Expression of homeo box genes during mouse development: A review

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10.1101/gad.2.7.773Access the most recent version at doi:
1988 2: 773-782Genes Dev.
P W Holland and B L Hogan
review.
Expression of homeo box genes during mouse development: a
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REVIEW
Expression of homeo box genes during
mouse development: a review
Peter W.H. Holland^ and Brigid L.M. Hogan^'^
^Department of Zoology, University of Oxford, South Parks Road, Oxford OXl 3PS, UK; ^Department of Cell Biology,
Vanderbilt University Medical School, Nashville, Tennesse 37232 USA
The aim of this review is to summarize briefly w^hat is
presently known about the spatial and temporal patterns
of homeo box gene expression during mouse develop-
ment. Since information about mouse homeo box genes
is accumulating very rapidly, this review does not offer a
complete compendium of facts about topics such as gene
sequence and organization. Instead, we have tried to as-
semble rather fragmentary published and unpublished
data about expression into a tentative framework which
may provide some clues as to the function of these
genes.
To simplify our analysis, we have concentrated
upon three different stages of mouse development; early
presomite gastrula (7-7.75 days post coitum*), early so-
mite neurula (8-8.5 days p.c), and a stage about two-
thirds through gestation (12.5 days p.c.) when most of
the organ systems have been established but are still un-
dergoing morphogenesis. Detailed accounts of the
anatomy of these embryos and the overall process of
mouse development can be found in Snell and Stevens
(1966),
Theiler (1972), Rugh (1968), and Hogan et al.
(1985,
1986).
The homeo box is a DNA sequence of about 180 bp,
originally identified within the coding region of several
Drosophila genes controlling embryonic development
(for review, see Gehring 1987). It is generally accepted
that the protein products of Drosophila homeo box
genes act as sequence-specific DNA binding proteins,
regulating gene expression. This suggestion is consistent
with the nuclear localization of several Drosophila
homeo box gene products (White and Wilcox 1984;
Beachy et al. 1985; Carroll and Scott 1985; Di Nardo et
al.
1985), with their reported in vitro DNA binding prop-
erties (Desplan et al. 1985), and with their predicted pro-
tein structure (Shepherd et al. 1984; Laughon et al.
1985).
Drosophila homeo box genes constitute a rather vari-
able gene family. DNA sequence comparisons, primarily
of the homeo box region
itself,
indicate that classes of
genes exist within which the genes share a more recent
evolutionary origin than that shared by the homeo box
gene family as a whole. The most extensive class, for
which Antennapedia (Antp) is the prototype, contains
several of the Drosophila homeotic genes (including
Antp, Sex combs reduced, Deformed, and infraabdo-
^To whom reprint requests should be addressed.
* Midday on the day of the vaginal plug is 0.5 day post coitum. However,
developmental ages can vary between embryos in a litter, and between
inbred and outbred strains of mice.
minal-J) see Gehring 1987). Other recognized classes of
Drosophila homeo box genes contain only one or a few
characterized genes. For example, engrailed
[en]
and in-
vected comprise the erz-like class (Poole et al. 1985; Co-
leman et al. 1987), whereas three other genes contain a
paired-likt homeo box (Bopp et al. 1986).
About 20 homeo box genes have so far been identified
in the mouse. Sequence comparisons indicate that most
of these genes are related to the Antp-like class of Dro-
sophila, whereas two have homeo box sequences more
similar to the en-likt genes. Current nomenclature
(Martin et al. 1987) designates each member of the first
class by a number after the prefix 'Hox-,' while the two
members of the second class have the prefix 'En-', Many
of the Hox- genes are organized into clusters, of which
the largest, Hox-1 and Hox-2, contain at least six genes
each and span over 70 kb on mouse chromosomes 6 and
11,
respectively. Genes within a cluster are numbered in
the order in which they were characterized. In addition
to the Hox- and En- genes, there are other more diver-
gent homeo box genes present in the mouse, but such
genes remain to be well characterized. More discussion
of the organization, and DNA sequence comparisons, of
mouse homeo box genes can be found in other recent
reviews and papers (Burglin et al. 1987; Colberg-Poley et
al.
1988; Fienberg et al. 1987; Hart et al. 1987; Lonai et
al.
1987; Martin et al. 1987).
It may be significant that close genetic linkage has
been established between some mouse homeo box genes
and known developmental mutations, e.g., between
En-1 and Dominant hemimelia (Hill et al. 1987; Joyner
and Martin 1987); £22-2 and Hemimelic extra toes, Ham-
mertoe, and reeler (Joyner and Martin 1987); the Hox-1
cluster and Hypodactyly (Mock et al. 1987; Rubin et al.
1987);
and the Hox-2 cluster and tail short (Munke et al.
1986;
Fienberg et al. 1987). However, it should be
stressed that allelism has not been demonstrated in any
of these cases.
Most studies of homeo box gene expression in the
mouse embryo have been concerned with steady-state
RNA levels. At present there are few specific antibodies
against mouse homeo box proteins available, and their
application to analyzing gene expression during embyro-
genesis has so far been limited to
En-1
(M. Frohman and
G. Martin, pers. comm.). However, antibodies have been
used to demonstrate that the protein products of mouse
Hox-1.1 (Kessel et al. 1987; Schulze et al. 1987), Hox-1.3
(Odenwald et al. 1987), and En-1 (M. Frohman and G.
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Holland
and Hogan
Martin, pers. comm.) are localized predominantly in the
nucleus, consistent with the in vitro DNA-binding prop-
erty demonstrated by the Hox-1.5 product (Fainsod et al.
1986).
By focusing on RNA studies we may be oversim-
plifying the issues, since it is possible that there are spa-
tial and temporal variations in transcription start sites,
splicing, and protein modification, for example. Never-
theless, we feel that it is worthwhile to take stock of
present information, and to see if it supports any useful
speculation about the nature of the developmental pro-
cesses that may be regulated by mammalian homeo box
genes.
We conclude that the patterns of expression seen
are consistent with a role for homeo box genes in the
determination of cell fate; an example of this role may
be to establish anteroposterior domains of positional
value in the mammalian embryo.
Early presomite gastrula (about
7.5-7.75
days post
coitum;
stage 11 of Theiler 1972)
Before gastrulation, the mouse embryo consists of two
epithelial sheets, the primitive ectoderm and the vis-
ceral endoderm (containing about 600 and 250 cells, re-
spectively), folded into a cup-shaped structure known as
the egg cylinder. Gastrulation begins when cells dela-
minate from the primitive ectoderm to generate a popu-
lation of individual mesodermal cells between the ecto-
derm and visceral endoderm. This process starts at the
posterior end of the tgg cylinder, in a region known as
the primitive streak (see Fig. 1 A) and continues for about
24 hr, during which time the anterior-posterior body
axis becomes apparent and the metamerism of the me-
soderm is established. Unfortunately, no detailed lin-
eage studies have yet been completed of gastrulation in
the mouse, but indirect experiments generally support
the idea that gastrulation movements and fate maps are
similar in mammalian and avian embryos (reviewed by
Snow 1985; Vakaet 1985). It is assumed that the first
mesoderm cells to delaminate through the primitive
streak end up beneath the neural folds and in the region
of the presumptive heart; they never aggregate into so-
mites,
but in the head form loose condensations known
as somitomeres. Mesoderm that delaminates later re-
mains at first in an apparently unorganized state (preso-
mitic mesoderm) before aggregating into visible somites
(Poelmann 1981; Meier and Tam 1982; Tam and Meier
1982;
Tam et al. 1982; Nakatsuji et al. 1986).
Although a number of studies have been made of
homeo box gene expression at the presomite stage of de-
velopment, the only positive evidence for differential
spatial expression comes from the in situ hybridization
studies of Gaunt (Gaunt et al. 1986; Gaunt 1987, 1988),
using probes for Hox-1.5 and Hox-3.1. He detects no ex-
pression of either gene before 7.5 days p.c. (for example,
at the stage shown in Fig. lA). Around 7.5-7.75 days,
however, expression of Hox-1.5 RNA is clearly detect-
able,
and is higher in posterior presomitic mesoderm and
ectoderm than in anterior tissues (Fig. 1B,C). Weaker ex-
pression is also seen in the allantois. This tissue is de-
rived from mesoderm which migrates from the posterior
end of the primitive streak and eventually contributes to
Figure 1. Presomite mouse embryos.
{A,B]
Sagittal section of
7.25-day post coitum embryo hybridized with antisense
Hox-1.5
probe. Mesodermal cells (mes) are begimiing to accu-
mulate between the posterior primitive ectoderm or epiblast
(ep) and the visceral endoderm
(end).
(A)
Anterior;
(P)
posterior;
(am) amnion.
{B]
Dark-ground illumination. No significant ex-
pression of any homeobox gene has so far been detected at this
stage by in situ hybridization.
{C,D)
Expression of
Hox-1.5
in
the
8-day
p.c.
embryo.
(B)
Bright-field and (C) dark-ground illu-
mination of sections hybridized with antisense
Hox-1.5
probe.
Note uniform expression in both the posterior presomite meso-
derm and overlying ectoderm. The grain density in the anterior
region is not significantly higher than with the positive sense
strand probe (see Gaunt 1987). (mes) Posterior mesoderm; (hf)
head fold neurectoderm with anterior mesoderm; (end) endo-
derm; (all) allantois; (am) amnion. Because the outer visceral
endoderm layer is so thin, it is not known whether this germ
layer is positive for expression or not. (Photomicrographs
kindly provided by Dr. S. Gaunt.) (See Gaunt 1987.)
extraembryonic blood vessels. Hox.3-1 RNA is also first
detected around 7.5 days p.c, predominantly localized to
the allantois. Careful in situ analysis has shown that
Hox-1.5 transcripts accumulate slightly earlier than
those of Hox-3.1 (Gaunt 1988), so that the two genes
show not only a spatial but also a temporal difference in
expression, even at this early stage.
Expression of Hox-2.1 and £22-2 could not be detected
in presomite embryos by in situ hybridization (Davis et
al.
1988; Holland and Hogan 1988). However, RNase
protection or Northern analysis, using RNA pooled from
a large number of embryos, has revealed very low levels
of Hox-2.1 and Hox-1.3 RNA at 7.5 days p.c. (Jackson et
al.
1985; Odenwald et al. 1987). Improvements in sensi-
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Homeo box genes during mouse development
tivity for in situ hybridization and use of antibody
probes would clearly facilitate analysis of these genes in
presomite embryos.
Early somite stage embryos (about 6 somites, 8-8.5
days post coitum; stage 12 of Theiler 1972)
As the embryo grows, the primitive streak extends and
the total amount of mesoderm tissue increases rapidly.
In the midline, some mesoderm cells differentiate into a
rod of tissue known as the notochord, while on either
side of the notochord paired somites gradually condense
in an anterior-to-posterior sequence. Cells furthest from
the midline do not contribute to metameric or repeating
structures and are known as the lateral plate mesoderm.
The intermediate mesoderm, which connects the so-
mites and the lateral plate, also shows metamerism, in
register with that of the somites. Eventually, the con-
densed somites will break up and give rise to a number
of different tissues. The part known as the dermatome
forms mesoderm in the skin, the myotome forms
muscle, and the sclerotome gives rise to vertebrae
around the spinal cord. During this process, the sclero-
tomes are referred to as prevertebrae. It is important to
note that not all the somites give rise to vertebrae; in the
mouse embryo the first four or five somites contribute
to part of the skull, so that the first vertebra (the atlas) is
derived from the fifth or sixth somite. It is also generally
accepted (but, in the absence of good lineage marking,
not rigorously proven) that each vertebra is derived from
the fusion of the posterior half of one sclerotome and the
anterior half of the next. These complications lead to
some ambiguities in numbering the somites as cervical,
thoracic, etc., so that in Figure 3 we have simply num-
bered them in straight sequence.
By the five- to six-somite stage, expression of a
number of homeo box genes can be detected clearly by in
situ hybridization (i.e., Hox-1.3, -1.5, -2.1, -3.1, -6.1,
En-2).
However, as shown in Figure 3, the spatial limits
of expression vary for the different genes. Of particular
interest is the expression of Hox-1.5, which a few hours
earlier was confined to posterior mesoderm and ecto-
derm and was about equal in both tissues (Fig. 1B,C). At
the early somite stage the spatial domains of Hox-1.5
expression appear to become spatially dissociated in me-
soderm and ectoderm. RNA levels decline significantly
in the mesoderm, but remain high in the ectoderm
where they extend anteriorly to the posterior end of the
neural folds (see open arrow in Fig. 2) (Gaimt 1987). This
position corresponds to the anterior limit of Hox-1.5 ex-
pression in later (10.5 days p.c.) embryos, in which the
neurectoderm has folded up into the neural tube and
brain. For Hox-1.5 the anterior limit is the midpoint of
one of the repeating neuromeric swellings in the mye-
lencephalon, just anterior to the otic vesicle (Fainsod et
al.
1987; Gaimt 1987). Subsequently, there is no signifi-
cant shift in this anterior boundary of Hox-1.5 in the
central nervous system (CNS) between 8.5 and 12.5 days
of development (Fig. 3).
The level of expression of Hox-2.1 appears to be lower
than Hox-1.5 but, in general, shows the same pattern at
this stage, although the anterior limit of hybridization in
the neural folds has not been so well defined (Holland
and Hogan 1988). In contrast, Hox-6.1, -1.3, -3.1, and
En-2 show very different patterns of RNA distribution to
Hox-1.5 and
-2.1
(Fig. 2). Hox-6.1 RNA is confined to the
posterior mesoderm, which has not yet condensed into
somites, to the overlying ectoderm, and to the allantois
(Sharpe et al. 1988). Similarly, Hox-1.3 expression is re-
ported to be confined to presomitic mesoderm (Dony
and Gruss 1987). En-2, on the other hand, shows a
narrow band of high-level expression in the neural folds
in the region of the foregut pocket. No expression is seen
in the underlying anterior mesoderm, or in other ecto-
dermal or mesodermal derivatives (Davis et al. 1988). As
development proceeds, En-2 expression is maintained in
structures derived from this narrow band of neural
tissue, namely in a large part of the metencephalon at
12.5 days p.c, and in the cerebellum, pons, and superior
and inferior colliculus at later stages still. In other
words, the domain of En-2 expression along the ante-
rior-posterior axis of the CNS does not change signifi-
cantly in spite of the extensive morphogenetic events
that take place between 8 and 12.5 days (and later).
Expression of Hox-3.1 has also been detected in early
somite stage mouse embryos by in situ hybridization.
RNA is reported to be localized to the posterior regions,
and apparently is equally abundant in neurectoderm,
presomitic mesoderm, and allantois (Gaunt 1988). In
slightly more advanced embryos, after the body axis has
'turned,' Hox-3.1 RNA is also detected in hindgut endo-
derm (Le Mouellic et al. 1988).
Embryos about two-thirds through gestation (12.5 days
p.C;
stage 20 of Theiler 1972)
This stage of embryogenesis has been studied most ex-
tensively for homeo box gene expression, because in
many cases it is the time when maximal transcript
levels are seen by Northern or RNase protection analysis
of total embryo RNA (Colberg-Poley et al. 1985; Jackson
et al. 1985; Awgulewitsch et al. 1986; Joyner and Martin
1987;
Rubin et al. 1987). By this time, all the major
systems of the body have been established. Briefly, the
somites posterior to somites 5 or 6 give rise to vertebrae,
skin mesoderm, and muscle blocks, as described in the
previous section. The lateral plate mesoderm has split
into two sheets; the somatopleure, which gives rise to
muscle and connective tissue in the body wall and the
limb buds, and the splanchnopleure, which gives rise to
mesodermal tissues enveloping the gut. Between the so-
mitic mesoderm and lateral plate mesoderm there is a
strip of mesoderm, loosely termed the intermediate me-
soderm, which gives rise to the segmented nephrotomes
of the pronephros, mesonephros, and metanephric
kidney. However, the precise origin of the mesoderm
that contributes to organs such as the lung, liver, and
stomach, and genital ridges is not clear; for convenience,
we will call it lateral plate mesoderm, although it may
be closer spatially to intermediate mesoderm (Le
Douarin 1964). Figure 3 shows the approximate levels
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Holland and Hogan
Allantois
Hind Gut
Pocket
Endoderm
Amnion
Heart
Fore Gut Poclcet
/ 5"
1
Pre-Somitic ^ ,^
/ I 1 IVIesoderr ' ^^'"^^^
/ 2.1 rm
I
Hox 6.1
Hox 1.5
Hox 2.1
En-2
Hox 3.1
6 5 4 3 2 1
J I I I » »
-Ectoderm
'iVIesoderm
Neurai \^\
Folds \ \
i\
n^^^^PTTTw
|lII!J!'!'?!r'?>i.v.v!«l»l«;v!v;.;i
Figure 2. Schematic representation of domains of expression of mouse homeo box genes in the six-somite neurula-stage mouse
embryo (approx. 8 days p.c). Mesoderm is stippled. No expression of En-2 is seen in the mesoderm. References: Hox-6.1, Sharpe et al.
(1988);
Hox-1.5, Gaunt (1987); Fainsod et al. (1987); Hox-2.1, Holland and Hogan (1988); En-2, Davis et al. (1988); Hox-3.1, Gaunt
(1988);
LeMouellic et al. (1988).
along the anterior-posterior axis at which the meso-
derm of visceral organs is thought to arise in the mouse
embryo. As discussed elsewhere (Flolland and Hogan
1988),
the evidence for this comes largely from cell-
marking studies on the chick embryo (Le Douarin 1964,
1982).
More precise localization of the origin of meso-
derm of the internal organs of the mouse is a major chal-
lenge for future lineage work.
By 12.5 days p.c. the central nervous system has
clearly differentiated into spinal cord and brain, which is
further subdivided anatomically into pros-, mes-, met-,
and myelencephalon (Fig. 3). During development, the
Figure 3. Schematic summary of mouse homeo box gene expression in the 12.5-day embryo. Vertical lines representing the limits of
expression of homeo box genes are determined by in situ hybridization of sagital sections of 12.5- to 13.5-day p.c. embryos. In some
cases the posterior limit is only approximate. Absence of expression, where clearly reported, is noted at the bottom of each panel. In
the right-hand part of the diagram, a cross means positive expression has been seen by in situ hybridization, and a minus sign means
no expression. A blank means expression has not been reported. For assignment of position along the anterior-posterior axis for
mesoderm of organ rudiments see Le Douarin (1964, 1982) and Holland and Hogan (1988). (Pros) Prosencephalon; (mes) mesenceph-
alon; (met) metencephalon; (myel) myelencephalon; (V, VII, VIII, DC, X) cranial nerves; (At) atlas; (Ax) axis. Data are from Colberg-
Poley et al. (1988); Davis et al. (1988); Dony and Gruss (1987); Fainsod et al. (1987); Gaunt (1987, 1988); Fienberg et al. (1987); Graham
et al. (1988); Holland and Hogan (1988); Le Mouellic et al. (1988); Sharpe et al. (1988); Toth et al. (1987); Utset et al. (1987); G.
Dressier, M. Frohman, S. Gaunt, A. Graham, P. Gruss, G. Martin, K. Mahon, (pers. comm.).
776 GENES & DEVELOPMENT
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Homeo box genes during mouse development
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GENES
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DEVELOPMENT 777
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Holland and Hogan
dorsal folds of the neural tube give rise to neural crest
derivatives, which form an important component of the
nervous system (Le Douarin 1982, 1986; Noden 1984).
The major neural crest derivatives in the spinal cord re-
gion are the dorsal root ganglia, which are metameri-
cally organized, as shown in Figure 2. In the head region,
the neural crest gives rise to much of the mesoderm in
the face, including that in the maxillary and mandibular
processes, to mesodermal components of organs such as
the thyroid and submaxillary glands, and to the non-
neuronal (glial) cells of the cranial ganglia. (The neu-
ronal cells of cranial ganglia are derived from neural
crests,
from ectodermal placodes, or from both.) These
ganglia and their associated cranial nerves are important
landmarks along the anterior-posterior axis of the head,
and their characteristic morphology and innervation
patterns make them easily distinguishable, unlike the
dorsal root ganglia.
A summary of mouse homeo box gene expression in
the 12.5 day embryo is shown in Figure 3. Unfortu-
nately, for many genes the information is incomplete,
since reports of in situ hybridization studies have often
concentrated on spinal cord and somite derivatives and
do not mention the peripheral nervous system (PNS) or
visceral organs. However, a number of general conclu-
sions can be reached.
In the CNS different homeo box genes show different
anterior-posterior domains of expression that do not
correspond to any obvious morphological boundaries.
This is particularly striking for En-2, which is localized
largely to the metencephalon (Davis et al. 1988), while
En-1 expression extends from the forebrain to the end of
the spinal cord (M. Frohman and G. Martin, pers.
comm.). Hox-1.5 has the next most anterior boundary
just anterior to the otic vesicle (Fainsod et al. 1987;
Gaunt 1987), while Hox-1.2, -1.3, -lA, -2.1, -2.6, and -6.1
all have anterior limits in the hind brain, posterior to the
otic vesicle (Dony and Gruss 1987; Krumlauf et al. 1987;
Toth et al. 1987; Graham et al. 1988; Holland and Hogan
1988;
Sharpe et al. 1988). Careful comparative studies
have yet to be done using probes for these genes on adja-
cent serial sections and correlating hybridization in the
CNS to morphological landmarks such as the cranial
ganglia and the transient neuromeric swellings in the
brain (Sakai 1987). Such studies should reveal, for ex-
ample, whether there is overlap between the domains of
expression of
£22-2
and Hox-1.5, or whether they directly
interface with each other. Hox-2.5 is expressed more
posteriorly, with an anterior limit around the level of
the first dorsal root ganglion (Fienberg et al. 1987). By
comparison, the anterior limit of Hox-1.1 in the spinal
cord is around the level of the third dorsal root ganglion
(K. Mahon, pers. comm.). Finally, Hox-3.1 is expressed
most posteriorly, beginning at about the level of the fifth
dorsal root ganglion (Utset et al. 1987; Holland and
Hogan 1988; Le Mouellic et al. 1988; G. Dressier and P.
Gruss,
pers. comm.). Although the anterior boundary of
many of the genes is distinct, the posterior boundary is
less so, and its position may alter during development
(see later). Although less well studied, the expression of
many mouse homeo box genes is also restricted in a dor-
soventral or mediolateral manner within the spinal cord
(Fienberg et al. 1987; Toth et al. 1987; Utset et al. 1987;
Holland and Hogan 1988; Le MouelHc et al. 1988).
Some, but not all, homeo box genes are also expressed
in the peripheral nervous system. This has been studied
in detail for Hox-2.1, where it appears that cells in the
dorsal root ganglia (derived from trunk neural crest), the
nodose ganglion (which receives cells from the neural
crest and from ectodermal placodes), and the myenteric
plexus of the gut (colonized by migration of neural crest
from the posterior hind brain) are positive by in situ hy-
bridization (Graham et al. 1988; Holland and Hogan
1988).
Other homeo box genes expressed in dorsal root
ganglia are Hox-1.1 (G. Dressier, K. Mahon, and P.
Gruss,
pers. comm.), Hox-1.2 (G. Dressier and P. Gruss,
pers.
comm.), Hox-1.4 (Toth et al. 1987), Hox-1.5
(Fainsod et al. 1987), Hox-2.6 (Graham et al. 1988), and
En-1 (which is also expressed in several cranial ganglia
including
V,
VII, VIII, and
X;
see Fig. 2) (M. Frohman and
G. Martin, pers. comm.). En-2 and Hox-3.1 do not appear
to be expressed in dorsal root ganglia (Davis et al. 1988;
Le Mouellic et al. 1988). Interestingly, no expression of
any homeo box gene has been reported for mesenchymal
components of the head (connective tissue, muscle, car-
tilage, or membrane bone), which derive from neural
crest and anterior mesoderm (Noden 1984), nor for me-
soderm of the heart, which is also derived from some of
the first mesoderm to delaminate from the ectoderm.
Figure 3 also summarizes expression of homeo box
genes in mesoderm derived from the somites (preverte-
brae),
intermediate mesoderm (mesonephros and metan-
ephros), or the lateral plate (lung and stomach). Several
general conclusions can be drawn. First, expression in
somites does not always mean expression in lateral plate
or intermediate mesoderm and vice versa. Second, al-
though anterior-posterior domains of expression in the
mesoderm sometimes correlate roughly with domains of
expression in the CNS (e.g., Hox-2.1], this is not always
the case. For example, the clear discrepancy between the
anterior limits of Hox-3.1 expression in the nervous
system and in the vertebrae has been noted indepen-
dently by a number of laboratories (Utset et al. 1987;
Holland and Hogan 1988; Le MoueUic et al. 1988; G.
Dressier and P. Gruss, pers. comm.). Third, although not
thoroughly examined, there is a possible correlation be-
tween the order of the Hox-1 genes on the chromosome
and the anterior limits of their expression in the somitic
mesoderm (see Fig. 3). Finally, as far as we are aware, no
spatially localized expression of homeo box genes has
been reported yet in the early limb-bud mesenchyme,
which is derived from lateral plate. Stage-specific ex-
pression of three human homeo box genes, HHO.cl,
HHO.cS,
and HHO.cl3 has been demonstrated by
Northern analysis of poly(A)"*" RNA from limbs of 6- to
8-week-old fetuses. The earliest stage examined corre-
sponds to approximately 13 days p.c. in the mouse when
both somite- and lateral plate-derived mesenchyme are
present (Mavilio et al. 1986; Simeone et al. 1987).
In conclusion, at 12.5 days p.c. there are clear spatial
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Homeo box genes during mouse development
domains of expression of homeo box genes in the CNS,
PNS,
and mesoderm. Different genes may be expressed
in different sets of embryonic structures, and over
dif-
ferent regional extents. This is most obvious along the
anteroposterior axis, but can also be seen along the me-
diolateral and dorsoventral axes. Furthermore, for some
genes,
the domains along these axes may be noncoinci-
dent between the CNS, PNS, and mesoderm.
Later stages of development
In a few cases, comparative in situ hybridization studies
have been made between 12.5- and 15.5-day embryos. For
Hox-2.1 the conclusion is that the overall level of homeo
box gene expression declines, and that, in the CNS at
least, the posterior limit of expression becomes more re-
stricted, while the anterior boundary remains the same
(Holland and Hogan 1988). It is unclear whether this
gradual change in the posterior boundary of Hox-2.1 re-
flects an underlying anterior-posterior gradient in ex-
pression, or whether at earlier stages expression is uni-
form throughout the spinal cord. Evidence in favor of a
gradient has been presented for Hox-1.5 (Gaunt 1987). A
similar sharpening of the domain of expression with
time has been reported for Hox-1.3 (Dony and Gruss
1987) and Hox-3.1 (Holland and Hogan 1988; Le
Mouellic 1988). If this is a general phenomenon for
mouse Antennapedia-likQ genes it may account for
some of the discrepancies between different laboratories
in assigning the posterior limits of gene expression at a
given time, since embryos vary in their rate of develop-
ment. Finally, it is interesting to note that the region-
specific patterns of Hox-2.1 and -3.1 are still evident in
the central nervous system of newborn mice (Awgule-
witch et al. 1986; Utset et al. 1987).
Conclusions
In this review we have considered only mouse homeo
box genes related to Dwsophila Antennapedia-likt and
engrailed-likQ genes. Information on expression is not
yet available for genes containing sequences more
closely related to the divergent Dwsophila homeo
boxes,
such as in
paired,
bicoid,
or even-skipped.
The general pattern emerging is that the mouse
homeo box genes examined so far are expressed in spa-
tial domains along the anteroposterior axis of the em-
bryo.
For at least some genes, these spatially restricted
regions become evident at the late gastrula or early so-
mite stage (Figs. 2 and 3). By mid to late gestation, each
gene is expressed in a characteristic pattern within a few
sets of embryonic structures, including the CNS, PNS,
somite derivatives, and visceral organs (Fig. 3). These
patterns of expression are consistent with, but of course
do not prove, a role for homeo box genes in establishing
domains of positional value in the mammalian embryo.
It is important to note that these patterns may exist in
nonsegmented regions of the mesoderm, and that each
domains appears initially to be continuous, with no evi-
dence of a discontinuous segmental periodicity. As more
genes are studied in detail, however, it is possible that
metameric patterning may emerge in the overlap of the
anterior limits of the different domains.
Although the data are still fragmentary, there may be
separate phases of homeo box gene expression and func-
tion in mammalian development. During the first, soon
after the start of gastrulation, a set of genes (including
Hox-1.5 and -3.1] are expressed in both mesoderm and
ectoderm. Their role may be to establish broad antero-
posterior divisions of the embryo. At present, we can
only speculate as to what signals activate these homeo
box genes, and control the initial limits of their expres-
sion. It is likely that either diffusible factors, or timing
mechanisms, acting around gastrulation are involved in
early regionalization (Hogan et al. 1985); such mecha-
nisms therefore may be the primary cue for homeo box
gene activation. It is possible that elucidation of the
time when several homeo box genes are first expressed
may help to understand the nature of primary regiona-
lizing cues in the mammalian embryo (Gaunt 1988).
By mid to late gestation, the patterns of expression are
more complex, often showing noncoincident anteropos-
terior domains, and different combinations of genes, in
different embryonic structures such as the CNS, PNS,
and somitic mesoderm (Fig. 3). In addition, although less
easy to analyze because of the extensive cell movements
that occur during development, it seems likely that axial
limits to expression are also present in the lateral meso-
derm. It is possible that these more refined patterns are
generated by combinatorial and interactive mechanisms
similar to those suggested for the regulation of Droso-
phila homeotic genes (Gehring 1987). For example, in
the CNS, the anterior limit of each homeo box genes
domain is generally sharp in early embryos and remains
relatively stable (allowing for cell migrations that ac-
company morphogenesis), while the posterior limit may
become more anterior with time. A remarkably similar
progressive anterior localization is shown by several
Drosophila homeotic genes expressed in the CNS (Akam
and Martinez-Arias 1985; Harding et al. 1985). An im-
portant area of investigation now is to elucidate the in-
fluences that establish and refine these patterns of ex-
pression, and to study the effects of experimental pertur-
bations on cell determination during embryogenesis.
Obviously, one approach is to misregulate genes in
transgenic mice. Another strategy is to combine analysis
of specific homeo box gene expression with tissue
grafting and in vitro culture. The latter approach is cer-
tainly more applicable to vertebrate embryos than to
Drosophila, and may give important insight into the
roles of germ layer interactions in development. A step
toward this goal in Xenopus is the demonstration that
influences from the mesoderm permit the expression of
a predetermined spatial pattern of expression of a homeo
box gene in the neurectoderm (Sharpe et al. 1987).
Not all details of the expression of mouse homeo box
genes in development are clearly consistent with a role
in regional diversification. In particular, expression at
stages later than mid gestation may be partly cell or
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Holland and Hogan
tissue specific, rather than region specific. For example,
Hox-2.1 RNA has a nonuniform distribution within the
mesoderm of embryonic visceral organs, and a possible
role in embryonic induction during organogenesis has
been suggested (Holland and Hogan 1988). Hox-1.4 ex-
pression is developmentally regulated in different cell
types during spermatogenesis, as revealed by in situ hy-
bridization (Rubin et al. 1986) and by Northern analysis
of RNA isolated from separated cells (Wolgemuth et al.
1987).
Expression of Hox-2.1 RNA has been seen in ma-
ture granulocytes in adult mice (Holland and Hogan
1988).
Similarly, homeo box gene expression may be
cell-type specific in the embryonic and adult CNS
(Odenwald et al. 1987; Toth et al. 1987; Davis et al.
1988),
and a role in cell determination and differentia-
tion during neurogenesis is a possibility, as suggested for
the Drosophila segmentation gene fushi tarazu (Doe et
al.
1988). To tackle these problems it is clearly essential
to study mouse homeo box gene expression at the single
cell level, which must await production of a wider range
of specific antibody probes.
Finally, the mechanisms by which homeo box genes
exert their putative roles also needs further investiga-
tion. Although it is now widely believed that the protein
products of homeo box genes act as DNA-binding pro-
teins with roles in regulating gene expression (Gehring
1987),
the ultimate target or effector genes have not been
identified in any organism. Since the products of these
effector genes must ultimately dictate region-specific
cellular properties, they may provide the critical link be-
tween regionalization and morphological form.
Acknowledgments
We thank many friends and colleagues for discussion and for
providing data prior to publication, in particular Drs. Gregory
Dressier, Denis Duboule, Michael Frohman, Steven Gaunt, An-
thony Graham, Peter Gruss, Alexandra Joyner, Gail Martin,
Katherine Mahon, Chi Nguyen-Huu, Paul Sharpe, Dennis Sum-
merbell, Leslie Toth, and Debra Wolgemuth. We also thank foe
Brock for drawing the diagrams, and Lydia Pearson and Vera
Murphy for preparing the manuscript. The authors' work cited
here was supported by the Medical Research Council of Great
Britain at the National Institute for Medical Research, Mill
Hill, London NW71AA.
Note added in proof
The axial limits of expression of mouse Hox-1.2, -1.3,
-1.4, -1.5, -3.1, and -6.1 recently have been accurately
compared by in situ hybridization at 12.5 days p.c. by S.J.
Gaunt, P.T. Sharpe, and D. Duboule, Development
(Suppl.) (in press), 1988.
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