Homeotic genes and the arthropod head: expression patterns of the labial, proboscipedia, and Deformed genes in crustaceans and insects.
ABSTRACT cDNA fragments of the homologues of the Drosophila head homeotic genes labial (lab), proboscipedia (pb), and Deformed (Dfd) have been isolated from the crustacean Porcellio scaber. Because the accumulation domains of the head homeotic complex (Hox) genes had not been previously reported for crustaceans, we studied the expression patterns of these genes in P. scaber embryos by using in situ hybridization. The P. scaber lab homologue is expressed in the developing second antennal segment and its appendages. This expression domain in crustaceans and in the homologous intercalary segment of insects suggests that the lab gene specified this metamere in the last common ancestor of these two groups. The expression domain of the P. scaber pb gene is in the posterior part of the second antennal segment. This domain, in contrast to that in insects, is colinear with the domains of other head genes in P. scaber, and it differs from the insect pb gene expression domain in the posterior mouthparts, suggesting that the insect and crustacean patterns evolved independently from a broader ancestral domain similar to that found in modern chelicerates. P. scaber Dfd is expressed in the mandibular segment and paragnaths (a pair of ventral mouthpart structures associated with the stomodeum) and differs from insects, where expression is in the mandibular and maxillary segments. Thus, like pb, Dfd shows a divergent Hox gene deployment. We conclude that homologous structures of the mandibulate head display striking differences in their underlying developmental programs related to Hox gene expression.
- SourceAvailable from: Maximilian J Telford[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: The biting mandible of the arthropods is thought to have evolved in the ancestor of the insects, crustaceans and myriapods: the Mandibulata. A unique origin suggests a common set of developmental genes will be required to pattern the mandible in different arthropods. To date we have functional studies on patterning of the mandibular segment of Drosophila melanogaster showing in particular the effects of the gene cap'n'collar (cnc), however, the dipteran head is far from representative of insects or of more distantly related mandibulates; Drosophila does not even possess a mandibular appendage. To study the development of a more representative insect mandible, we chose the red flour beetle Tribolium castaneum and investigated the function of the Tribolium orthologs of cap'n'collar (Tc-cnc) and the Hox gene Deformed (Tc-Dfd). In order to determine the function of Tc-cnc and Tc-Dfd, transcripts were knocked down by maternal RNA interference (RNAi). The effects of gene knockdown were examined in the developing embryos and larvae. The effect of Tc-cnc and Tc-Dfd knockdown on the expression of other genes was determined by using in situ hybridization on Tribolium embryos. RESULTS: Our analyses show that Tc-cnc is required for specification of the identity of the mandibular segment of Tribolium and differentiates the mandible from maxillary identity. Loss of Tc-cnc function results in a transformation of the mandible to maxillary identity as well as deletion of the labrum. Tc-Dfd and the Tribolium homolog of proboscipedia (Tc-mxp = maxillopedia), Hox genes that are required to pattern the maxillary appendage, are expressed in a maxilla-like manner in the transformed mandible. Tribolium homologs of paired (Tc-prd) and Distal-less (Tc-Dll) that are expressed in the endites and telopodites of embryonic appendages are also expressed in a maxilla-like manner in the transformed mandible.We also show that Tc-Dfd is required to activate the collar of Tc-cnc expression in the mandibular segment but not the cap expression in the labrum. Tc-Dfd is also required for the activation of Tc-prd in the endites of the mandible and maxillary appendages. CONCLUSIONS: Tc-cnc is necessary for patterning the mandibular segment of Tribolium. Together, Tc-cnc and Tc-Dfd cooperate to specify mandibular identity, as in Drosophila. Expression patterns of the homologs of cnc and Dfd are conserved in mandibulate arthropods suggesting that the mandible specifying function of cnc is likely to be conserved across the mandibulate arthropods.EvoDevo 11/2012; 3(1):25. · 3.91 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Developmental competence is the response of a cell(s) to information. Determination of adult labial identity in Drosophila requires Proboscipedia (PB) and Sex combs reduced (SCR); however, co-ectopic expression of PB and SCR is not sufficient for induction of ectopic adult labial identity, because the developmental information supplied by PB and SCR is suppressed. The evolutionarily conserved LASCY, DYTQL, NANGE motifs, and the C-terminal domain of SCR are sequence elements that mediate some, or all, of the suppression of ectopic proboscis determination. Therefore, the developmentally competent primordial proboscis cells provide an environment devoid of suppression, allowing PB and SCR to determine proboscis identity. SCR derivatives lacking suppression sequences weakly induce ectopic proboscis transformations independently of PB, suggesting that SCR may be the activity required for induction of adult labial identity, as is the case for larval labial identity. A possible explanation for PB independence of SCR in determination of adult and embryonic labial identity is PB operates as a competence factor that switches SCR from determining T1 identity to labial identity during metamorphosis. Lastly, labial determination is not conserved between SCR and murine HOXA5, suggesting that SCR has acquired this activity during evolution.Development Genes and Evolution 10/2013; · 1.70 Impact Factor
Article: Hox genes regulation in vertebrates.[Show abstract] [Hide abstract]
ABSTRACT: Hox genes encode transcription factors defining cellular identities along the major and secondary body axes. Their coordinated expression in both space and time is critical for embryonic patterning. Accordingly, Hox genes transcription is tightly controlled at multiple levels, and involves an intricate combination of local and long-range cis-regulatory elements. Recent studies revealed that in addition to transcription factors, dynamic patterns of histone marks and higher-order chromatin structure are important determinants of Hox gene regulation. Furthermore, the emerging picture suggests an involvement of various species of non-coding RNA in targeting activating and repressive complexes to Hox clusters. I review these recent developments and discuss their relevance to the control of Hox gene expression in vivo, as well as to our understanding of transcriptional regulatory mechanisms. Developmental Dynamics, 2013. © 2013 Wiley Periodicals, Inc.Developmental Dynamics 07/2013; · 2.59 Impact Factor
Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 10224–10229, August 1999
Homeotic genes and the arthropod head: Expression patterns of
the labial, proboscipedia, and Deformed genes in crustaceans
ARHAT ABZHANOV AND THOMAS C. KAUFMAN*
Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, IN 47405
Communicated by Edward B. Lewis, California Institute of Technology, Pasadena, CA, June 25, 1999 (received for review March 22, 1999)
Drosophila head homeotic genes labial (lab), proboscipedia (pb),
and Deformed (Dfd) have been isolated from the crustacean
Porcellio scaber. Because the accumulation domains of the
head homeotic complex (Hox) genes had not been previously
reported for crustaceans, we studied the expression patterns
of these genes in P. scaber embryos by using in situ hybrid-
ization. The P. scaber lab homologue is expressed in the
developing second antennal segment and its appendages. This
expression domain in crustaceans and in the homologous
intercalary segment of insects suggests that the lab gene
specified this metamere in the last common ancestor of these
two groups. The expression domain of the P. scaber pb gene is
in the posterior part of the second antennal segment. This
domain, in contrast to that in insects, is colinear with the
domains of other head genes in P. scaber, and it differs from
the insect pb gene expression domain in the posterior mouth-
parts, suggesting that the insect and crustacean patterns
evolved independently from a broader ancestral domain sim-
ilar to that found in modern chelicerates. P. scaber Dfd is
expressed in the mandibular segment and paragnaths (a pair
of ventral mouthpart structures associated with the stomo-
deum) and differs from insects, where expression is in the
mandibular and maxillary segments. Thus, like pb, Dfd shows
a divergent Hox gene deployment. We conclude that homol-
ogous structures of the mandibulate head display striking
differences in their underlying developmental programs re-
lated to Hox gene expression.
cDNA fragments of the homologues of the
Homologues of the Drosophila homeotic complex (HOM-C)
genes from various arthropods have been the subject of intense
research and comparative analysis (1–10). A goal of these
studies has been to understand the role of these genes in the
morphological evolution of this group. The function of Hox
gene products as selective transcription factors is apparently
highly conserved in animals (1, 2), and the expression patterns
of these genes have been used as stable molecular markers for
judging evolutionary relationships, such as homologies, of
structures along the anteroposterior axis (5, 7, 8). Neverthe-
less, for the Hox genes to serve as useful tools for phylogenetic
comparisons, a better understanding of their particular evo-
lutionary history is needed. Moreover, observations on their
expression patterns should ideally be coupled with tests for
developmental function (6, 10, 11). Thus far, such comparative
analyses have been done primarily on Hox genes in insects, on
‘‘trunk’’ Hox genes in the crustacean Artemia franciscana, and,
most recently, in the chelicerates (3–7). Some models for the
roles of Hox genes in the evolution of arthropods have already
been proposed (2, 4, 5). However, the vast phylogenetic
distances between and among the groups studied and the small
assortment of genes analyzed make it difficult to create a
complete picture of the evolution of Hox genes in arthropods.
In Drosophila, the Hox genes specify structures along the
anteroposterior body axis; three are necessary for the correct
development of head segments and appendages: the labial
(lab), proboscipedia (pb), and Deformed (Dfd) genes. The
expression patterns of these genes in insects show small,
distinct, and usually nonoverlapping expression domains cov-
ering one to two segments. This pattern is in sharp contrast to
the expression patterns of their homologues in vertebrate (lab,
pb, and Dfd are homologous to the genes of vertebrate classes
1, 2, and 4 respectively) and chelicerate embryos, where
expression domains are broadly overlapping and many seg-
ments long (7, 8, 12). The fact that the trunk genes Ultrabitho-
rax, abdominal-A, and Abdominal-B are also expressed in
extended overlapping domains in insects, chelicerates, and
vertebrates suggests that the insect head genes have undergone
evolution to limit their expression domains. Unfortunately, in
the Mandibulata, which includes the Insecta, Crustacea, and
Myriapoda, no data exist for Hox head gene expression
patterns for any group other than Insecta.
We have chosen the crustacean Porcellio scaber, order
Isopoda, as a noninsect model organism to study the expres-
our model organism belongs to the subclass Malacostraca
(higher crustaceans) and is as derived as insects are in its body
plan and tagmatization, relative to phylogenetically more basal
groups. Moreover, the interpretations of the expression pat-
terns reported here are based on the assumptions that: (i) the
Insecta is monophyletic, with the order Thysanura a basal
group; (ii) the Mandibulata are monophyletic with the Crus-
tacea, a sister group of the Insecta; and (iii) the Chelicerata is
an outgroup to the Insectan–Crustacean clade (based on refs.
13–17; reviewed in refs. 18 and 19).
The six-segmented mandibulate head is believed to be a
primitive character shared by all three extant mandibulate
groups (20–22). This feature allows reliable identification of
homologous segments and appendages, and it allows predic-
tions regarding expression patterns and possible developmen-
tal functions of the Hox genes. Based on the available insect
and chelicerate expression pattern data, we proposed two
conflicting predictions: (i) because malacostracan crustaceans
and insects are related mandibulate groups with morpholog-
ically similar head structures, their Hox expression patterns
should be similar, if not identical; or (ii) the crustacean
patterns could be intermediate between those seen in cheli-
cerates and in insects (6–8). Whereas much is known about the
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: a1, first antennal (segment); a2, second antennal
(segment); EN, engrailed; Hox, homeotic complex (genes); mx1, first
maxillary (segment); Ps, P. scaber.
Data deposition: The sequences reported in this paper have been
deposited in the GenBank database [accession nos. AF148935 (P.
scaber lab), AF148936 (P. scaber pb), and AF148937 (P. scaber Dfd)].
*To whom reprint requests should be addressed. E-mail: kaufman@
expression patterns for the trunk genes of the crustacean A.
franciscana (4), no expression patterns of the head genes are
known for any crustacean group, although partial sequences
have been published (5, 23). Here, we describe the expression
patterns of lab, pb, and Dfd in the crustacean P. scaber. Our
observations show that neither of the two predictions pre-
sented above is borne out. Rather, a combination of these
results and our previously published data on the expression
pattern of the head Hox gene Sex combs reduced (Scr) (24)
demonstrates that the expression patterns of these genes in this
crustacean are divergent from those seen in insects. Moreover,
the observed crustacean pattern does not appear to represent
a chelicerate–insect intermediate. We conclude that the di-
vergent crustacean?insectan expression patterns reveal that
unexpectedly dissimilar developmental processes likely under-
lie the specification of homologous and morphologically sim-
ilar mandibulate mouthparts.
MATERIALS AND METHODS
Cloning of the cDNA Fragments and Sequence Analysis.
Total mRNA was isolated from two broods (about 50 em-
bryos) of P. scaber embryos by using the TRIzol reagent (Life
Technologies) and following the manufacturer’s instructions.
The cDNA was screened by PCR with degenerate primers
designed against conserved amino acid regions of the head
Hox protein (see Fig. 2). Multiple independent clones pro-
duced only repeat copies of the same sequence for the lab, pb,
and Dfd genes. All primer pairs and PCR protocols have been
previously described (6, 10, 11). A related PCR screen for
homeoboxes that used degenerate ELEKEF- and WFQNRR-
encoding primers produced single unique copies of the lab, pb,
and Dfd homeoboxes matching those cloned with the more
specific primers. A total of 39 and 27 homeobox fragments
have been cloned from the cDNA pool and from genomic
DNA, respectively. For example, the Hom-C fragments cloned
from cDNA include 12 Ubx, 9 pb, 3 Scr, 3 Abd-A, and 5 lab
homeobox segments with nucleotide sequences identical to the
previously cloned orthologous copies of these genes. A short
cDNA fragment of the Hox3 class homologue was also cloned
and will be described elsewhere. The sequences were com-
pared with our homeobox sequences from miscellaneous ar-
thropods (unpublished data), cloned in the laboratory, and
with those available in the National Center for Biotechnology
Whole-Mount in Situ Hybridization, Microscopy, and Pho-
tography. In situ hybridization was performed as previously
described by Panganiban et al. (26), but with modifications
(10), and with the exception that protease K treatment was
10–15 min instead of 1 hr. The size of the in situ probes ranged
from 230 bp (pb) to 260 bp (lab). Probes of similar size have
been used successfully to reveal specific expression domains of
a number of arthropod genes (4, 6, 8, 10, 11, 24). All proce-
dures for mounting and photographing embryos have been
described (10, 27).
Development of the P. scaber Head as Revealed by Antibody
to Engrailed (EN) Protein. To assign the P. scaber head
appendages to specific segments (Fig. 1A), we used the mono-
clonal antibody Mab4D9, which recognizes EN, to indicate the
posterior of the segmental borders (28, 29). The antibody
revealed six segments in the embryonic head of P. scaber:
ocular, first antennal, second antennal, mandibular, first max-
illary, and second maxillary (Fig. 1B). The most anterior
region of the head, the labrum, develops as a pair of small
appendage-like structures that fuse medially at about the
65–70% stage of development. In early embryos, the EN stripe
of the ocular segment is not complete and is interrupted on the
ventral side by the labrum. The labrum itself does not express
EN and, in this respect, appears to be continuous with the
stomodeum. The first antennal segment bears a pair of small
uniramose antennae, which are reduced in the adult. The
second antennae are the largest pair of the appendages on the
head. The stomodeal opening protrudes at the level of the
posterior first antennal (a1) segment and extends to the
posterior of the second antennal (a2) segment, which results in
a ventral interruption of the EN stripes of the a1 and a2
segments. The broad mandibular EN band is seen in the
developing posterior mandibular appendages. EN is expressed
similarly in the first and second maxillary segments.
The Orthologues of the lab, pb, and Dfd Genes. Fig. 2 shows
an alignment of the predicted partial amino acid sequences for
the recovered P. scaber genes lab, pb, and Dfd and their
homologues from several insect orders and from A. fran-
ciscana. Because of high levels of Porcellio Hox sequence
specific HOM-C gene class. Analysis of the alignment in Fig.
2 reveals that the lab homologues from Drosophila melano-
are highly similar immediately downstream of the YKWM
motif and in most of the homeobox; however, the N-terminal
arm of the homeodomain and the ‘‘variable’’ region just
upstream are not conserved among the orthologues. All pb
homologues share essentially identical homeobox and N-
terminal arm regions (see Fig. 2). Porcellio has the largest
variable region of all the sequences shown. Porcellio Dfd is very
similar to its homologues in insects and A. franciscana. Its
variable region appears to be more similar to that of insects
than to that of Artemia.
Hox Gene Expression Patterns. The expression patterns of
lab, pb, and Dfd were obtained by using whole mount in situ
hybridization to reveal transcript distribution. Expression can
be detected from the 30% to the 80% stage but is best seen at
the 50–60% stage. All embryos shown in Fig. 3 are at the
50–60% developmental stage. At this stage the embryo begins
dorsal closure and its appendages develop unique morpholo-
gies. Additionally, the first pair of the thoracic appendages
begins to transform into the mouthpart maxillipeds (24).
Expression of P. scaber lab is clear in the developing a2
segment and its appendages (Fig. 3A). The embryo in Fig. 3A
shows no expression in the first antennae, mandibles, or any
ventral view, with anterior at the top. (A) A scanning electron
micrograph of a developing (45–50%) embryo (?50.) The segments
indicated are as follows: a1, first antennae; a2, second antennae; lb,
labrum; mn, mandibles; pg, paragnaths; mx1, first maxillae; mx2,
second maxillae; T1?mxp, first thoracic appendages?maxillipeds (B)
The head of the embryo stained with Mab4D9 anti-EN antibody. The
segments indicated are as follows: oc?lb, ocular?labral; a1, first
antennal; a2, second antennal; mn, mandibular; mx1, first maxillary;
mx2, second maxillary; T1, first thoracic segments.
Head appendages and segments of the crustacean P. scaber,
Evolution: Abzhanov and KaufmanProc. Natl. Acad. Sci. USA 96 (1999) 10225
expression is continuous within the a2 segment and circles the
stomodeum (Fig. 3A). Very strong expression just anterior to
the stomodeum and in the posterior part of the labrum either
suggests that the anterior boundary is ventrally parasegmental
or that parts of the aforementioned organs originate from the
a2 segment. Fig. 4A shows the P. scaber lab expression domain
as a colored overlay on a scanning electron micrograph.
The expression domain of P. scaber pb appears as a narrow
stripe in the posterior a2 segment; it is reminiscent of the
ventral portion of the EN stripe in that segment (Fig. 1 and Fig.
3C). Like the EN stripe, the P. scaber pb stripe is interrupted
medially by the stomodeum. Careful examination of the em-
bryonic head reveals additional strong expression in a small
group of cells in the posterior-lateral portion of the a2
appendages (Fig. 3D). These antennal cells form a small
bud-like structure, possibly representing a rudimentary anten-
nal exopod (Fig. 3D). The P. scaber pb stripe does not extend
into the remainder of the a2 appendage and is restricted to the
ventrolateral portion of the a2 segment. Based on the segment
morphology and direct comparison to P. scaber lab stained
embryos, we conclude that P. scaber pb expression in the a2
segment overlaps that of P. scaber lab. The exact extent of the
overlap between the domains of the two genes is difficult to
ascertain because of the lack of good morphological markers
in the posterior a2 segment. Embryos that were stained for
several more hours showed additional weak expression in the
paragnaths, ventral mouthpart structures associated with the
stomodeum, but this expression appears to be mesodermal
(Fig. 3C). No other expression domains can be seen, even in
overstained embryos. A summary of the pb expression do-
mains is shown in Fig. 4B.
In embryos of all stages that were examined, the P. scaber
Dfd homologue is expressed in the developing paragnaths (Fig.
1; and Fig. 3 E and F; see Discussion). Weak expression in the
mandibles is mesodermal. There is also P. scaber Dfd expres-
sion in the ventral portion of the mandibular segment, which
may be parasegmental; it extends from the mid-mandibular
segment into the anterior half of the first maxillary (mx1) (Fig.
3E). A much weaker expression domain in the mesoderm can
be recognized in the mandibular appendages (Fig. 3E). There
is not the detectable accumulation in the mx1 appendages or
posterior half of the mx1 segment that one might have pre-
dicted based on the expression of Dfd in the homologous
maxillary segment of insects (6). These data are summarized
in Fig. 4C.
The pattern of P. scaber Scr expression has been described
(24). We note here only that colinearity of expression is
is in the posterior of the mx1 segment, whereas the posterior
border of Dfd resides in the anterior of this same segment. It
is possible that there is some overlap of expression in the
middle of mx1 but a demonstration of this point awaits further
Two comparative analyses of insect and chelicerate Hox
expression patterns have been published recently (7, 8). These
analyses are intriguing, but they point to the necessity for
studies on crustacean and myriapod Hox head genes to gain a
better understanding of their evolution in arthropods (7, 8). To
partially fill this need, we have determined the embryonic
expression patterns for the genes lab, pb, and Dfd in P. scaber,
a malacostracan crustacean, and compared our results with
data from insects and chelicerates. In general, we found the P.
scaber expression patterns to be well-defined and discrete, but
not identical to those of insects or chelicerates (Fig. 5).
Comparison of Insect and Crustacean Hox Expression
Domains. lab expression in the Drosophila head is in the
intercalary segment, a small metamere that is devoid of
appendages, located posterior to the antennal segment and
anterior to the mandibular segment. The exact role of lab in
this segment is unclear, because mutants do not show an
obvious homeotic transformation. Nevertheless, the gene is
important for the formation of the embryonic and adult head
in Drosophila, because mutants do show defects in the devel-
opment of cephalic structures (for review, see ref. 30). lab
expression in the intercalary segment is conserved in all of the
insect species examined thus far (for review, see ref. 6).
Comparison of the P. scaber lab expression domain to that
of insects reveals both conservation and change. P. scaber
expression is restricted to the first postantennular metamere,
i.e., the a2 segment, which, based on morphological and
molecular (EN expression) data, is thought to be homologous
to the intercalary (22, 29–32). Moreover, innervation of this
first postantennular segment is associated with the tritocere-
lab is expressed in a homologous segment in insects and
crustaceans suggests that it was recruited for and might be
involved in conserved and homologous developmental pro-
cesses. However, in Porcellio, lab is expressed in the a2
appendages, whereas the adult insect intercalary segment is
limbless. Interestingly, the embryos of some insects develop
small transitory appendages on this segment, which might be
atavisms to a more primitive state (34). It is not clear, however,
four insect species and the crustaceans A. franciscana and P. scaber (some sequences were not available). All sequences are compared with their
Drosophila counterparts. Dashes indicate sequence identity, breaches in the sequences indicate introduced gaps. Species are indicated in the first
column: Dm, Drosophila melanogaster; Of, Oncopeltus fasciatus; Ad, Acheta domestica; Td, Thermobia domestica; Ps, P. scaber; Af, A. franciscana.
Gene names are indicated in the second column: LAB, labial; PB, proboscipedia; DFD, Deformed.
Alignments of the deduced head Hox protein sequences. Hexapeptide ? variable region and homeodomain sequences are aligned for
10226 Evolution: Abzhanov and KaufmanProc. Natl. Acad. Sci. USA 96 (1999)
whether lab was directly involved in appendage loss in the
insects; mutations in this gene do not cause limb growth from
the intercalary (30). Nevertheless, it is possible that in crus-
taceans, lab contributes to the unique morphology of the
second as compared with the first pair of antennae. For
example, in P. scaber, the first antennae are greatly reduced in
size, whereas the second antennae are large and more leg-like.
The pb gene is located upstream of lab in the HOM-C and
is expressed in insects in the appendages of the maxillary and
labial segments, where it has been shown to specify the
posterior mouthparts in Drosophila and Tribolium castaneum
(3, 35). Oddly, the main expression domain of pb in Drosophila
is not colinear with that of lab and Dfd. In all of the insects
surveyed, the anterior boundary of Dfd, the next upstream
HOM-C gene relative to pb, is in the mandibular segment (10),
and, based on the position of pb in the complex, one might
expect its anterior border of expression to be at or anterior to
this point. To rationalize this fact, it has been suggested that
an ancestral insect pb gene lost its colinear expression pattern
and gained a new, appendage-specific role in the maxillae and
labium (35). Studies of the expression patterns of pb in several
insect orders revealed another, albeit weaker, expression do-
main in the ventral portion of the intercalary (6). The signif-
icance of this domain is not clear, and in some groups,
including Drosophila, it was found to be mesodermal. In the
apterygote insect T. domestica, there is epidermal pb expres-
sion in the intercalary, but it is weak, appears late, and is
transient (6, 35). These latter, more anterior patches of
expression may be remnants of the posited ancestral, more
extensive expression domain.
The pb expression domain of P. scaber was found to be quite
dissimilar from the insect pattern. In this crustacean, pb
accumulation is restricted to the posterior part of the a2
segment and includes neither the mx1 nor the mx2 segments or
appendages (Figs. 3A and 4B). The first and second maxillae
of crustaceans are homologous to the maxillae and labium of
insects, respectively (30). As pb is required for maxillary and
labial appendage development in insects, it clearly cannot be
performing a similar function in the P. scaber embryo. At this
point, it is difficult to discern a possible developmental func-
tion for pb in P. scaber embryos.
of the Drosophila genes lab, pb, and Dfd, as revealed by in situ
hybridization. Abbreviations as in Fig. 1. Ventral view of embryos with
anterior at the top in all panels. (A and B) 50–60% Stage embryos
showing the lab expression pattern; whole embryo (A) and close-up of
embryonic head (B). (C and D) 50–60% Stage embryos showing pb
expression in close-up views of the embryonic head revealing details
of the expression domain. Arrow points to the pb expression in the
posterior-lateral second antennae (D). (E and F) 50–60% Stage
embryos showing Dfd mRNA distribution with dissected embryonic
head (F) showing strong Dfd expression in the paragnaths. (A and E,
?50; B, C, D, and F, ?100.)
Embryonic expression patterns of the P. scaber homologues
domains of expression in the ectoderm are shown in red: (A) lab is
expressed in the second antennal segment and its appendages. (B) pb
is expressed in the posterior second antennal segment. (C) Expression
of Dfd is limited to the ventral mandibular segment and paragnaths.
(D) Scr mRNA distribution in the first maxillary appendages, second
maxillary segment, and appendages and distal half of T1 leg (24).
Summary of Hox expression domains in P. scaber. The main
Evolution: Abzhanov and KaufmanProc. Natl. Acad. Sci. USA 96 (1999)10227
In D. melanogaster, the expression domain of Dfd includes
the mandibular and maxillary segments and appendages. It has
been shown genetically to be required for the normal devel-
opment of both mandibles and maxillae in Drosophila (30). As
noted above, Dfd expression has been found to be very similar
in all insect groups studied, including the basal insect T.
domestica (10, 11), and the mandibles and maxillae of insects
are homologous to the mandibles and first maxillae (maxillu-
lae) of crustaceans (31).
Comparison of the Dfd expression domains in P. scaber and
insects reveals that the crustacean domain is smaller (Figs. 3E
and 4C). P. scaber Dfd is expressed strongly in the paragnaths
and the mandibular segment, but not the mandibular append-
ages. The paragnaths are associated with the stomodeum, but
their exact embryonic origin is obscure. Some authors have
concluded that these structures are sternal protrusions of the
mandibular segment associated with the mouth, reduced ap-
pendages associated with the mandibles, or even structures
homologous to the insect hypopharynx (31, 37, 38). Whatever
their allegiance, paragnaths are found in a diversity of crus-
taceans (37). Thus, P. scaber Dfd is not expressed in the
mandibles or maxillae (only mesodermal expression is de-
tected in mandibles), where Dfd function is required in insects,
suggesting that Dfd has a different developmental function in
is expressed in the ectoderm of the crustacean mandibles
Comparison with Chelicerate Hox Gene Expression Do-
mains. A comparison of the crustacean?insect and chelicerate
patterns of Hox gene expression is made difficult by the
uncertainty of segmental homologies between the two groups.
The more traditional view, based on anatomy and patterns of
innervation, concludes that the segment associated with the
deuterocerebral ganglion of the central nervous system is
greatly reduced or absent in modern chelicerates (31). If this
conclusion is correct, the more anterior ocular?protocerebral
segment would be homologous to the same segment in insects
and crustaceans, whereas the next posterior segment in the
spider would correspond to the intercalary?tritocerebral seg-
ment of insects and to the second antennal segment of
crustaceans. Thus, the homologue of the insect antennal and
the crustacean first antennal segment would be absent in
chelicerates (31). More recently, this question of head–
segment homology has been revisited by using the patterns of
Hox gene expression as a basis for determining the presence
or absence of the deuterocerebral segment in chelicerates (7,
8). These authors, using the spider Cupiennius salei, the mite
Archegozetes longisetosus, and several head Hox genes as
probes, have concluded that this segment is present (7, 8). For
the purposes of this discussion we will take this point of view.
However, we must stress that the use of only the expression
patterns of the Hox genes as a primary reference for anatom-
ical homology is inappropriate. Indeed, we show in this work
that boundaries of expression patterns can and do change
during evolution, and only some general tendencies, such as
colinearity, persist. Nonetheless, in chelicerates, lab is ex-
pressed anteriorly in the developing pedipalps (homologous to
the appendages of the intercalary segment). This expression
extends posteriorly to the fourth pair of walking limbs, which
would correspond to the first pair of the thoracic appendages
in insects and crustaceans (maxillipeds in P. scaber) (7, 8).
Therefore, the anterior boundary of lab expression appears to
be conserved among chelicerates, crustaceans, and insects. It
is notable, however, that the posterior limit of expression is not
conserved. The integumentary expression of lab is limited to
the intercalary or second antennal segment in insects and
crustaceans, respectively, whereas in chelicerates, expression
extends posteriorly a further four full segments (Fig. 5A).
pedipalps to the third pair of walking legs, where it is accu-
mulated in the appendages (7). This boundary is colinear with
both lab and Dfd, and is thus similar to the relative expression
domains of these genes in vertebrates, annelids, and Porcellio
(39–41); but it is dissimilar to that seen in insects (6). Thus, the
anterior boundary of pb expression in the a2 segment of P.
scaber appears to resemble that seen in chelicerates rather than
that in insects. However, it should also be noted that, as for lab,
the expression domain of chelicerate pb extends further pos-
teriorly than that in insects and crustaceans (Fig. 5A).
Dfd expression has been examined in both C. salei and A.
longisetosus. In C. salei, Dfd is expressed in all walking legs (7).
The mite shows a similar expression domain in the L1–L4 legs,
with additional accumulation covering all opistosomal (ab-
dominal) segments except the most terminal ones (8). The
anterior boundary of Dfd accumulation appears to be con-
served and located in the mandibular (insects) and homolo-
gous L1 (chelicerates) segments (Fig. 5A). If one assigns the
paragnaths to the mandibular segment, the anterior boundary
of Dfd expression then appears to be similar, albeit not
identical, in P. scaber vis-a-vis insects and chelicerates. How-
aries in the Crustacea (P. scaber), Insecta, and Chelicerata (refs. 6–8;
unpublished data). Detailed expression pattern of the Scr homologue
of P. scaber has been described elsewhere (24). Columns represent
homologous segments. Parasegments and mandibulate segments (as in
Crustacea) are labeled at the top of the diagram and chelicerate
segments are labeled at the bottom. Bars indicate Hox gene expression
patterns. Black bars indicate primary domains (strong expression) and
expression). (B) Model for evolution of the homeotic gene pb expres-
sion pattern. Schematic head segments and structures are shown for
insects, P. scaber, and a hypothetical mandibulate ancestor. Segmental
expression of pb homologues is shown in dark gray. The segments are
labeled according to the accepted conventions in the respective taxa.
Abbreviations as in Figs. 1–4, except that in the schematic Insecta
head, ic marks the intercalary segment and lab indicates the labial
(A) Diagram of the Hox gene expression pattern bound-
10228Evolution: Abzhanov and Kaufman Proc. Natl. Acad. Sci. USA 96 (1999)
ever, both the chelicerate and insect Dfd domains are clearly
broader than those seen in the crustacean.
Hox Genes and the Evolution of Mandibulate Head Struc-
tures. As noted above, the extended and broadly overlapping
expression domains in chelicerates are reminiscent of those in
vertebrates and are probably closer to an ancestral state. In
contrast, the expression domains in insects and crustaceans are
more resolved and segment-specific (Fig. 4). Based on mor-
phological and recent molecular evidence, the Crustacea
belongs to the monophyletic group Mandibulata, which is a
close sister group to the Insecta (13–19). Thus, crustaceans
represent an ideal case for study of the evolution of the
homologous head Hox gene expression patterns and possible
functions in the homologous structures of insects.
insect Hox genes demonstrates that there is conservation of
segment affinity (e.g., lab) and spatial colinearity (e.g., lab, pb,
Dfd, and Scr) of expression (Figs. 4 and 5A; and ref. 24). In
addition, the anterior boundaries of the lab and Dfd genes
appear to be conserved in insects, crustaceans, and cheli-
cerates. However, there is also divergence of the observed
expression domains (e.g., pb and Dfd) (Figs. 4 and 5A).
Consequently, substantial variation in the deployment of the
Hox genes, and presumably in the developmental processes
regulated by them, can be seen in homologous and morpho-
logically similar crustacean and insect head structures. Genes
involved in the development of mandibles and posterior
mouthparts in insects are expressed in novel, though still
colinear domains. For example, in insects, the maxillary and
labial mouthparts express pb, whereas in P. scaber, the homol-
ogous appendages both express and probably depend on Scr,
a different head homeotic gene (refs. 6, 11, 24; also see Fig. 4).
We hypothesize that the mandibulate head evolved prior to
the establishment of the defined head Hox gene expression
domains, which have been recruited to their current regions
and developmental functions independently in crustaceans
and insects (Fig. 5B). This model involves an intermediate
hypothetical mandibulate ancestor that did not have segment-
specific expression domains and probably resembled the pat-
tern of expression seen in modern chelicerates. The specifi-
cation of individual segments and mouthparts in such an
animal would depend on the redundant and?or fractional
functions of multiple Hox genes, and would be facilitated by
the subsequent evolution of more distinct expression domains
(Figs. 4 and 5). That is, the head Hox genes functioned in a
manner analogous to the genes of the D. melanogaster Bithorax
complex. To test this model and to better understand the
evolution of the Hox genes and head structures, further studies
across different crustacean and myriapod groups will be
We thank R. Turner for the scanning electron microscopy; M.
Peterson and A. Popadic for providing primers and protocols; Thierry
Rigaud for identifying the woodlouse species and culture suggestions;
N. Patel for the EN?INV antibody; Dee Verostko for administrative
assistance; and G. Scholtz, Kevin Cook, S. Glueck, and T. Powers for
valuable comments on the manuscript. This work was supported by the
Howard Hughes Medical Institute. T.C.K. is an Investigator of the
Howard Hughes Medical Institute.
Carroll, S. B. (1995) Nature (London) 376, 479–485.
Warren, R. & Carroll, S. B. (1995) Curr. Opin. Genet. Dev. 5,
Beeman, R. W., Stuart, J. J., Brown, S. J. & Denell, R. E. (1993)
BioEssays 15, 439–444.
Averof, M. & Akam, M. (1995) Nature (London) 376, 420–423.
Akam, M. (1995) Philos. Trans. R. Soc. Lond. B 349, 313–319.
Rogers, B. T. & Kaufman, T. C. (1997) Int. Rev. Cytol. 174, 1–84.
Damen, W. G. M., Hausdorf, M., Seyfarth, E-A. & Tautz, D.
(1998) Proc. Natl. Acad. Sci. USA 95, 10665–10670.
Telford, M. J. & Thomas, R. H. (1998) Proc. Natl. Acad. Sci. USA
Dev. Genet. 15, 19–31.
Rogers, B. T., Peterson, M. D. & Kaufman, T. C. (1997)
Development (Cambridge, U.K.) 124, 149–157.
Peterson, M. D., Rogers, B. T., Popadic, A. & Kaufman, T. C.
(1999) Dev. Genes. Evol. 209, 77–90.
Manak, J. R. & Scott, M. P. (1994) Development (Cambridge,
U.K.) Suppl. 1994, 61–71.
Friedrich, M. & Tautz, D. (1995) Nature (London) 376, 165–167.
Osorio, D., Averof, M. & Bacon, J. P. (1995) TREE 10, 449–454.
Ballard, J. W., Olsen, G. J., Faith, D. P., Odgers, W. A., Rowell,
D. M. & Atkinson, P. W. (1992) Science 258, 1345–1348.
Boore, J. L., Collins, T. M., Stanton, D., Daehler, L. L. & Brown,
W. M. (1995) Nature (London) 376, 163–165.
Boore, J. L., Lavrov, D. V. & Brown, W. M. (1998) Nature
(London) 392, 667–668.
Gilbert, S. F. & Raunio, A. M. (1997) Embryology: Constructing
the Organism (Sinauer, Sunderland, MA).
Zrzavy, J., Hypsa, V. & Vlaskova, M. (1997) in Arthropod
Relationships, eds. Fortey, R. A. & Thomas, R. H. (Chapman &
Hall, London), pp. 97–108).
Nielsen, C. (1989) Animal Evolution: Interrelationships of the
Living Phyla (Oxford Univ. Press, Oxford).
Telford, M. J. & Thomas, R. H. (1995) Nature (London) 376,
Scholtz, G. (1997) in Arthropod Relationships, eds. Fortey, R. A.
& Thomas, R. H. (Chapman & Hall, London), pp. 317–332.
Mouchel-Vielh, E., Rigolot, C., Gibert, J-M. & Deutsch, J. S.
(1998) Mol. Phylogenet. Evol. 9, 382–389.
Abzhanov, A. & Kaufman, T. C. (1999) Development (Cam-
bridge, U.K.) 126, 1121–1128.
Whitington, P. M., Leach, D. & Sandeman, R. (1993) Develop-
ment (Cambridge, U.K.) 118, 449–461.
Panganiban, G., Nagy, L. & Carroll, S. (1994) Curr. Biol. 4,
Gorman, M. J. & Kaufman, T. C. (1995) Genetics 140, 557–572.
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Pole, S. J., Ellis,
Scholtz, G. (1995) Zoology 98, 104–114.
Kaufman, T. C., Seeger, M. A. & Olsen, G. (1990) Adv. Genet. 27,
of Invertebrates (Univ. of Chicago Press, Chicago), pp. 21–25,
Schmidt-Ott, U. & Technau, G. M. (1992) Development (Cam-
bridge, U.K.) 116, 111–125.
Rogers, B. T. & Kaufman, T. C. (1996) Development (Cambridge,
U.K.) 122, 3419–3432.
Tamarelle, M. (1984) Int. J. Insect Morphol. Embryol. 13, 331–
Denell, R. E., Brown, S. J. & Beeman, R. W. (1996) Semin. Cell.
Dev. Biol. 7, 527–538.
Peterson, M. D. (1998) Ph.D. thesis (Indiana Univ., Blooming-
Kaestner, A. (1970) Invertebrate Zoology (Interscience, New
York), Vol. 3.
Schram, F. R. (1986) Crustacea (Oxford Univ. Press, Oxford).
Hanken, J. (1993) Am. Zool. 33, 448–456.
Keynes, R. (1994) Annu. Rev. Neurosci. 17, 109–132.
Matthew, J. K., Master, V. A., Lokhorst, D. K., Nardelli-
Haefliger, D., Wedeen, C. J., Martindale, M. Q. & Shankland, M.
(1997) Dev. Biology 190, 284–300.
Evolution: Abzhanov and KaufmanProc. Natl. Acad. Sci. USA 96 (1999)10229