embryos. This placesftz in the 'pair-rule'
class of segmentation mutants, a class which also includes genes such as paired, even-skipped, odd-skipped
and so forth2L The names convey the effects of mutations in the pair-rule loci on the segmentation pattern.
Since segmentation genes other thanftz are not (so far
as is known) in clusters of homoeotic genes, ftz seems
remarkable in being 30 kb downstream from Antp
(Fig. 3). Does this mean thatftz is a closet homoeotic?
One mutant allele offtz, ftz Rpl, found by Ian Duncan,
has a recessive phenotype in which fewer than half the
segmental boundaries are missing, suggesting reduced but not eliminated ftz function ~2. The unusual
attribute offtz Rpl is that it also has a dominant effect:
the posterior halteres are transformed into posterior
wing, and there are slight changes in the adult
abdominal segmentation as well. The phenotype is
reminiscent of the (postbithorax)pbx phenotype of the
bithorax complex, but pbx is recessive t. The ftz Rpl
mutation shows that an abnormalftz allele is capable
of inducing a homoeotic transformation, but this does
not mean thatflz is a normal homoeotic gene. Rather,
the phenomenon may provide us with a first example
of an interactive link between the homoeotic loci and
the segmentation loci.
Becauseftz is a small locus, more is known about the
structure of the gene and its products than is known
about the giant Antp and Ubx loci. The gene was
located by mapping an insertion mutation (a 4.9 kb
piece of DNA was inserted)and a translocation breakpoint (associated with flz Rpl )12
' . The gene has been
sequenced and the primary structure of the encoded
protein predicte&. The RNA splicing pattern was
deduced by comparing the sequence of a cDNA clone
to the genomic sequence. The encoded protein, 413
amino acids long, has an unusual composition. The
amino terminal part (about 250 amino acids) is more
than 10% proline and 10% tyrosine and ends at the
point where the single intron interrupts the gene. The
next part of the protein, a domain of 60 amino acids, is
about 30% basic amino acids and is not rich in either
proline or tyrosine. The C-terminal part is again 10%
proline and 10% tyrosine and is also about 15% glutamine. Theftz Rpx translocation results in replacement
of the C-terminal part with a few novel amino acids, so
that a truncatedj)rotein is encoded. Since the mutant
phenotype offtz~Pl/ftz- embryos is less extreme than
that of ftz-/ftz- embryos, the truncated protein
probably does have partial function ~2.
In situ h y b r i d i z a t i o n to t i s s u e s
different). In situ hybridization revealed that flz
transcripts are present even before the cells have
formed and that they are localized in stripes around
the embryo. The stripes appear to correspond to the
priv~ordia of alternate segments of the embryo. Both
ect¢,dermal and mesodermal progenitor cells contain
ftz transcripts. On the basis of these results, it appears
that the nuclei in syncytial blastoderm-stage embryos
become aware of their spatial arrangement and a
subset of them respond by synthesizingftz products.
An interesting subtlety is that initially transcripts are
not arranged in well-defined stripes, but are instead
more uniform. The striped pattern appears subsequently. The gradual emergence of the final pattern
suggests that regulatory interactions occur at the
initial time offlz expression. Presumably the striped
pattern of ftz transcripts, and quite likely similar
patterns of expression of other segmentation genes, is
the way in which the segmental differentiation
process begins. More precise segmental identities
would arise in response to the differential expression
of homoeotic genes such as A nip.
Antp transcripts are found in embryos, larvae and
pupae ~~:~. The segmental distribution of the RNAs
corresponds to the segments that genetic studies"~)7
show require Anlp function. It is not yet known
whether the two major A nip transcripts differ in their
spatial distribution. They do vary in relative amount
during development 9. An unexpected finding is that
Antp transcripts, like Ubx transcripts ~:~, are most
abundant in the neural tissue~:~-2LThe neural ganglia
are segmented, so the activity ofA ntp in neural ceils is
not surprising but why the highest concentration of
RNA is in neural cells is not known. Antp transcripts
are found early in development in abdominal segments but the abdo~vinal segments do not have detectable Antp RN,\ at later stages ~. As in the case offlz,
!he location of the transcripts becomes progressively
more restricted. The absence nf BX-C function, which
transforms the abdominal segr~,cnts into thoracic segments, also results in stable and abundant accumulation of A ntp transcripts in the transformed abdominal
segments 2~. Thus, BX-C gene activity is capable of
affecting the location of A ntp transcripts.
The major finding from the in situ hybridization
studies is that the accumulation of RNA encoded by a
regulatory gene is limited to tissues in which the gene
has been shown to function. An alternative would
have been, for example, that a homoeotic gene is
expressed everywhere but its activity is restricted by
the presence or absence of modulating cofactors.
The availability of molecular clones had made it
possible to observe the distribution offlz and Antp
transcripts using in situ hybridization to sectioned T h e h o m o e o d o m a i n
An early surprise was the finding that an Ant/)
tissue ~:)']4.The technique reveals the accumulation of
transcripts and therefore reflects the balance between cDNA clone hybridized to the ftz locus9. Further
synthesis and turnover. The results for flz were investigation revealed cross-hybridizationbetweenftz
especially striking. The body segments first appear in protein-coding DNA and DNA within the 3' exons of
the embryo after about 5 hours of development, but An~O and Ubx (Fig. 2)2627. DNA sequencing revealed
flz, which regulates the segmental pattern, is first that the 60 amino acid middle-domain of theflz protein
active at the blastoderm stage 3 hours earlier. The is nearly identical to a predicted protein sequence
blastoderm-stage embryo is initially a syncytium of encoded in the 3' exons of Ubx and Antp (Refs 27, 28).
nuclei lying along the surface of the embryo. Later, In addition, similar cross-hybridizing sequences are
cell membranes form around the nuclei to give a found elsewhere in the ANT-C and BX-C, including
monolayer of cells that appear tO be homogeneous positions that probably correspond to the DefirnneA
(except for the germ line or 'pole' cells which look gene of the ANT-C and the infraabdominal-2 gene of
T I G - March 1985
Splice Site
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TCG ~
CGC ACC CGT C&G ACG TAC &CC C(~ T/~
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~
l.l~(l SER L~U SER GI~ KRG
CAG ATC /lAG &TC TGG TTC C/~ ~ C CC~ COC ATG ~J~G TCG ~ G ~ G C~T
GIJ4 l i e LYS IL II TRP P ~
Gill /LJSN/~G ~tG ~
LYS SEll LYS LYS ASP
Fig. 4. Sequenceof the ftz ~ i n ,
showingthe locationof
the RNA splicejunction that separates the homoeoboxsequence
from the upstream codingsequence. Underlinedamino acidsare
amserved in the Antp honweodomain. Verticalline indicatesstart
of homoeodomain.
the BX-C26. The 60 amino acid protein sequence is
commonly referred to as the homoeodomain; the DNA
sequence as the homoeobox. Theftz homoeodomain is
shown in Fig. 4. Whether all the genes that contain the
sequence are homoeotic (is ft,?) is an unsettled issue.
Also, only some of the homoeotic genes appear to
encode a homoeodomain, although this could be a
result of our inability to detect poorly-conserved
coding sequences by DNA hybridization. About seven
homoeodomain coding sequences have been found in
D. me/anogaster - fewer than the number of homoeotic
loci (at least 15). The homoeodomain is also encoded
by genes found in higher vertebrates, including
humans =. Two copies of the coding sequence have
been found to be within 3 Vb of each other in the
human genome~, suggesting that developmentregulating gene clusters also exist in humans.
The homoeodomain is highly basic, which suggests
that it may be employed in RNA or DNA interactions.
More specific support for this idea comes from the
homology of part of the homoeodomain with the DNA
binding domains of certain bacterial proteins z2. Three
bacterial DNA-binding proteins have been crystallized and in each case the part of the protein that
contacts the DNA is composed of two a-helical
sections with a sharp mrn between them 3°. Certain
amino acids (or types of amino acid) are required in
particular framework positions in the structure. For
example, small residues such as glycine are needed in
the tight turn between the helices. Non-framework
residues are involved in DNA sequence recognition
and therefore differ in proteins of different function.
The homology between a 20 amino acid part of the
homoeodomain and the bacterial helix-turn-helix
structure is in the framework residues.
Since there is quite a lot of variation between the
putative DNA binding domains of different bacterial
proteins 3°, it is hard to say whether the homoeodomain
(and yeast mating-type) homologies with the helixturn-helix structure are significant. A clue that shows
one of the critical frarnework amino acids to r e W i r e W ~ ~
rant for flz function comes from a temperature-sensitive (ts) allele of ftz. In the ts allele, an alanine that is
one of the most conserved amino acids in the proposed
helix-turn-helix structure is changed into a valine2z.
The structural homology and the ts allele suggest that
the possibility of the homoeodomain being a DNA
binding sequence is worth investigating, as are alternatives such as an RNA-binding function. The recent
observation of Ubx-encoded protein(s) in the nuclei of
imaginal discs and embryonic neural cells is consistent
with DNA (or RNA) binding function for homoeotic
gene products31'32. DNA binding activity is one way
that the homoeotic proteins could influence the expressions of other genes. However, the central
question (really a cell biology problem) is how gene
activity results in the spatial localization of structures.
A DNA-binding model is a long way from explaining
how bristles are always found in the same places.
Puzzles and prospects
E. Lewis proposed that the different homoeotic
genes of the BX-C evolved by a process of duplication
and divergence ~. As yet, the homoeodomain is the
only molecular evidence that different homoeotic
genes are related structurally. It is increasingly clear,
however, that the homoeotic genes are part of an
interacting network of genes that together create the
normal pattern of development. Such regulatory
genes are plausible targets of mutations that lead to
new patterns and, eventually, to new species. Genetic
analysis is the key to identifying the components of the
regulatory gene hierarchy. Molecular analysis
supplies the fine details of structure and sensitive
probes for gene products. With the tools that are
currently being prepared, it will soon be possible to
study the molecular mechanisms through which the
regulatory genes exert their influence. Both genetic
and molecular techniques will help to reveal how
various genes interact. We need to know why genes
are clustered (history or function?), why genes as
different as Antp andftz encode proteins that share a
domain, the reason for giant transcription units, how
the position-specific differences in homoeotic gene
expression are controlled and how these differences
lead to a choice of a differentiation pathway. The
extraordinary evolutionary conservation of the
homoeodomain suggests that at least some of the
answers have ancient roots and may have very general
importance.
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Acknowledgements
cytology and genetics. Genetics 105, 581-600
I would like to thank Drs Thomas Kaufman, fan
18 Kafatos, F. (1972) The cocoonase zymogen cells of silk
moths: a model of terminal cell differentiation for Duncan, Allen Laughan and Sean Carroll for
specific protein synthesis. Curt. Topics Dev. Biol. 7, thoughtful discussions relevant to this review.
125-146
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Cloning and transcriptional analysis of the segmenta- Developmental Biology. Universi~' of Colorado at Boulder,
Campus Box 347, Boulder. CO 80309, USA.
tion gene fushi tarazu of Drosophila. Cell 37, 825-831