Clusters of Master Control Genes
Regulate the Developmentof Higher Organisms
Edward B. Lewis, PhD

THIS article is an account of how basic
research on the nature of the gene has
led to the discovery of two clusters ofmaster control genes that play a central
role in programming the developmentof the fly. These clusters, known as thebithorax complex (BX-C) and Antennapedia complex (ANT-C), are believed tohave evolved from an ancestral gene bya process of tandem duplication and divergence in function by mutation. So
successful during evolution have cognates of these clusters been in programming development that they are now
found in vertebrates, including humans,
as well as in invertebrates.
The strategy of achieving complexity
by duplicating and then diversifying isused at the developmental level, as well
as at the genetic level. Thus, early inembryonic life, most higher organisms

consist of little more than a tandem array of duplicated body segments. During the course of development, the segments diverge from one another and diversify to produce the variety of forms
that these organisms achieve. It now
seems likely that it is precisely the genesof the BX-C and ANT-C, or their cog¬
nates in other organisms, that initiate
and control the diversification of the bodysegments.
This article is concerned primarily
with the BX-C, how it came to be dis¬covered, and how we think it functions

during development.

From the Division of Biology, California Institute ofTechnology, Pasadena.

The 1991 Albert Lasker Medical Award was preNew Yorksented to Dr Lewis on September 27,1991,in

City.
Reprint requests to Division of Biology, California Institute of Technology, Pasadena, CA 91125 (Dr Lewis).

EARLY HISTORY

The view of genes as beads on a string,randomly arranged by function, was for¬ever altered in 1925 by Sturtevant's dis¬covery that the function of a gene isdependent on its position in the chro¬mosome. ' He came on this phenomenonof "position effect" in the course of an¬alyzing one of the first cases of unstablegenes in Drosophila, the famous Bareyed mutant, found by Tice in 1913. Sturtevant showed that the occurrence of
two exceptional types of progeny fromhomozygous Bar flies—non-Bar (nor¬mal-eyed) and double-Bar (tiny-eyed)
types—was associated with crossingover of an unusual kind. He assumed
that non-Bar types had lost the Bar geneand that double-Bar was a tandem du¬plication of that gene. (A detailed ac¬
count of this early work and of relevant
crossing-over mechanisms is given by
Maeda and Smithies.2)
In 1936, using the then recently dis¬covered giant salivary gland chromo¬
somes of the Drosophila larva, Bridges3
and Muller et al4 showed, independently,that the Bar mutation is itself a tandem
duplication of seven bands, and doubleBar is a tandem triplication, rather thana duplication, for those bands. The or¬igin of this triplication can be easily pic¬
tured if, for present purposes, we rep¬resent Bar as having the sequence
ABCABC and if we introduce a crossingover between two homologous ABC re¬gions that are unequally paired. Thus,
ABCABCx -> ABCABCABC

ABCABC
Non-Bar is, of course, readily ac¬
counted for as the reciprocal crossoverproduct, ABC.

From homozygotes for the triplica¬
a similar kind of crossing-overevent is expected to generate a single
copy of ABC and a fivefold tandem rep¬
etition of ABC. Just such a quintuplication was generated by Rapoport5 whocalled it quadruple-Bar, since at thetime he would not have known that Bar
itself was a duplication. Later, Rapo¬
port obtained a sevenfold to ninefold
repetition from homozygous quadrupleBar females, as described by Lindsley
and Grell.6 These early studies showed
for the first time that, starting with atandem duplication, multiple linear rep¬
etition can be generated by successive
rounds of crossing-over.
In a second development, Bridges7
called attention to several types of sal¬
ivary gland chromosome banding pat¬terns, which he interpreted as duplica¬
tions that have become established in
the species. Genetic evidence for suchduplications was however lacking.
In 1940, Oliver8 reported that hecould recover apparent reverse muta¬
tions associated with crossing-over be¬
tween two lozenge eye mutants of
Drosophila. An alternative interpreta¬tion was that the reversions were notmutations but instead were wild-typecrossovers between two separate but
closely linked loci. Failure to find thereciprocal crossover having both mu¬
tants in the same chromosome left thevalidity of the latter interpretation in
limbo.
Another pair of rough-eyed mutants,
Star(S) and asteroid (ast), proved more
tractable to analysis. (This mutant,originally called Star-recessive,9 was
found by E. Novitski, who sent it to me
in 1937, suggesting that I test to see if
it was an alíele of S. We were undertion

Fig 1. —Photographs of adult wild-type and homeotic mutant flies. A, Wild-type male (genotype: heldout, in order to produce spread wings). B, Dorsal view of afour-winged fly homozygous for the triple mutant combination anterobithorax bithorax3 postbithorax. The third thoracic segment is transformed into one resembling
the second, resulting in two sets of nearly identical thoraces and wings. C, Ventral view of an eight-legged fly of the genotype bithoraxoid I Df, where Df is a de¬ficiency for the bithoraxoid gene. D, Dorsal view of an Antennapedia male (genotype: Antpy" I Pc3 [where the Pc mutant enhances the Antp phenotype]).

graduates at the time, he at Purdue
University and I at the University of
Minnesota, where C. P. Oliver had
kindly given me space in his laboratory
to work on Drosophila. Novitski and I

had worked together on Drosophila
while in high school in Wilkes-Barre,
Pa.) From the trans hétérozygote, S +I + ast, not only wild-type crossovers
but the reciprocal double mutant, S ast,
could be derived as well.10 Moreover,there is a striking position effect.
Whereas S + / + ast animals are nearly
eyeless and have wing defects, S ast I ++ animals are wild-type except for aslightly roughened eye identical withthat of S / + animals.10·11 Thus, only
when the two wild-type alíeles (sym¬bolized by + signs) are located together
in the same chromosome or in eis dothey function normally. The eis hét¬érozygotes for lozenge mutants were
eventually obtained by Green andGreen12; whereas the trans hétérozy¬gotes have small eyes, the eis have nor¬
mal eyes. A comparison of the eis andtrans phenotypes has come to be known

as the cis-transtest, and the position

effect, if present, as the cis-trans effect(see Hayes13 for a discussion of the his¬

tory of these concepts).

The S and ast loci proved to be withindouble-banded structure known as
21E1-2 near the tip of the second chro¬mosome.14 Not only had Bridges7 inter¬
preted such structures as tandem du¬plications that had become establishedin the species, he had actually cited
21E1-2 as the type example. Although
this correlation of genetic and cytological observations seemed to lend sup¬port to the tandem gene duplication hy¬
pothesis, we still do not know the true
significance of such doublet structures,and proof that they consist of separate,
homologous bands has not been forth¬acoming.

THE BX-C

In 1946, a deliberate search was be¬

gun to find additional cases of the S-ast
type to test the theory that a common
mode of gene evolution would involve
tandem gene duplication. The resultant

genetic redundancy would allow one of

the genes to evolve a new function while
the other gene continued to carry out
the old function.11
Of several cases that we explored,
the most illuminating from a develop¬
mental standpoint was that of a series ofmutants that transform the third tho¬racic segment (T3) of the fly toward thesecond (T2). The type mutant, bithorax(bx), was discovered by Bridges in 1915
(as reported by Lindsley and Grell6); it
is a weak and variable mutant. Fortu¬
nately, a very strong and constant mu¬
tant, bxi, was found later by Stern (alsoas reported by Lindsley and Grell6) andis the one that has been most useful indissecting the bx function and contrib¬
uting to the creation of the four-winged
fly (Fig 1, B). The details of the early
work on these mutants need not con¬cern us herein except to note that three
types of mutants, bx, Ultrabithorax(Ubx) (formerly bxD), and bithoraxoid(bxd) mapped to three closely linked locion the genetic map. Cytologically, theymapped to two adjacent doublet struc-Fig 2.—A correlation of the cytological and genetic maps of the bithorax complex. The bithorax complex maps to the two doublets in 89E of the third chromosomeof the salivary gland chromosomes. Dominant gain-of-function mutants are In the row above that of the loss-of-function mutants. Ubx indicates Ultrabithorax; abx,
anterobithorax; bx, bithorax; Cbx, Contrabithorax; bxd, bithoraxoid; pbx, postbithorax; iab, infra-abdominal; Hab, Hyperabdominal; Mcp, Miscadastral pigmen¬
tation; and Tab, Transabdominal.
tures that often fuse into one struc¬ture, presumably as the result of pair¬ing of partially homologous bands (Fig2).15 Again, Bridges' prediction that such
structures represent tandem duplica¬
tions seemed well supported.

From the start, we found dramatic
cis-trans effects between almost every
double mutant combination tested.11
Furthermore, these effects were soon
found to be conspicuously polarized.
Thus, if we let a and b represent any
two recessive mutants that are qualita¬
tively different in phenotype, then in
every case in which a b I + + is wildtype and a + I + b is mutant, the animal
shows either the a phenotype or the bphenotype, but not both. For example,
bx3 +1 + pbx (postbithorax) has a weakpbx phenotype (posterior T3 trans¬
formed toward posterior T2) but no traceof the bx phenotype (anterior T3 trans¬
formed toward anterior T2). Only muchlater were such polarized effects dis¬covered in the opérons of bacteria.16
ANALYSIS BY GENE DELETION
A new dimension to the analysis camewith the derivation of x-ray-induced de¬letions for the BX-C, or for portions
thereof. Thus, animals lacking all of the
BX-C (homozygous for a deletion of thetwo 89E doublets) die as late embryos
but not before they have developed the
intricate cuticular and trachea! systems
that enable the body segments to be
distinguished from one another. Suchanimals show a striking transformationof the abdominal segments transformed
into a row of thoraciclike segments.15
Thus, complete loss of the BX-C results
in a more extreme homeotic transfor¬
mation than that produced by the spon-taneous mutants, all of which turned
out to represent only partial losses ofgene function.

WHAT DO THE BX-C GENES DO?
In 1964, we reported that mutants of
the BX-C are autonomously expressedin somatic mosaics. " We therefore knewthat the developmental abnormalities
were not due to substances, such as hor¬mones, that diffuse readily between

cells. Our mosaic studies further dem¬
onstrated that even late in development,a bx3 mutant expressed the bx trans¬
formation even in a single cell of an oth¬erwise wild-type fly.18 Thus, the wildtype gene could be inferred to function
throughout development, at least until
the pupal stage.
It has always been somewhat treach¬erous to deduce the normal function ofa gene from the effects on the organismof mutations in that gene. The problem
is especially severe when the mutations
result in morphological changes. To as¬
sess the normal or wild-type functionsof the BX-C, we adopted a new proce¬dure. We constructed a set of genotypes
in which the BX-C was entirely lacking
except for the presence of one dose ofvarious wild-type regions of the com¬
plex. In this way, we could show that allof the regions studied are involved ininitiating, or in some cases suppressing,
the formation of specific organs andstructures, a more profound and basic
type of developmental function than had
been inferred from considering the homeotic effects shown by the mutants.16,18
Analysis of the function of the wildtype BX-C genes during development
revealed two basic properties ofthe sys¬tem. First, the genes are arranged in

the chromosome in the same order inwhich they are expressed along the anaxis of the organism (Figteroposterior3) 15,19 T^jg colinearity has now beenfound in all clusters of the BX-C type inwhich the order in the chromosome has
been established, as will be discussed
herein. Second, the morphological evi¬
dence showed that once expressed in agiven segment, the gene tends to re¬main expressed in more posterior seg¬ments (Fig 4).15 Thus, the more poste¬rior a segment in the organism, the moreadvanced in development it is as far as
genes of the BX-C are concerned, theground state being that of a thoracic

state.15

Further subdivision of the complex
only by means of over¬lapping deletions but by rearrangementof breakage points in the complex.15·20
The latter have proved useful in iden¬
tifying a set of cis-regulatory regionsthat direct the control of gene expres¬sion in each of the abdominal segmentsfrom A2 to A9, inclusive. A loss-of-function mutation in one of these regions,
was achieved notdesignatedinfra-abdominal (iab),
segment involved to trans¬
form to the next most anterior type ofsegment, ie, toward the thoracic orcauses theground state.

Before recombinant DNA methodol¬
ogy was applied to the BX-C, we inter¬

preted the wild-type action of the BX-C

genes in terms of the operon model16 forgene clusters in prokaryotes. Thus,
many of the genetic properties of the
BX-C, including polarized cis-trans ef¬
fects and the presence of dominant con¬stitutive mutants (Fig 2), appeared tobe remarkably similar to the genetic
properties of opérons. This led us tosuggest that the BX-C might constitutesystem in which the func¬

discussed, proves to be entirely differ¬ent from that of the operon. Our earlier
concept of the function ofthe BX-C genes
was clearly too narrow. Quite different

an inducibletion of its genes would be to "repress
certain systems of cellular differentia¬
tion and thereby allow other systems tocome into play."17 However, the molec¬
ular basis of the BX-C, which will be

and only partially understood mecha¬
nisms induce the expression ofthe BX-C
genes, and we now suppose that thesegenes can activate, as well as repress,
systems of cellular differentiation.
One of the most challenging problems
remaining will be to identify the genesthat the BX-C genes control. Such"downstream" or "target" genes arethose that we assume will bring about
the differentiation of tissues and organsof the third thoracic and abdominal re¬gions of the organisms.

THE MOLECULAR GENETICS OF
THE BX-C
In 1978, Prof David Hogness and his
collaborators at Stanford University be¬
gan a molecular analysis of the BX-C.
They used chromosome walking andjumping to clone the BX-C.20 The cur¬rent status of the molecular map of thecomplex is shown in the lower half of
Fig 4 based on this early work and sub¬sequent work.21·22 Three domains offunc¬tion have come to be recognized: Ubx,
abdominal- (abd-A), and Abdominal-B (Abd-B), named after the proteinclass coded by each domain.18 The Ubx

Fig 3.—Summary of Drosophila bithorax complex
functions and their spatial expression. The parasegmental (PS) morphological function and protein
expression for PS5 to PS14 (segments T3 to A9)
are shown in parentheses and are inclusive. The
extent of Ultrabithorax function and protein expres¬sion is shown in green; that of abdominal- (abd-A)
in blue, Abd-BI in yellow, and Abd-BII In orange; bx
indicates bithorax; bxd, bithoraxoid; and iab, infraabdominal.

I
f200

I

I

I

I

I

I

I

domain has four exons and a very large
intron within which lie the anterobithorax (abx) and bx loci. As the result ofalternative splicing, a family of proteins
results.23 For the sake of simplicity, only
the largest transcription unit is shownin Fig 4.
The colinearity rule, first noticed formorphological effects, as already dis¬cussed, has been fully supported by stud¬ies of the expression of proteins comingfrom each of the three domains of the
BX-C using immunohistochemical tech¬niques (Fig 4). Thus, in wild-type em¬bryos, the Ubx proteins are first de¬tected in posterior T2 and anterior T324·25(or in terms of the parasegmental [PS])units26 of the embryonic ectoderm, in
PS5). The expression declines but re¬mains in the abdominal segments.25·27
Analysis of mutant embryos indicatesthat the bx and bxd regions direct Ubx
protein expression proteins in PS5 andPS6, respectively.26·27

The level of abd-A protein in wild-

type embryos remains elevated in thesesegments and those following up to andincluding PS13.28·29 Double labeling with
antibodies to Ubx and abd-A proteins
has shown that Ubx proteins, although

reduced in amounts, are on in different
cell types than is the abd-A protein.29
Analysis of mutants in the iab-2, iab-3,

I

I

I

I

h100

I

I

I

I

-100

H

M
iab-4

Abd-Bs

abd-

->^.
Abd-B

Ubx

òxd

-/^,
aixM

Ubx

Fig 4.—Colinearity of order of gene expression in the adult fly and the order on the molecular map of the c/s-regulatory regions. The bithorax complex encompasses
300 kb (from +200 kb to 100 kb, where the start of the walk was 0.0). Below the map, which is approximately to scale, are five characterized transcription units;
three containing homeoboxes, Abd-B, abd-A, and Ubx; and two noncoding, iab-4 and bxd; iab indicates infra-abdominal; pbx, postbithorax; bxd, bithoraxoid; bx,
bithorax; abx, anterobithorax; abd, abdominal; and Ubx, Ultrabithorax.
-

alleis the morphological expression. It
confirms and establishes the rule thatthe more posterior the segment, themore functions controlled by the cisregulatory regions become expressed up
to and including PS13. Thereafter, there
is loss of detectable expression of all but
the small Abd-B protein.
The colinearity rule also applies toRNAs coming from the tab regions of
the complex, as shown by Sánchez-Her¬
rero and Akam.34 They point out thatthese transcripts may be functionless,as discussed for the bxd case by Lipshitz
et al,35 or the iab transcripts "might func¬tion in eis by some unprecedented mech¬anism."34

Fig 5.—Correlation of homeotic gene expression in
the Drosophila embryo with that of the mouse em¬bryo. The anterior ends of the embryos are to theleft. The Drosophila homeotic genes form two clus¬
ters in the right arm of the third chromosome, theseparation being between the Antp and Ubx genes.
The homologous mouse genes are in a single clus¬
ter; among the four such clusters only that of Hox2
in chromosome 11 is shown. The genes are colorcoded; however, the homeobox sequences of theAntp, Ubx, and abd-A genes of Drosophila ate sosimilar that homology with the mouse genes isbased only on the expression patterns. In the
Drosophila and mouse embryos, the anterior
boundary of expression of each gene is shown by
the corresponding color for each gene. For simplic¬
ity, the posterior boundary for a given color is notshown since it would usually continue beyond thenext most posterior gene's anterior boundary. Only
expression in the cuticle of the Drosophila embryo
is shown, while in the mouse, expression is shownin the nervous system and in the prevertebrae. lab
indicates labial; pb, Proboscipedia; Dfd, De¬formed; Scr, Sex-combs reduced; Ubx, Ultrabithorax; and abd, abdominal.

and iab-4 regions show that abd-A pro¬tein is expressed in a colinear fashion
for PS7, PS8, and PS9.29·30
The Abd-B region produces a large
and small protein.3032 Our analysis of
the distribution of these proteins has
been indirect since our monoclonal an¬
tibody is directed to an epitope in the 3'exon common to both proteins of thisregion. To infer their distribution, there¬
fore, we have used mutants that elim¬
inate the messenger RNA for either thesmall or the large protein. The latter isfirst detected in the posterior fourth ab¬
dominal segment (PS10), while the small
protein coming from the more distally lo¬
cated promoter is first detected in the pos¬terior eighth abdominal segment (PS14).
Using rearrangement break points dis¬
rupting the iab-5 to iab-9 regions, inclu¬
sively, we have shown that the pattern ofexpression of the large protein is colinearwith the order of those regions.33
Thus, the expression of proteins fromthe three domains of the complex par-THE ANT-C
It is likely that the BX-C was once
part of a single cluster that included
another larger complex, the ANT-C.36
The latter lies in the same chromosome
arm but at a considerable distance prox¬
imal to the BX-C. The type mutant for
which this complex was named was firstfound in Drosophila affinis by Sturtevant.37 The phenotype is that of an an¬
tenna transformed toward a leg. A mu¬tant with a strong transformation of thistype (Fig 1, D) was induced with x-rays
in 1948 by Yu as described by Lindsley
and Grell.6 In the same year, Les Calves
reported the occurrence of a neutroninduced mutant that he named Aristapedia and suggested that it was a dom¬
inant alíele of a recessive mutant andcauses the antenna to transform towarda tarsus, rather than a full leg. From his
determination of its cytological locationin 84A, the location of the ANT-C (andof Aristapedia in 89C16), it is now clearthat it is in fact an alíele of the Antennapedia locus.
Extensive genetic and cytologicalanalyses of these and other related mu¬tants by Kaufman and colleagues38 es¬
tablished the existence of this second
cluster of homeotic mutants and theircolinearity (Fig 5). Extensive molecularanalyses have been made, particularlyof the Antp gene itself.39·40

REGULATION OF THE GENES OF
THE BX-C IN TRANS
A class of genes that initiates the axesof the body and divides the body into
segments has been identified by Nüs¬
slein-Volhard and Wieschaus41 based on
massive screenings for mutants affect¬ing early development Drosophila.42·43
Some of these genes appear to act as

major traws-regulators of the BX-C andANT-C. A possible relationship of one
such gene, hunchback (hb), to the BX-C
was first detected genetically when, Regulator-of-pbx (Rg-pbx) a dominant transregulator of the postbithorax function of

the BX-C,44 was found by Bender et al45
to be a gain-of-function mutant of the hb
gene. Garcia-Bellido and Capdevila^had
earlier made extensive studies of therole of Rg-pbx in regulating the BX-C.
Recently, Qian et al47 have shown the hb
protein binds to a specific motif in the
intron of the Ubx gene and interacts torepress transcription of Ubx in regions
anterior to the onset of its normal tran¬
scription in PS5 of that gene. The ex¬pression of Ubx has been inferred to be
iraws-regulated by still other genes ofthe segment-initiation class.43·48"51 Thus,
the genes that initiate segment forma¬tion seem to represent a major and per¬haps primary set of regulators of the
BX-C and ANT-C.
Several genes act as negative regu¬
lators of the BX-C; that is, if such a geneis removed or inactivated, many if not
all of the genes of the complex become
activated, resulting in all of the seg¬ments of the body being converted to¬ward the eighth-abdominal segment.16
The first of these genes was Polycomb,
found by P. H. Lewis as reported by
Lindsley and Grell.6 Zink and Paro52 haveshown that the protein coded for by thenormal Polycomb gene is specifically as¬sociated in the salivary gland chromo¬
somes to the BX-C and ANT-C regions,as well as other sites. Zink et al53 haveshown in an elegant way that it binds toupstream (5') sequences of the Antp
gene. Paro and Hogness54 find that thesequence of the Polycomb gene showssome homology, not as might have been
expected to a DNA-binding class of pro¬teins but to a nonhistone chromosomal
protein. Hence, the remarkably pre¬cise binding of Polycomb protein tobands in the salivary gland chromo¬
somes associated with the BX-C andANT-C suggests that the protein iscomplexing with one or more otherproteins that do bind to those bands.
If so, the Polycomb class of genes18
would be involved in maintaining a
repressed state in regions of the em¬bryo where it is not activated.55·56 Ingham and Whittle57 have shown that
another gene, trithorax, probably al¬leile to Regulator-of-bx (Rg-bx), acts
as a positive regulator. Ingham58 sug¬gests that it may be involved in main¬taining the activated state of the
BX-C genes.

THE HOMEOBOXSupport from molecular studies forthe hypothesis that the BX-C and the
ANT-C owe their origin to repeated tan¬dem gene duplication finally came withthe discovery of the homeobox by McGinnis et al59 and independently by Scottand Weiner.60 This region of 160 basepair codes for 60 amino acids that con-

stitute the DNA binding or "homeodomain" of each of the proteins codedby the BX-C and ANT-C genes. It is
now thought likely that these proteinsbind to downstream genes and thereby
regulate them.
So similar are the homeobox se¬quences between adjacent genes that it
is exceedingly improbable that theyarose by convergent evolution. Only the
homeodomain of the protein shows ob¬
vious conservation, possibly because
most ofthe specificity required for DNA
binding lies in the homeodomain. We
would expect, however, that many
motifs in enhancerlike elements of the
cis-regulatory regions may be con¬served. At present, there are insuffi¬
cient data on DNA sequence in theds-regulatory regions to test for con¬servation of such motifs.

HOMEOBOX GENE CLUSTERS IN
OTHER ORGANISMS
Genetic and morphological evidence
for cognates of the homeobox-containing genes of the ANT-C and BX-C havealso been discovered in beetles, where
they appear to lie within a single homeotic complex (HOM-C).61·62 (In thesilkworm Bombyx, a fascinating set ofmutants have striking parallels to manyof the mutants of the BX-C and ANT-C.
They were designated as alíeles of an E
locus within which, however, crossingover and cis-trans effects have been re¬ported,63"65 suggesting that they are infact a gene complex; however, in theabsence of a genetic or molecular map,the colinear rule obviously cannot be
tested. One of the E mutants produces
an eight-legged moth [a bronze sculp¬
ture of which is in an ancient temple inJapan]. The exact cognate of that mu¬
tant in Drosophila is bxd [Fig 1, C].
Another of the E mutants was reported
to lack gonads and led us to examine its
cognates in Drosophila, namely iab-4
mutants. When homozygous, the lattermutants are viable and appear virtually
wild-type, but internally they too lackgonads in both sexes.16)
Astonishingly, mice and humans not
only have cognates of the BX-C andANT-C genes in a single HOM-C, but
the complexes occur in four sets, each ina different chromosome.6669 Moreover,colinearity of spatial order of expres¬sion and molecular map order extends
over the entire complex in the murine(Fig 5) and in the human cases. Other
less extensively characterized cluster¬ing of cognates of the HOM-C type oc¬
cur in sea urchins,70 nematodes,71·72
bees,73 frogs,74 and chickens.75 Omitted
are a number of other organisms in which
cognates have been found for only one
or two genes of the HOM-C type.76·77

In Drosophila, it is not yet known
whether temporal order of expression is
also colinear with map order. However,there is evidence that colinearity is tem¬poral as well as spatial from experiments
involving (1) chicken76 and murine78 limb
buds; (2) expression of transcripts from
HOM-C in the central nervous systemand pre vertebrae of the mouse embryo79;
and (3) induction of the HOM-C by retin¬ole acid in human cell cultures.80
WHY HAVE THE HOM-C
REMAINED INTACT?
A fundamental question is why genesof the HOM-C have remained linked foran estimated 500 million years or more,assuming they arose before the separa¬tion of the vertebrates and inverte¬
brates. Even though they presumably
evolved by tandem gene repetition, it ishighly unlikely that the linkage would
have persisted in so many organisms
unless it confers a selective advantage.
Clearly, in Drosophila, the existenceof two separate complexes, which still
remain tightly linked, suggests that atleast the Antp and Ubx genes do nothave to be adjacent in this organism.
Whether other genes, such as Ubx andabd-A, can be separated and still func¬tion normally is uncertain. Several re¬arrangements with break points near
the boundary of the Ubx and abd-A do¬mains have been detected by their sup¬pression of transvection81·82 in a screenthat recovers break points that are unselected, with respect to having any mu¬tant effect.19 These rearrangements
show a striking phenotype; namely, de¬velopment of tiny sense organs on thelarval abdominal segments (organs nor¬mally found only on the larval thoracic
segments), an effect that corresponds
to a weak bxd phenotype. Bender et al83
have shown that such break points map
near the boundary of the Ubx and abd-A
domains; however, from their bxd phe¬notype, it is clear that they still lie in the
Ubx domain. Thus, if a break point canfall at or near that boundary and lack adetectable phenotype, our screen has

yet to recover one.

A possible clue to understanding why
gene clusters of the BX-C and ANT-C
type have remained intact comes fromstudies of gene regulation in anothercluster; namely, that coding for the fam¬
ily of ß-globin peptides of hemoglobin.
Regulation of the genes of this cluster
has been shown to depend on enhancer
elements residing in the cis-regulatory
regions surrounding the protein-coding
regions of the genes. Nickol and Felsen¬
feld84 and Choi and Engel85 find that theadult and embryonic ß-globin genes inchickens share an enhancer.
In the BX-C, Celniker et al33 haveevidence, based on morphology, that theiab-5 region, which normally regulates
the expression of the Abd-B gene, may
be regulating expression of both abd-A
and Abd-B. They suggest, therefore,that in wild-type, the abd-A and Abd-B
genes share one or more enhancers lo¬cated in the iab-5 region. Duncan18 has
discussed this and other cases in thecomplex where cis-regulatory elements
seem to be "bifunctional." If enhancer
sharing does occur in the BX-C and if it
confers a selective advantage, then thegenes involved will of course tend toremain linked.

WHY FOUR SETS OF COMPLEXESIN MICE AND HUMANS?
As just noted, homeobox (HOX) clus¬
ters have been duplicated in the genomeof mice and humans four times, each ina different chromosome in both organ¬
isms. In humans, four clusters are des¬ignated HOX1, HOX2, HOX3, andHOX4 and are located in chromosomes
7, 17, 12, 2, respectively. Boncinelli etal67 have speculated that having four
sets might provide "a finer-grained spa¬tial control" than would be needed inorganisms such as Drosophila, whichhave only one set and perhaps fewer
requirements for setting up the bodysegments than have the higher verte¬
brates.
MEDICAL IMPLICATIONS
We can look forward to an excitingtime when the functions of the genes ofthe four human HOX clusters are de¬termined. Human genetic abnormalities
at virtually any stage of development,
but especially those traceable to abnor¬
malities of early embryonic or fetal de¬velopment, would be expected from mu¬tations in genes of the HOX clusters.
The results of genetic experiments onother vertebrates strongly support suchconjecture.
Recently, Chisakaand Capecchi86 used
the new technique of gene targeting to
produce a disrupted Hoxl.5 gene of themouse. Homozygotes for this recessivealoss-of-function mutation are found to

produce a constellation of defects thatthey point out is remarkably similar tothe human congenital disorder DiGeorge
syndrome. The obvious implications arethat such a syndrome might result froma mutation in the human cognate of themouse gene and that other congenital
disease syndromes might result frommutations in other HOX genes.
Balling et al87 overexpressed the mu¬rine Hoxl.l (Hox2.3 of the HOX2 clus¬ter) gene in transgenic mice and ob¬
served craniofacial abnormalities. Kes¬sel et al,88 studying more severely af¬
fected mice from the same experiment,observed dramatic homeotic effects on
the cervical vertebrae. It follows that again-of-function mutation in the humancognate of Hoxl.l is expected to show
broadly similar types of defects, depend¬ing on the penetrance of the alíele.
In Drosophila, it is well known thatsuch gain-of-function mutants are notuncommon (Fig 2). The first example in
the BX-C was the Contrabithorax (Cbx)mutant, which causes T2 to transformtoward T3, notably turning the wings
into haltères.16·17 All of the Antennapedia mutants are also now known to be
an overexpression of the wild-type genein the antenna (Fig 1, D). We would
expect that when mutants of HOX genes
in humans are found they will often beof the gain-of-function type.
The HOX genes may also be impli¬
cated in the genesis of malignancies.
Blatt et al89 have reported a possible
example involving a murine myeloid leu¬
kemia cell line in which a Hox2.4 genehas been disrupted by a retroviral in¬
sertion. Their finding is confirmed byKongsuwan et al90 who have studied thesame cell line and who suggest thatHox2.4 may interfere with terminal dif¬ferentiation; then, in combination withactivation by the same provirus of an¬other gene in the same cell line, condi¬
tions would be met for development of
the transformed cell. Blatt and Sachs91
also report that deletion of the Hox4-lgene occurs in another murine myeloid
leukemia cell line; this gene is the cog¬nate of Hox2.7 (Fig 5). They have dis¬cussed a number of mechanisms thatmight involve such a deletion in the gen¬eration of the malignancy.
To summarize the medical relevanceof the discovery of HOX clusters in hu¬
mans, we can expect that germinal mu¬
tations occurring within these clusters
are likely to result in abnormalities atmany stages of human development. On
the basis of preliminary findings in mu¬
rine leukemia cell lines, it is conceivablethat somatic mutations in the HOX clus¬
ters may potentially play a significant
role in the generation of tumors, bothbenign and malignant. However, thesearch for DNA lesions in potential casesof germinal or somatic mutations pre¬sents formidable technical problems, notonly because of the large size of suchclusters but because of their fourfold

redundancy.
This study was supported in part by research
grant HD06331 from the National Institutes of
Health, Bethesda, Md, and by grants from theAmerican Cancer Society, Atlanta, Ga, the Marchof Dimes, White Plains, NY, and the Lucille P.
Markey Charitable Trust, Miami, Fla.
I thank Susan Celniker, PhD, and Welcome

Bender, PhD, for a critical reading of the manu¬
script and Peter Chang, James Hardwick, Helen
Park, and Seema Sharma, for assistance in the

preparation of it. While at Caltech, a number ofcolleagues have directly contributed to our re¬search on the bithorax complex, namely, Welcome
Bender, PhD, Marie-Paz Capdevila, PhD, Susan
Celniker, PhD, Loring Craymer, PhD, Madeline
Crosby, PhD, Ian Duncan, PhD, Antonio GarciaBellido, PhD, William Gelbart, PhD, Alain Ghysen, PhD, Lily Jan, PhD, Margit Lohs-Schardin,
PhD, and Shige Sakonju, PhD. Finally, I want tostress the close cooperation that we have had over
the years with Prof David Hogness, PhD, and hiscolleagues at Stanford University and Prof Wel¬
come Bender, PhD, at Harvard Medical School. It
Professor Hogness' foresight to launch themolecular analysis of the bithorax complex in 1978
in his laboratory.
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