Interactions of the Tribolium Sex combs reduced and proboscipedia Orthologs in Embryonic Labial Development

Corresponding author: Robin E. Denell, Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506-4901., rdenell{at}ksu.edu (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The role of Hox genes in the development of insect gnathal appendages has been examined in three insects: the fruitfly, Drosophila melanogaster; the milkweed bug, Oncopeltus fasciatus; and the red flour beetle, Tribolium castaneum. In each of these organisms, the identity of the labium depends on the homeotic genes Sex combs reduced (Scr) and proboscipedia (pb). Loss of pb function in each of the three insects results in homeotic transformation of the labial appendages to legs. In contrast, loss of Scr function yields a different transformation in each species. Interestingly, mutations in Cephalothorax (Cx), the Tribolium ortholog of Scr, transform the labial appendages to antennae, a result seen in the other insects only when both pb and Scr are removed. We show here that the Tribolium labial appendages also develop as antennae in double mutants. Further, we demonstrate that expression of the Tribolium proboscipedia ortholog maxillopedia (mxp) is greatly reduced or absent in the labium of Cx mutant larvae. Thus, in the wild-type labial segment, Cx function is required (directly or indirectly) for mxp transcription. A similar interaction between Scr and pb during Drosophila embryogenesis has been described recently. Thus, this regulatory paradigm appears to be conserved at least within the Holometabola.

OUR current understanding of the importance of Hox genes and Hox gene complexes in the patterning of phylogenetically diverse animals has resulted in considerable interest in how changes in Hox gene expression and function may have contributed to morphological evolution. In this context, the development of insect gnathal segments is a particularly informative area of study. Insects show a remarkable diversity of mouthpart morphology. The ancestral type, mandibulate, is specialized for chewing (SNODGRASS 1935 ). The mandibular segment elaborates a grinding appendage, while the maxillary and labial segments form serially homologous palps. Various insect taxa independently have evolved modifications associated with sucking and sometimes piercing. Roles of Hox genes in the establishment of gnathal segment identities have been described in two insects that are rather specialized with respect to gnathal morphology: the fruit fly, Drosophila melanogaster (STRUHL 1982 ; PATTATUCCI et al. 1991 ; PERCIVAL-SMITH et al. 1997 ), and the milkweed bug, Oncopeltus fasciatus (ROGERS and KAUFMAN 1997 ; HUGHES and KAUFMAN 2000 ). In addition, we have been studying the orthologous genes in the red flour beetle, Tribolium castaneum (SHIPPY et al. 2000B ; CURTIS et al. 2001 ), which has relatively unspecialized mandibulate mouthparts.

In all three insects, the Hox genes proboscipedia (pb) and Sex combs reduced (Scr) or their orthologs are expressed in the labium and are necessary for its normal development. (Their Tribolium orthologs, maxillopedia (mxp) and Cephalothorax (Cx), respectively, were originally ascertained genetically and given different names.) Loss of pb/mxp function in each of the three insects results in a homeotic transformation of the labial appendage to leg. (In Drosophila, pb plays this role only during imaginal development and is dispensable with respect to normal larval development.) In contrast, loss of Scr/Cx function yields a different transformation in each species. In Drosophila, examinations of hypomorphic alleles and mutant clones indicate that loss of Scr function results in a transformation of the adult proboscis to maxillary palp (STRUHL 1982 ; PATTATUCCI et al. 1991 ). In Oncopeltus nymphs, depletion by RNA interference of transcripts encoded by the Scr ortholog yields a labial appendage with a mixed leg/antennal identity (HUGHES and KAUFMAN 2000 ). Finally, in Cx mutant larvae the labium is transformed into antenna. This single mutant phenotype is unexpected since a labial to antennal transformation is observed in Drosophila and Oncopeltus only when both pb and Scr functions are removed (PERCIVAL-SMITH et al. 1997 ; HUGHES and KAUFMAN 2000 ). Work in Drosophila suggests that appendages develop as antennae when all Hox gene function is eliminated (STRUHL 1981 ; PERCIVAL-SMITH et al. 1997 ). This principle, which may be a general paradigm for all insects, is demonstrated even more dramatically by studies in Tribolium (STUART et al. 1991 ; BROWN et al. 2000 ) and Oncopeltus (HUGHES and KAUFMAN 2000 ). Consistent with this principle, we show here that in mxp Cx double mutants, labial appendages are transformed to antennae. However, the identical transformation in Cx single mutants remains to be explained. We can envision at least two scenarios. First, Mxp protein might not be functional in the absence of Cx. Alternatively, mxp may not be transcribed in the labial segment of Cx mutants. In this report, we demonstrate that mxp expression is greatly reduced or absent in the labium of Cx individuals. Thus, in the wild-type labial segment, Cx function is required (directly or indirectly) for mxp transcription. A similar interaction between Scr and pb during Drosophila embryogenesis has been described recently (RUSCH and KAUFMAN 2000 ), suggesting that this is a regulatory paradigm conserved at least among the Holometabola.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Tribolium stocks and mutagenesis:
Mutants described in this work were maintained as balanced stocks on whole-wheat flour supplemented with 5% brewer's yeast in a 28° humidified chamber. mxp^Stm is a gain-of-function dominant allele (SHIPPY et al. 2000B ), and mxp^Stm Cx⁵/Ag^Pin males were irradiated to isolate an mxp revertant null allele as described by BROWN et al. 2000 . Cx⁵ was separated from mxp^Stm by recombination, which occurred at a frequency of 1/1782.

SEM analysis of mutant larvae:
Homozygous first instar larvae were fixed in a 1:3 solution of dimethyl propane:ethanol at 4° for 1–3 days, washed in ethanol, and critical point dried (LEWIS et al. 2000 ). Fixed larvae were examined by scanning electron microscopy (SEM) with a Hitachi (Mountain View, CA) S-570 scanning electron microscope at the Russell Laboratories SEM facility.

Double-stranded RNA interference:
Double-stranded RNA (dsRNA) was prepared as previously described (SHIPPY et al. 2000B ). dsRNA was injected into 0- to 1-hr-old wild-type Tribolium embryos.

In situ hybridization to whole-mount embryos:
Digoxigenin (Dig)-labeled riboprobes were prepared using a Dig RNA labeling kit according to manufacturer's recommendations (Boehringer Mannheim, Indianapolis). The mxp riboprobe was hydrolyzed for 35 min according to a previously published protocol (PANGANIBAN et al. 1994 ). The Cx riboprobe was not hydrolyzed. Cx⁵/Ey and Ga-1 embryos 0–4 days old were dechorionated in 50% bleach and fixed for 40 min in a 1:1 solution of 4% formaldehyde, 1x PEM (100 mM PIPES, 2 mM MgSO₄, 1 mM EGTA, 1% Triton X-100):heptane. After removal of the vitelline membranes, the fixed embryos were treated essentially according to the protocol of HAUPTMANN and GERSTER 1994 with the following prehybridization modifications. Embryos were washed in PBST (PBS, 0.1% Tween-20), treated with 50 µg/ml proteinase K for 3 min at 37°, and then washed twice for 10 min in 2 mg/ml glycine. Samples were equilibrated in PBST, postfixed in 5.5% formaldehyde in PBST, and finally washed four times for 10 min each in PBST. Dig-labeled riboprobes were detected by a secondary antibody conjugated to alkaline phosphatase. Individual embryos were mounted in 80% glycerol in PBS and documented by light microscopy with a Zeiss (Thornwood, NY) Axiophot and Nomarski DIC optics.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Morphology of the gnathal appendages in wild-type and mutant Tribolium larvae:
The gnathal appendages of the Tribolium first instar larva are shown in Fig 1A. The labial appendages are composed of proximal coxapodites that are fused at the ventral midline and distal telopodites that develop as palps. The labial limb buds originate as a pair of protrusions flanking the ventral midline and then migrate ventrally and anteriorly to a position nested between the maxillary appendages. The coxapodites of the maxillary appendages bear both an endite and a telopodite (which closely resembles a labial palp).

View larger version (158K): In this window In a new window Download PPT slide   Figure 1. Phenotypic analysis of Cx and mxp single and double mutants. The first instar larval gnathal appendages of mutants (C–E) and RNAi phenocopy (F) are shown in ventral view, with anterior toward the top. (A and B) Wild type. The proximal segments of the labial appendages (li) are fused at the ventral midline. The two distal segments (palps) are unfused. The maxillary appendages (mx) contain proximal endites as well as distal palps. The maxillary palps are larger than the labial palps but have similar patterning. Normal antennae (arrowhead) and legs (arrow) are shown in B. (C) Cx5 homozygote. The labial appendages are transformed to antennae (arrowhead). (D) mxp14 homozygote. Labial and maxillary palps are transformed to legs (arrows). (E) mxpStmR8 Cx5 homozygote. The mxp Cx double mutant larva displays maxilla to leg transformations (arrow) similar to the mxp14 homozygote shown in D and labium to antennal transformations (arrowhead) similar to the Cx5 homozygote shown in C. (F) RNA depletion using a mixture of Cx and mxp double-stranded RNA. The phenotype of this larva is similar to that of the double mutant mxpStmR8 Cx5 shown in E.

The Tribolium ortholog of proboscipedia is mxp (SHIPPY et al. 2000A ). In larvae homozygous for null mxp alleles, the labial telopodites are transformed to legs (Fig 1D), although, unlike thoracic legs, the coxapodites remain fused at the ventral midline (SHIPPY et al. 2000B ). In addition, the maxillary telopodites are transformed to legs, but the endites are unaffected. Mutations in Cx, the Tribolium ortholog of Scr, also affect the labial appendages. The Cx⁵ allele is a strong loss-of-function variant, and, when homozygous or hemizygous, results in transformation of the labial appendages to antennae (BEEMAN et al. 1993 ) (Fig 1C). In these mutants, the labial appendages do not migrate toward the ventral midline nor do the coxapodites fuse. Further, the labial and first thoracic segments fuse, changing the orientation of the T1 legs to resemble that of the gnathal appendages. In wild-type embryos mxp and Cx are both expressed in the gnathal appendages (SHIPPY et al. 2000B ; CURTIS et al. 2001 ). mxp is expressed in the maxillary and labial appendages (Fig 2A), whereas Cx is expressed in the labium (Fig 2B).

View larger version (130K): In this window In a new window Download PPT slide   Figure 2. mxp and Cx expression in wild-type and Cx null embryos. In situ hybridization of mxp (A, C, and D) and Cx (B) specific riboprobes to whole-mount embryos are shown in ventral view, with anterior toward the top. (A) mxp transcripts appear in the maxillary and labial appendages of wild-type embryos. (B) In wild-type embryos, Cx transcripts appear throughout the labial segment including the appendage. Thus, mxp and Cx are coexpressed in the labial appendages. (C and D) mxp transcripts are absent from the labial limbs in both young (C) and midstage (D) Cx5 homozygous embryos.

Examination of Cx mxp double mutants:
maxillopedia-Stumpy (mxp^Stm) is a dominant mutation that was isolated in an EMS mutagenesis and is associated with abnormalities of the adult antennae (BEEMAN et al. 1989 ). Reversion experiments demonstrate that Stm is a gain-of-function mxp allele, since revertants act as loss-of-function mxp alleles (SHIPPY et al. 2000B ). Cx⁵ is a strong loss- of-function variant (BEEMAN et al. 1993 ; Fig 1C) that arose during a gamma-ray mutagenesis on a chromosome bearing mxp^Stm.

To generate mxp Cx double mutants, we irradiated mxp^Stm Cx⁵/Ag^Pin heterozygotes, mated them to wild-type beetles, and screened the progeny for individuals in which the dominant antennal effect of mxp^Stm was reverted. Two revertants, denoted mxp^StmR7 and mxp^StmR8, were isolated. mxp^StmR8 is a strong loss-of-function or null allele of mxp by the criterion that it completely fails to complement mxp¹⁴ (itself a strong loss-of-function allele), resulting in heterozygous larvae that show strong transformations of labial and maxillary appendages to legs. In contrast, mxp^StmR7 is a hypomorphic mxp allele since in heterozygous combination with mxp¹⁴ it gives only partial transformations of labial and maxillary appendages. As expected, the revertant-bearing chromosomes also fail to complement Cx variants, although they do complement mutations at other loci in the Homeotic complex such as the Tribolium Deformed ortholog (data not shown).

We have examined the phenotype of larvae homozygous for the mxp^StmR8 Cx⁵ chromosome (Fig 1E). As in mxp single mutants, the maxillary appendages are transformed to legs. The labial appendages develop as antennae and appear indistinguishable from those of Cx⁵ homozygotes bearing normal alleles of mxp. Thus, in the labium, loss of Cx is epistatic to loss of mxp.

To demonstrate that the phenotype of the double mutant is due solely to lack of Cx and Mxp proteins we also examined embryos in which Cx and mxp transcripts were depleted via RNA interference (RNAi). Tribolium null mutant phenotypes for mxp (SHIPPY et al. 2000B ) and Cx (CURTIS et al. 2001 ) have previously been phenocopied by this technique. We injected 400 eggs with both mxp and Cx double-stranded RNAs. Of the 74 embryos that formed larval cuticles, 22 resembled the mxp Cx double mutants described above in that the maxillary appendages are transformed to legs (demonstrating interference with maxillopedia function), and the labial appendages are transformed to antennae (Fig 1F). These results further support the conclusion that Cx mutations are epistatic to mxp mutations with respect to labial identity.

Maxillopedia expression in Cx mutants:
RUSCH and KAUFMAN 2000 have recently demonstrated that Sex combs reduced is a positive regulator of proboscipedia during Drosophila embryonic development. If the same relationship were true during Tribolium embryonic development, we would predict that mxp expression would be extinguished in the labium of Cx mutants. For this purpose, we utilized a Cx⁵ allele that had been separated from mxp^Stm by recombination. From a cross between Cx⁵ heterozygotes, Cx⁵ homozygous embryos can be identified near midembryogenesis by the failure of the labial appendages to migrate and fuse. We performed in situ hybridization with a mxp-specific riboprobe. This probe detected mxp transcripts in the maxillary appendages of Cx⁵ homozygotes, but not in the labial appendages (Fig 2C and Fig D). By a similar approach, we have also observed a great reduction in mxp transcripts in the labial appendages of embryos homozygous for Cx⁶, another severe loss-of-function variant (data not shown). These results indicate that normal expression of mxp in the labium depends on the presence of Cx there.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the hexapods examined to date, development of a normal labium depends on the wild-type function of two Hox genes, Scr and pb (summarized in Table 1). In Drosophila, examinations of hypomorphic alleles and mutant clones indicate that loss of Scr function in adults results in a homeotic transformation of the proboscis (labium) to maxillary palp (STRUHL 1982 ; PATTATUCCI et al. 1991 ). Null mutations of pb transform the proboscis to a pair of prothoracic tarsi (KAUFMAN 1978 ). When clones are missing both Scr and pb activities, the adult labium develops antennal structures (PERCIVAL-SMITH et al. 1997 ). These and other observations led PERCIVAL-SMITH et al. 1997 to propose that antennal development is the default state in the adult labium in the absence of Scr and Pb proteins. Scr (in the absence of Pb) promotes development of tarsi, while Pb (in the absence of Scr) results in maxillary identity. Both of these proteins are necessary for formation of a proboscis.

The situation is somewhat different during embryonic development. Even though pb is expressed in the embryonic labium, it is entirely dispensable with respect to the normal development of the labial sense organs (PULTZ et al. 1988 ). The phenotype of the larval labial sense organs in Scr mutants is controversial. The original interpretation was that the labium is transformed to a maxillary identity (see PATTATUCCI et al. 1991 ), a change paralleling that described in adults. Alternatively, PEDERSON et al. 1996 suggested that there is a loss of labium-specific features, but no transformation to maxillary identity. Thus, when both Scr and pb activities are missing, the default state with respect to embryonic labial cuticular development appears to be either maxillary or a generic gnathal identity. In either case, Hox gene regulation of labial fate appears very different during larval vs. adult development.

HUGHES and KAUFMAN 2000 have used RNAi to generate phenocopies of mutants of pb and Scr, as well as of the double mutant in the milkweed bug O. fasciatus. This Hemipteran has highly specialized mouthparts in which the mandibular and maxillary appendages are modified to form long thin stylets that lie in a groove formed by the extended, fused labial appendages. As noted earlier, in most insects the maxillary and labial appendages resemble one another, and both typically express pb (ROGERS and KAUFMAN 1997 ). However, in Oncopeltus, in which the maxillary appendages more closely resemble those of the mandibular segment, maxillary expression of the pb ortholog is highly reduced. In addition, there is evolutionarily novel expression of Scr in the maxillary segment. ROGERS and KAUFMAN 1997 found that, despite its specialized morphology, the labial segment expresses pb and Scr, thus resembling the case in Drosophila and Tribolium. When RNAi is used to deplete pb function in the bug, the distal labium is transformed to leg. Scr depletion results in appendages with mixed leg and antennal identity. Finally, depletion of both pb and Scr function results in transformation of the labial appendages to antennae. HUGHES and KAUFMAN 2000 have suggested that, in the lineage leading to Oncopeltus, pb has gained a function, partially redundant with Scr, in specifying leg development. This explains the partial transformation to leg when Scr activity is depleted even though a homeotic leg can clearly develop in the absence of pb function. We can conclude from this work that, in addition to an apparently novel function for pb in leg development, the bug pb and Scr orthologs are both necessary to promote normal labial development.

Implications of Tribolium mxp Cx phenotype:
The mxp Cx double mutant provides insight into the mechanisms underlying the phenotypes of mxp and Cx single mutants. In the absence of mxp function, both the maxillary and labial telopodites are transformed to legs. In the mxp Cx mutant, the maxillary appendages are still transformed to legs, but the labial appendages are transformed to antennae. This suggests that Cx function is required for transformation to leg identity in the labial appendages but not in the maxillary appendages of mxp single mutants. The apparent dispensability of Cx in the maxillary segment is not particularly surprising given that it is expressed in only the posterior compartment. TcDeformed (TcDfd; the only Hox gene predicted to be present in the maxillary segment of mxp Cx mutants) is probably responsible for transformation to leg identity there. Note that these results suggest that the homeotic legs of mxp mutants do not arise from expression of the same Hox genes present in the thoracic leg primordia. Neither Cx nor TcDfd is normally expressed in the epidermis of thoracic legs (BROWN et al. 1999 ; CURTIS et al. 2001 ). Rather, identity of the thoracic appendages depends on the normal function of the likely Antennapedia ortholog prothoraxless (ptl; BEEMAN et al. 1993 ). In Drosophila, leg development occurs as a default state when antennal development is repressed by homeotic genes. Many homeotic genes (including Antp and Scr) can repress antennal identity when ectopically expressed (see YAO et al. 1999 ). Thus, in beetles, a normal role of Cx and TcDfd in repressing antennal development in the labial and maxillary appendages, respectively, would explain the development of legs in mxp mutants.

The identical labial transformations seen in Cx single mutants and mxp Cx double mutants suggested that mxp is not functioning in the labial appendages in the absence of Cx and led us to examine mxp expression in Cx⁵ mutant embryos. We observed mxp expression in the maxillary but not labial appendages of these mutants. Thus, when Cx is absent, mxp function in the labium is affected at the transcriptional rather than post-transcriptional level.

A conserved regulatory pathway:
The reduction or loss of mxp expression in the labial segment of Cx mutant embyros suggests that Cx positively regulates mxp (either directly or indirectly) in the wild-type labium. A similar relationship between Scr and pb in Drosophila has recently been reported (RUSCH and KAUFMAN 2000 ; MILLER et al. 2001 ). Since pb has no apparent function in Drosophila embryos, Kaufman and co-workers have proposed that this regulatory interaction (and in fact the entire pb embryonic expression pattern) are evolutionary remnants of an ancestral regulatory network (RUSCH and KAUFMAN 2000 ; MILLER et al. 2001 ). Our results suggest that, indeed, an ancestral Scr gene positively regulated expression of an ancestral pb gene at least as early as the divergence of the Holometabola. Determining whether this regulatory relationship evolved even earlier will require functional examination of Scr and pb orthologs in more basal insects. The only such data currently available are from the RNA interference experiments in Oncopeltus (HUGHES and KAUFMAN 2000 ). Since Oncopeltus pb appears to retain some function when Scr is depleted, HUGHES and KAUFMAN 2000 have suggested that Scr is not required for pb expression in the milkweed bug labium. However, an ancestral regulatory relationship between Scr and pb might well have been lost during the evolution of Oncopeltus's highly derived mouthparts. Data from a basal mandibulate insect might provide more insight into the evolutionary origin of this interaction.

Comparison of embryonic and adult phenotypes:
As described above, the Tribolium Cx single mutant phenotype (transformation of larval labial appendages to antennae) differs from the phenotypes of Drosophila Scr mutant larvae and adults. Understanding the underlying regulatory pathways helps resolve this discrepancy. In both Drosophila and Tribolium embryos, Scr/Cx positively regulates pb/mxp (RUSCH and KAUFMAN 2000 ; MILLER et al. 2001 ; this work). However, pb lacks an embryonic function in Drosophila, and some other gene is presumably responsible for the residual gnathal identity in the labial segment of Scr mutant larvae. In adult flies, pb functions to specify the maxillary palps. Scr hypomorphs and mitotic clones lacking Scr cause transformation of the adult labial appendages to maxillary palp (STRUHL 1982 ; PATTATUCCI et al. 1991 ). However, when clones lack both Scr and pb, cells assume antennal identity (PERCIVAL-SMITH et al. 1997 ). These results suggest that, in contrast to the embryonic paradigm, pb is present and functional during Drosophila adult development even in the absence of Scr. ABZHANOV et al. 2001 recently demonstrated that expression of pb in the labial imaginal disc does not require Scr function. In fact, to some extent, the reverse appears to be true. Scr expression is greatly reduced in the distal portions of pb null labial imaginal discs (ABZHANOV et al. 2001 ).

In Drosophila, embryonic development is highly specialized and results in a limbless larva. Thus, comparisons between adult development in Drosophila and embryonic development in other insect species have revealed conserved features that would be missed in comparisons limited to embryonic development (BEEMAN et al. 1989 ; SHIPPY et al. 2000B ). It is interesting that the regulatory interaction between the Scr and pb orthologs is conserved in Tribolium and Drosophila embryos, but not in Drosophila adults. This may be an exception to the general principle that Drosophila adult development is less derived. Alternatively, adult pb regulation may be similar in beetles and flies. Analysis of the Cx mutant phenotype in Tribolium adults would give insight into this question. Unfortunately, this information is not yet available. Cx null mutations are larval lethals, and no technique for inducing mitotic clones in Tribolium currently exists. With the advent of germ-line transformation for Tribolium (BERGHAMMER et al. 1999 ), experiments using inducible RNA interference techniques (FORTIER and BELOTE 2000 ) may allow such questions to be addressed.

	ACKNOWLEDGMENTS

We thank Teresa Shippy for critical reading of the manuscript. This work was supported by grants from the National Science Foundation and the National Institutes of Health, as well as by a Human Frontier Science Program postdoctoral fellowship to M.A.D. M.A.D. and D.L.L. thank Dr. Sean Carroll for use of reagents and facilities. This study is contribution no. 02-63-J from the Kansas Agricultural Experiment Station.

Manuscript received December 19, 2000; Accepted for publication September 27, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ABZHANOV, A., S. HOLTZMAN, and T. C. KAUFMAN, 2001  The Drosophila proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendage genes. Development 128:2803-2814[Abstract/Free Full Text].

BEEMAN, R. W., J. J. STUART, M. S. HAAS, and R. E. DENELL, 1989  Genetic analysis of the homeotic gene complex (HOM-C) in the beetle Tribolium castaneum. Dev. Biol. 133:196-209[Medline].

BEEMAN, R. W., J. J. STUART, S. J. BROWN, and R. E. DENELL, 1993  Structure and function of the Homeotic gene complex (HOM-C) in the beetle, Tribolium castaneum. BioEssays 15:439-444[Medline].

BERGHAMMER, A. J., M. KLINGLER, and E. A. WIMMER, 1999  A universal marker for transgenic insects. Nature 402:370-371[Medline].

BROWN, S. J., S. HOLTZMAN, T. KAUFMAN, and R. DENELL, 1999  Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant. Dev. Genes Evol. 209:389-398[Medline].

BROWN, S. J., M. DECAMILLIS, K. GONZALEZ-CHARNECO, M. DENELL, and R. BEEMAN et al., 2000  Implications of the Tribolium Deformed mutant phenotype for the evolution of Hox gene function. Proc. Natl. Acad. Sci. USA 97:4510-4514[Abstract/Free Full Text].

CURTIS, C. D., J. A. BRISSON, M. A. DECAMILLIS, T. D. SHIPPY, and S. J. BROWN et al., 2001  Molecular characterization of Cephalothorax, the Tribolium ortholog of Sex combs reduced. Genesis 30:12-20[Medline].

FORTIER, E. and J. M. BELOTE, 2000  Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila.. Genesis 26:240-244[Medline].

HAUPTMANN, G. and T. GERSTER, 1994  Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 10:266[Medline].

HUGHES, C. L. and T. C. KAUFMAN, 2000  RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head. Development 127:3683-3694[Abstract].

KAUFMAN, T. C., 1978  Cytogenetic analysis of chromosome 3 in Drosophila melanogaster: isolation and characterization of four new alleles of the proboscipedia (pb) locus. Genetics 90:579-596[Abstract/Free Full Text].

LEWIS, D. L., M. DECAMILLIS, and R. L. BENNETT, 2000  Distinct roles of the homeotic genes Ubx and abd-A in beetle embryonic abdominal appendage development. Proc. Natl. Acad. Sci. USA 97:4504-4509[Abstract/Free Full Text].

MILLER, D. F., B. T. ROGERS, A. KALKBRENNER, B. HAMILTON, and S. L. HOLTZMAN et al., 2001  Cross-regulation of Hox genes in the Drosophila melanogaster embryo. Mech. Dev. 102:3-16[Medline].

PANGANIBAN, G., L. NAGY, and S. B. CARROLL, 1994  The role of the Distal-less gene in the development and evolution of insect limbs. Curr. Biol. 4:671-675[Medline].

PATTATUCCI, A. M., D. C. OTTESON, and T. C. KAUFMAN, 1991  A functional and structural analysis of the Sex combs reduced locus of Drosophila melanogaster. Genetics 129:423-441[Abstract].

PEDERSON, J., D. KIEHART, and J. MAHAFFEY, 1996  The role of HOM-C genes in segmental transformations: re-examination of the Drosophila Sex combs reduced embryonic phenotype. Dev. Biol. 180:131-142[Medline].

PERCIVAL-SMITH, A., J. WEBER, E. GILFOYLE, and P. WILSON, 1997  Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster. Development 124:5049-5062[Abstract].

PULTZ, M. A., R. J. DIEDERICH, D. L. CRIBBS, and T. C. KAUFMAN, 1988  The proboscipedia locus of the Antennapedia complex: a molecular and genetic analysis. Genes Dev. 2:901-920[Abstract/Free Full Text].

ROGERS, B. T. and T. C. KAUFMAN, 1997  Structure of the insect head in ontogeny and phylogeny: a view from Drosophila. Int. Rev. Cytol. 174:1-84[Medline].

RUSCH, D. B. and T. C. KAUFMAN, 2000  Regulation of proboscipedia in Drosophila by the homeotic selector genes. Genetics 156:183-194[Abstract/Free Full Text].

SHIPPY, T. D., S. J. BROWN, and R. E. DENELL, 2000a  maxillopedia is the Tribolium ortholog of proboscipedia. Evol. Dev. 2:145-151[Medline].

SHIPPY, T. D., J. GUO, S. J. BROWN, R. W. BEEMAN, and M. S. HAAS et al., 2000b  Analysis of maxillopedia expression pattern and larval cuticular phenotypes of wild-type and mutant Tribolium. Genetics 155:721-731[Abstract/Free Full Text].

SNODGRASS, R. E., 1935 Principles of Insect Morphology. Cornell University Press, Ithaca, NY.

STRUHL, G., 1981  A homeotic mutation transforming leg to antenna in Drosophila. Nature 292:635-638[Medline].

STRUHL, G., 1982  Genes controlling segmental specification in the Drosophila thorax. Nature 79:7380-7384.

STUART, J. J., S. J. BROWN, R. W. BEEMAN, and R. E. DENELL, 1991  A deficiency of the homeotic complex of the beetle Tribolium. Nature 350:72-74[Medline].

YAO, L.-C., G.-J. LIAW, C.-P. PAI, and Y. H. SUN, 1999  A common mechanism for antennato-leg transformation in Drosophila: suppression of homothorax transcription by four HOM-C genes. Dev. Biol. 211:268-276[Medline].

This article has been cited by other articles:

				H. Marques-Souza, M. Aranda, and D. Tautz Delimiting the conserved features of hunchback function for the trunk organization of insects Development, March 1, 2008; 135(5): 881 - 888. [Abstract] [Full Text] [PDF]

				T. D. Shippy, C. D. Rogers, R. W. Beeman, S. J. Brown, and R. E. Denell The Tribolium castaneum Ortholog of Sex combs reduced Controls Dorsal Ridge Development Genetics, September 1, 2006; 174(1): 297 - 307. [Abstract] [Full Text] [PDF]

				T. C. Kaufman, D. W. Severson, and G. E. Robinson The Anopheles Genome and Comparative Insect Genomics Science, October 4, 2002; 298(5591): 97 - 98. [Abstract] [Full Text] [PDF]