Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

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

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

THE homeotic selector genes of the fruit fly, Drosophila melanogaster, assign developmental fate to cells appropriate to their location along the anterior-posterior axis (DENELL et al. 1996 ). Mutations in these genes often produce transformations of one body region to another. Homeotic genes are very ancient; a single cluster arose very near the origin of the Eumetazoa (FINNERTY 1998 ). In D. melanogaster, this cluster has been split into two complexes: the Antennapedia complex (ANTC; KAUFMAN et al. 1990 ) and the bithorax complex (BXC; LEWIS 1978 ). In recent years homeotic genes have been cloned from a number of organisms to study how changes in expression and/or targets of homeotic genes may have played an important role in the morphological evolution of animals (AVEROF and PATEL 1997 ; ROGERS et al. 1997 ).

Drosophila and other higher flies are specialized with respect to embryonic cephalic development. The head and gnathocephalic segments involute through the presumptive mouth and contribute predominately to internal larval structures. The effects of homeotic mutations on the larval head differ dramatically from the transformations produced in adult heads. This observation led DIEDERICH et al. 1991 to argue that the embryonic functions of the ANTC genes affecting these segments have been modified from those typical of most insects, and that in fact the adult mutant abnormalities associated with these genes are a better guide to their ancestral regulatory roles.

The ANTC gene proboscipedia (pb) is a particularly striking example of the dichotomy between adult and embryonic homeotic mutant phenotypes. pb is unique among Drosophila homeotic genes in that it is completely dispensable for normal embryonic development (PULTZ et al. 1988 ). This lack of embryonic function is somewhat surprising since an ancestral pb gene existed as part of the Homeotic complex before the separation of protostomes and deuterostomes (RUDDLE et al. 1994 ) and presumably shared with its homeotic neighbors a role in assigning developmental fate during embryogenesis. In contrast, adult flies lacking pb gene function exhibit dramatic transformation of the proboscis, or labium, to prothoracic legs, as well as a reduction in size of the maxillary palps that may represent a transformation toward antennae (BRIDGES and DOBZHANSKY 1933 ; KAUFMAN 1978 ).

Studying the function of a pb ortholog in a less derived insect could provide valuable insight into whether an ancestral pb gene had an embryonic function, and if so what that function may have been. The red flour beetle, Tribolium castaneum, exhibits a mode of embryogenesis more typical of insects and is currently the only non-Drosophilid insect for which full-scale genetic studies are feasible. In work published elsewhere, we have cloned the Tribolium ortholog of pb and have demonstrated that it corresponds to the genetically ascertained maxillopedia locus (SHIPPY et al. 2000 ).

maxillopedia (mxp) was first defined by the spontaneous, recessive-viable mutation mxp¹ (formerly mxp), which weakly transforms maxillary and labial appendages toward legs in both adults and larvae (HOY 1966 ). BEEMAN et al. 1989 identified a group of additional mxp mutations that are believed to include nulls, hypomorphs, and gain-of-function (GOF) alleles. The putative null mxp alleles cause strong transformation of larval maxillary and labial palps to legs and lethality about the time of the first larval molt. Thus, unlike pb, mxp function is required for proper identity of larval as well as adult mouthparts. It is also striking that GOF mutations of mxp, most of which produce dominant transformations of the legs and/or antennae to palps, are quite common, whereas only one pb GOF mutation has been described (CRIBBS et al. 1992 ).

Here we classify available mxp alleles based on genetic analysis and larval mutant phenotype. Furthermore we demonstrate that both null and hypomorphic mxp alleles are phenocopied by RNA interference. We describe the expression pattern of mxp transcripts in wild-type embryos and show that it closely resembles the pattern described for Pb protein (PULTZ et al. 1988 ). In addition, we demonstrate that several GOF mxp mutations are associated with ectopic mxp expression and, in some cases, loss of expression in all or part of the normal domain.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Beetle strains and genetic analysis:
Beetle cultures were maintained at 30° as described by BEEMAN et al. 1989 . For egg collection, beetles were transferred to Gold Medal flour (General Mills) supplemented with 5% brewer's yeast. Strains used were mxp¹⁷⁰/A^Es; mxp¹⁹/A^Es; Lu^R1/Ey; mxp¹, apt, pas³⁰/mxp¹, apt, pas³⁰; mxp⁸/A^Es; mxp^lp/A^Es; mxp^X9, A^Es/Hw; mxp^Ng/A^Es; mxp^Stbd/A^Es; mxp^Stb/Ag; mxp^Dch-1/A^Es; mxp^Dch-4/A^Es; mxp^Dch-3/Ey; mxp^Stm/mxp^Stm; mxp^Stm/A⁸³ and mxp^StmR1/A^Es. For analysis of gene expression, strains were first outcrossed to wild type and then either self-crossed or outcrossed a second time. Novel expression patterns observed only in self-cross progeny were interpreted as representing the homozygous mutant class.

Documentation of larval phenotypes:
Eggs were collected from balanced stocks or crosses of mxp mutants and then incubated at 30° until hatching. Hatched larvae were transferred into 9:1 lactic acid:ethanol and heated at 60° for 1–3 days to clear. Larvae to be photographed were mounted under a coverslip in PVLP mounting medium (8.4% polyvinyl acetate, 22% lactic acid, 22% phenol). Nomarski images from an Olympus BX50 microscope were captured with a Kontron Progres 3012 digital camera. Contrast, brightness, and sharpness were adjusted using Adobe Photoshop software.

RNA-mediated interference:
Double-stranded RNA (dsRNA) was synthesized as described by BROWN et al. 1999 except that transcription was performed using MEGAscript SP6 and T7 polymerase kits (Ambion) according to the manufacturer's instructions. Two mxp templates were used for RNA synthesis: a nonhomeobox-containing 903-bp subclone from the 3' end of the mxp transcript, and a 1.5-kb partial cDNA that includes the homeobox (SHIPPY et al. 2000 ). dsRNA were diluted with injection buffer (5 mM KCl, 0.1 mM KPO₄, pH 6.8, and green food dye) and microinjected into 0–4-hr Tribolium embryos as described by BROWN et al. 1999 . The nonhomeobox dsRNA (8.5 µg/µl) was diluted to 7.7 µg/µl, while the homeobox dsRNA (1.87 µg/µl) was diluted to 0.93 µg/µl. Injected embryos were incubated at room temperature in a humidified, oxygenated chamber. Approximately 1 week after injection, hatched larvae were treated with lactic acid and ethanol (9:1) and examined for abnormalities in cuticle morphology.

Analysis of gene expression:
In situ hybridization was performed as described by BROWN et al. 1994B . A digoxygenin-labeled antisense riboprobe was transcribed from a 3.8-kb homeobox-containing XhoI-HindIII fragment of mxp genomic DNA (SHIPPY et al. 2000 and unpublished results). Engrailed protein was detected with the crossreacting Mab4D9 antibody (PATEL et al. 1989 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Analysis of mxp mutant alleles: We have used genetic analysis and larval cuticular phenotype to classify existing and newly identified mxp mutant alleles (see Table 1 for summary). The alleles fall into four categories: null alleles, hypomorphic alleles, those in which gain-of-function effects accompany loss of normal function (GOF/LOF), and those with normal function accompanied by GOF effects.

Null alleles: Several radiation-induced alleles of mxp identified by BEEMAN et al. 1989 cause recessive larval lethality with strong transformation of the maxillary and labial palps to legs and have been proposed to be null alleles. We have analyzed two putative null alleles: mxp¹⁷⁰ and mxp¹⁹. To determine whether these alleles truly represent mxp nulls, we utilized Lucifer Revertant 1 (Lu^R1), a deficiency which removes the mxp locus. This deficiency will be described in detail elsewhere, but for the purposes of this article it is important to note that it deletes genes on either side of mxp. Lu^R1 fails to complement alleles of TcDeformed (TcDfd) and has been shown molecularly to lack Tclabial (our unpublished results). The phenotypes of mxp¹⁹ (Fig 1B) and mxp¹⁷⁰ (data not shown) homozygotes and hemizygotes (data not shown) are identical, suggesting that mxp¹⁹ and mxp¹⁷⁰ are null alleles. Furthermore, mxp¹⁹ is associated with a single base pair deletion within the mxp coding region that is predicted to result in a truncated, nonfunctional protein (SHIPPY et al. 2000 ). We will use the notation mxp^- to represent a null allele of mxp.

View larger version (174K): In this window In a new window Download PPT slide   Figure 1. Effect of mxp mutations on larvae. All images (except G and H) are ventral views with anterior at the top. (A) In the wild-type larval head each maxillary appendage (M) comprises a basal coxopodite with a ventral (medial) endite and a distal telopodite (palp). The labial appendages (L), which have no visible endites, are fused at the base and nest between the maxillary appendages. Numerous sensilla of various forms are found on the distal tips of the maxillary and labial palps. (B) A homozygous null (mxp19/mxp19) larva shows complete transformations of maxillary and labial palps to legs (M** and L**). (C) An mxp19/+ larva appears morphologically normal except that an abnormally long spike is present on each maxillary palp (M'). (D) An mxp1/mxp1 larva exhibits distal transformation of maxillary palps to leg (M*). In this individual, the labial palps appear normal. (E) The labial palps of an mxplp/mxplp larva are completely transformed to legs (L**). The maxillary palps occasionally show distal spikes or distal transformations to leg, but are normal in this individual. (F) An mxpDch-4/mxpDch-4 larva exhibits distal transformations of maxillary palps to leg(M*). In this individual, one maxillary palp also has a distal spike (M*'). The labial palps are sometimes distally transformed to legs, but in this individual they are normal. (G) A lateral view of a wild-type larva (anterior to the left) shows a normal T1 leg. (H) An mxpDch-3/+ larva has a much shorter than normal T1 leg. (I) An mxpDch-3/mxp19 larva exhibits complete transformation of maxillary palps to leg (M**), but only distal transformation of labial palps to leg (L*). (J) The maxillary appendages of an mxpDch-1/mxpDch-1 larva are distally transformed to leg, but also have a distal spike (M*') similar to the null heterozygote in C. The labial appendages also have a distal spike (L'). (K) Injection of mxp dsRNA into wild-type eggs results in transformations of the maxillary and labial palps similar to those produced by the null mxp allele shown in B. (L) Injection of lower concentrations of mxp dsRNA (approximately eightfold lower than in K) resulted in partial transformations of maxillary and labial palps to legs (M* and L*). One maxillary palp is normal in this individual.

The maxillary appendages of Tribolium are composed of a basal coxopodite, a distal telopodite (palp), and an endite lobe that projects ventrally from the coxopodite (Fig 1A). Only the telopodites are transformed to legs in mxp^- mutants; the endites are unaffected (Fig 1B). The coxopodites of wild-type labial appendages are fused at the ventral midline and lack discernable endites. Except for their slightly smaller size, the labial telopodites closely resemble the maxillary telopodites (Fig 1A). In mxp^- mutants, the labial telopodites are completely transformed into legs, but the coxopodites remain fused (Fig 1B). The appearance of the transformed larval appendages in null homozygotes and hemizygotes is variable. While some of these individuals have morphologically normal legs (Fig 1, compare B to G) extending from the maxillary and labial segments, the transformed appendages of the remaining larvae are warped and shortened to varying degrees (not shown). Interestingly, the thoracic legs of these individuals (where mxp is not normally expressed) are also warped. Warped legs are sometimes observed in other (non-mxp) mutant strains, suggesting that this phenotype may be a nonspecific developmental abnormality.

To date, no haploinsufficient mxp phenotype has been reported. We have observed, however, that more than half of larvae heterozygous for a null mxp allele have an abnormally long spike on one or both maxillary palps (Fig 1, compare A and C). This phenotype appears to be an alteration of a normally paddle-shaped sensilla to a longer tapered structure.

Hypomorphic alleles:
BEEMAN et al. 1989 interpreted the original mxp mutation, mxp¹ (HOY 1966 ), as a hypomorphic allele. Indeed, we have observed that mxp¹/mxp^- larvae and adults (data not shown) have more severe transformations than mxp¹/mxp¹ individuals (Fig 1D). Several additional hypomorphic alleles have been induced by -irradiation. mxp⁸ homozygotes die at the first larval molt, but show incomplete transformations of mouthparts, suggesting that partial mxp function is retained. The terminal portions of the palps are longer than normal, and tarsal claws are often observed (data not shown). Occasionally, palps terminate in a spike identical to that observed in null heterozygotes. mxp^labiopedia (mxp^lp; BEEMAN et al. 1989 ) is unique in that the labial palps of homozygous larvae are much more strongly transformed than the maxillary palps (which occasionally bear a spike or claw, but more often appear morphologically normal; Fig 1E). mxp^lp is apparently a hypormorphic allele since at least partial mxp function is present in the maxillary appendages. mxp^X9 was identified in a mutagenesis of a chromosome carrying the Abdominal allele Extra-sclerite (A^Es; BEEMAN et al. 1989 ). The A^Es mutation suppresses recombination in the homeotic gene complex (HOMC), preventing separation of the two mutations, and is a recessive early embryonic lethal. Thus, the larval phenotype of mxp^X9 homozygotes cannot be determined. To determine whether mxp^X9 is associated with LOF at the mxp locus, we examined its phenotype when heterozygous with a null mxp allele. mxp^X9/mxp^- heterozygotes die about the time of the first larval molt showing strong maxillary and labial transformations to leg similar to null alleles (data not shown). However, mxp^X9/mxp^lp heterozygotes are viable to adulthood and show only labial to leg transformations (data not shown). mxp^-/mxp^lp heterozygotes die as larvae (data not shown), indicating that mxp^X9 is a hypomorphic allele.

GOF alleles that fail to complement the adult phenotype of mxp¹:
BEEMAN et al. 1989 identified a number of mxp alleles that fail to complement mxp¹ and, in addition, show dominant effects. These alleles were interpreted as having GOF as well as complete or partial LOF effects. One mutation, mxp^{Notched gena} (mxp^Ng), produces notches in the anterior head capsule of the adult. Both mxp^Ng homozygotes and mxp^Ng/Lu^R1 hemizygotes (data not shown) die as larvae and show complete maxillary and labial transformations to leg, suggesting that mxp^Ng has lost all normal mxp function. As observed for other mxp nulls, some mxp^Ng homozygotes show warping of both gnathal and thoracic legs. Apparently, mxp^Ng also has dosage-dependent larval GOF effects since homozygotes show larval head capsule abnormalities. However, no abnormal expression of mxp is observed in progeny of an mxp^Ng/A^Es self-cross (see below). If mxp is ectopically expressed in the larval head, it is at a level below that detectable in our assays.

Other dominant mxp mutations cause shortening of the antennae and/or legs in what is thought to be a transformation to palp (BEEMAN et al. 1989 ). Alleles in this class include mxp^Dachs-4 (mxp^Dch-4), mxp^Dch-3, mxp-Stubby (mxp^Stb, formerly mxp^D2), mxp^Dch-1, and mxp-Stuboid (mxp^Stbd, formerly mxp^D3).

The mxp^Dch-4 allele was recovered from a -ray mutagenesis screen (BEEMAN et al. 1989 ). mxp^Dch-4 homozygotes normally die as larvae, but BEEMAN et al. 1989 reported the discovery of an mxp^Dch-4 homozygous escaper that showed striking transformation of adult tibia and tarsi to palps, as well as shortened antennae. Heterozygous adults are less severely affected, but legs and antennae are noticeably shortened. No effect on larval legs or antennae has been observed. mxp^Dch-4 homozygous larvae show only mild LOF effects with variable expressivity (Fig 1, compare F to A). In the most severe cases, the distal tips of the maxillary and labial palps are transformed to tarsal claws. The maxillary appendages may also have the distal spike seen in mxp null heterozygotes. Some individuals have both a tarsal claw and a distal spike on the same maxillary palp, demonstrating that the spike is not simply an abnormal tarsal claw. Changes in the mxp expression pattern consistent with the mxp^Dch-4 phenotype have been noted in mxp^Dch-4 heterozygotes and homozygotes (see below).

mxp^Dch-3, a previously undescribed -ray-induced mutation, is unique in that it causes only the prothoracic (T1) legs to be shortened. This effect is seen in both adults (data not shown) and larvae (Fig 1, compare G and H). The mxp^Dch-3 mutation results in pseudolinkage of the HOMC with the LG9 locus pearl (R. W. BEEMAN, unpublished results), indicating that mxp^Dch-3 is associated with an LG2 to LG9 translocation. mxp^Dch-3 homozygotes die before cuticularization (presumably due to the LG9 breakpoint), but mxp^Dch-3/mxp^- heterozygotes exhibit maxillary and to a lesser extent labial transformations to leg (Fig 1, compare I and A). These phenotypes are consistent with changes in the mxp expression pattern associated with the mxp^Dch-3 allele (see below).

mxp^Stb (BEEMAN et al. 1989 ) was recovered from an EMS mutagenesis and causes shortening of the antennae as well as slight shortening of the legs in adult heterozygotes. The larval antennae and legs do not appear to be affected. No homozygous class can be differentiated among progeny of an mxp^Stb heterozygous self-cross, suggesting that either these individuals die early in embryogenesis or mxp^Stb has no mxp recessive LOF phenotype. mxp^Stb/mxp^- larvae, however, exhibit transformations of maxillary and to a lesser extent labial palps to legs (data not shown), indicating that mxp^Stb is associated with partial LOF. Among 531 progeny of an outcross of mxp^Stb/+ males, only one mxp^Stb male was recovered. However, both male and female mxp^Stb individuals are obtained from a balanced stock. Thus, mxp^Stb behaves as an X-linked gene and so is apparently associated with an LG2 to X translocation.

mxp^Dch-1 (SOKOLOFF 1982 ) is a -ray-induced allele that causes pseudolinkage between LG2 and LG9 (BEEMAN et al. 1986 ). Homozygotes die about the time of the first larval molt and show moderate maxillary, but not labial, transformations to leg. The transformed maxillary appendages usually have both a distal spike (as seen in null heterozygotes) and a tarsal claw (Fig 1, compare J to A). The labial palps of mxp^Dch-1 homozygotes often have a distal spike as well. The legs and antennae of homozygous and heterozygous larvae appear normal, despite the presence of short legs and antennae in adult heterozygotes.

mxp^Stbd was induced by -irradiation (BEEMAN et al. 1989 ). Adult heterozygotes have shortened antennae and slightly shortened legs. mxp^Stbd homozygous larvae show moderate maxillary and weak labial transformations to leg (data not shown), but no apparent effect on larval antennae or legs.

GOF mutations that complement the adult phenotype of mxp¹: Three mutations that map to the HOMC and exhibit dominant antennal effects, Antennapalpus (Apl), Spatulate (Spa), and Stumpy (Stm), were predicted by BEEMAN et al. 1989 to be GOF mxp alleles. Apl and Spa, but not Stm, complement the larval lethality of mxp nulls. All three mutations were reported to complement the recessive adult phenotype of mxp¹ (BEEMAN et al. 1989 ). Reversion experiments indicate that Stm is an mxp allele. Two events that completely reverted the dominant Stm phenotype were recovered among 31,500 irradiated chromosomes. Each reversion (StmR1 and StmR6) fails to complement the adult phenotype of mxp¹ and results in lethality when heterozygous with an mxp null. The reversions do not appear to affect multiple loci, since homozygous larvae display only the mxp null phenotype (data not shown). Thus, we will hereafter refer to Stm as mxp^Stm. mxp^Stm was identified in an EMS mutagenesis, but reduces the frequency of crossing-over in the vicinity of the HOMC, suggesting it may be a chromosomal rearrangement. We show below that mxp^Stm is associated with ectopic mxp expression, which is eliminated by reversion.

RNA interference:
RNA-mediated interference (RNAi), a technique first used in the nematode Caenorhabditis elegans, has been adapted recently for use in Drosophila and Tribolium (KENNERDELL and CARTHEW 1998 ; BROWN et al. 1999 ; MISQUITTA and PATERSON 1999 ). dsRNA corresponding to the 3' end of the mxp cDNA was injected into wild-type Tribolium embryos. Of 103 hatching larvae, 100 exhibited transformations of the maxillary and labial palps to legs similar to those seen in mxp null larvae (Fig 1, compare B and K), indicating that mxp function had been severely reduced, if not eliminated. Injection of a lower concentration (approximately eightfold less) of an mxp dsRNA that included the homeobox produced less severe phenotypes, similar to those seen in mxp hypomorphs (Fig 1, compare L to D and I). The presence of the homeobox did not appear to result in crossreactivity with other homeobox-containing genes. The phenocopies of mxp mutant phenotypes produced by RNAi verify the conclusion of BROWN et al. 1999 that RNAi in Tribolium should be useful for predicting the LOF phenotypes of genes for which no mutants exist.

mxp expression in wild-type embryos:
Expression of mxp was examined via in situ hybridization using a digoxygenin-labeled antisense riboprobe containing the mxp homeobox (SHIPPY et al. 2000 ). In most respects, the expression pattern is quite similar to that observed for Pb protein (PULTZ et al. 1988 ). Neither Pb nor mxp is expressed before or during the blastoderm stage. In Drosophila, pb transcripts are detected on developmental Northerns at 2–4 hr, which corresponds with the stages of gastrulation and germband elongation. mxp expression in Tribolium also appears prior to morphological signs of segmentation, as the newly formed germ rudiment begins to elongate. Tribolium embryogenesis is quite different from that of Drosophila, in that the segments form one at a time rather than virtually simultaneously (BROWN et al. 1994A ). The visible formation of each segment is presaged by expression of the Tribolium Engrailed homolog (TcEn) in what will become the posterior compartment of that segment. Thus, TcEn serves as a useful indicator of embryonic stage, as well as providing a framework in which to interpret other expression patterns. mxp transcripts are first detected in a small group of mesodermal cells at the ventral midline of the mandibular segment in embryos with four to five Engrailed stripes (Fig 2A). As the embryo develops, expression in the mandibular segment broadens into two clusters (Fig 2B). By the time nine Engrailed stripes have formed, staining in the mandibular mesoderm is quite intense, and weak transient expression appears at the ventral midline of the maxillary segment (Fig 2C). As germband elongation continues, mxp expression intensifies in the mesoderm of the developing mandibular appendages. At this time, faint mxp expression is detected in the maxillary and labial limb buds (Fig 2D). By the time segmentation is complete, strong mxp expression is detected in both ectoderm and mesoderm of the maxillary and labial limb buds (Fig 2E and Fig F). Faint expression is still detected in the mesoderm of the mandibular limbs. As germband retraction begins, mxp expression is detected in two clusters of cells flanking the ventral midline of the intercalary segment (Fig 2G). These cells lie below the ventral surface of the embryo and are most likely part of the developing central nervous system (CNS). Staining in the maxillary and labial appendages intensifies still further as the labial palps assume their larval position nested between the bifurcated maxillary appendages (Fig 2H). mxp expression is visible in both the telopodites and endites of the maxillary appendages. At this stage, mxp is also expressed in a subset of cells in the ganglia of each segment from the maxillary segment through the posterior tip of the embryo.

View larger version (120K): In this window In a new window Download PPT slide   Figure 2. Wild-type mxp expression pattern during embryogenesis. Embryos in A–F are double-stained with mxp riboprobe (purple) and a crossreacting Engrailed (En) antibody (brown). The embryos in G and H were not stained with En antibody. An arrow points to the mandibular En stripe in A–F and its approximate position in G. All views are of the ventral surface of an embryo with anterior to the left. (A) mxp is first expressed in the mandibular mesoderm. The mandibular (Mn), maxillary (Mx), and labial (L) segments are denoted by brackets. (B) The mandibular mesoderm expression resolves into two distinct clusters. (C) Faint expression is detected in the maxillary segment. (D) Expression in the maxillary and labial segments becomes restricted to the developing limb buds. The staining in the posterior region is nonspecific. (E and F) mxp expression intensifies in the maxillary and labial appendages as they lengthen. (G) Two clusters of cells beneath the surface of the intercalary (Ic) segment begin to express mxp. (H) During germband shortening, mxp is expressed very strongly in the maxillary and labial appendages and in a segmentally repeated pattern in the CNS.

The epidermal expression pattern of mxp is quite similar to that of Pb if the maxillary and labial lobes of Drosophila are considered equivalent to the maxillary and labial appendages of Tribolium embryos.

mxp expression in mutant embryos:
We have also analyzed embryonic mxp expression in a number of mutant strains (see Table 2 for summary of results). mxp/balancer heterozygotes were mated to wild-type beetles. Heterozygous progeny, recognizable at the adult stage by the absence of the marked balancer chromosome (and in some cases by the presence of an mxp allele-specific dominant phenotype), were either outcrossed again to generate one-half wild-type and one-half heterozygous embryos, or mated inter se to generate one-quarter wild types, one-half heterozygotes, and one-quarter homozygotes. Embryos from both crosses were subjected to in situ hybridization with antisense mxp riboprobe. Several mxp mutant alleles were associated with changes in the mxp embryonic expression pattern. In the outcross, mutant heterozygotes were identified by abnormal expression patterns, whereas mutant homozygotes from the self-cross were recognized by patterns not observed in the outcross progeny.

mxp^Dch-4: The early mxp expression pattern in the gnathal segments of mxp^Dch-4 heterozygotes (data not shown) appears similar to that seen in wild-type embryos. After germband extension, ectopic mxp expression appears in the tips of the developing antennae and thoracic limbs (Fig 3A). mxp expression in the mandibular segment is noticeably reduced relative to wild type. Later, expression in the labial, maxillary, and antennal segments intensifies, but expression in the thoracic appendages fades slightly (Fig 3B). CNS staining is faint relative to wild type (data not shown). Since the homozygotes appear to lack all CNS expression (see below), we interpret this fainter staining in heterozygotes to reflect expression only from the wild-type mxp allele.

View larger version (113K): In this window In a new window Download PPT slide   Figure 3. mxp expression in mxpDch-4 and mxpStm embryos. All images are ventral views with anterior to the left. (An) Antennal; (Ic) intercalary; (Mx) maxillary; (L) labial; (T1, T2, T3) first, second, and third thoracic segments, respectively. (A) In an mxpDch-4 heterozygote, faint mxp expression is visible in the normal domain. In addition, strong expression is visible in the tips of the antennal, maxillary, labial, and thoracic appendages. (B) During germband retraction, mxp expression remains strong in the antennal, maxillary, and labial appendages, but fades in the thoracic appendages. (C) In an mxpDch-4 homozygote, all normal mxp expression is absent (compare to Fig 2G). Expression is seen, however, in the tips of the antennae, maxillary and labial palps, and legs. (D) mxpStm is associated with ectopic mxp expression in the antennae. Expression in heterozygotes and homozygotes is apparently identical.

In mxp^Dch-4 homozygotes, mxp expression is never detected in the mandibular or intercalary segments (Fig 3C) and is also absent from the CNS (data not shown). In fact, there is no early mxp expression in any gnathal segment. However, mxp expression in the tips of the maxillary and labial appendages appears coincident with ectopic expression in the tips of the thoracic limbs and antennae. Although the legs and antennae of mxp^Dch-4 heterozygous and homozygous larvae appear normal, ectopic expression in the distal portions of thoracic legs corresponds to the transformation of that portion of the adult leg to palp in homozygous adult escapers. The expression of mxp in the tips of head, gnathal, and thoracic appendages may represent novel regulation of the mxp gene, even within the normal domain (i.e., mxp expression restricted to tips of maxillary and labial appendages). The very weak maxillary and labial transformations in mxp^Dch-4 homozygotes (see above and Fig 1F) indicate that this tip-specific expression is nearly sufficient for proper gnathal development. Interestingly, it is the distal tips of the palps (where mxp is expressed) that are visibly transformed.

mxp^Dch-3: In mxp^Dch-3 heterozygotes, ectopic expression of mxp in the first thoracic segment (T1) appears simultaneously with normal expression in the mandibular mesoderm (Fig 4A). As the germband elongates, additional ectopic expression appears at the ventral midline in T2 and T3 (Fig 4C). During development of the thoracic legs, mxp expression remains strong in T1, but is greatly reduced in T2 and T3 (Fig 4E). Correspondingly, the larval T1 legs are shortened (see Fig 1H). mxp expression in the gnathal segments appears similar to, but fainter than, that in wild type (compare Fig 2 and Fig 4). Likewise, the pattern of mxp expression in the CNS (data not shown) is similar to, but fainter than, that in wild-type embryos. As with mxp^Dch-4/+, reduced signal in the normal domain apparently reflects expression from the single wild-type allele.

View larger version (93K): In this window In a new window Download PPT slide   Figure 4. mxp expression in mxpDch-3 embryos. A, C, and E are mxpDch-3/+ embryos; B, D, and F are mxpDch-3/mxpDch-3 embryos. All embryos are stained for mxp (purple), and all but A are also stained for En (brown). The mandibular (Mn), maxillary (Mx), labial (L), and first thoracic (T1) segments are denoted in B by brackets. An arrow points to the position of the mandibular En stripe in each embryo (position in A is approximate). All embryos are viewed from the ventral side with anterior to the left. (A) In mxpDch-3 heterozygotes, ectopic mxp expression in T1 appears just as expression in the mandibular mesoderm arises. (B) An mxpDch-3 homozygous embryo of about the same age as A lacks mandibular mesoderm expression. (C) Later in embryogenesis, ectopic mxp expression is also seen in T2 and T3 of an mxpDch-3 heterozygote. (D) Faint expression begins to appear in the labial segment of an mxpDch-3 homozygote as ectopic expression becomes visible in T2 and T3. (E) Expression in the normal mxp domain is fainter in an mxpDch-3 heterozygote than in wild-type embryos (see Fig 2E), except in the labial segment. (F) In an mxpDch-3 homozygote, mxp is expressed strongly in the labial segment and T1 and faintly in T2 and T3. It is not clear whether the observed labial expression represents wild-type or novel expression.

Although mxp^Dch-3 homozygotes die before hatching (see above), we could recognize this class among developing embryos by abnormalities of the normal mxp expression pattern. The pattern of mxp expression in the thoracic segments of mxp^Dch-3 homozygotes is similar to that observed in heterozygotes (Fig 4B and Fig D), but the mandibular and maxillary aspects of mxp expression are absent (Fig 4F). There is no evidence of mxp expression in the central nervous system. Although mxp is expressed in the labial appendages of mxp^Dch-3 homozygous embryos, the labial palps of mxp^Dch-3/mxp^- individuals are partially transformed to leg (see above and Fig 1I).

mxp^Stm: Homozygous mxp^Stm embryos were obtained from a homozygous stock, and heterozygous embryos were generated by an outcross to wild type. Homozygotes and heterozygotes were indistinguishable and showed ectopic expression in the antennae (Fig 3D). All other aspects of the expression pattern appeared normal. Since the original mxp^Stm mutation is homozygous lethal, it is possible that the homozygous mxp^Stm stock acquired a wild-type allele of mxp while retaining the mutant allele. The normal expression in the wild-type mxp domain might reflect the presence of this additional copy.

mxp^StmR1: mxp^StmR1 is a revertant of mxp^Stm that lacks the gain-of-function antennal transformation and acts as a typical null mxp allele (see above). No ectopic antennal expression is seen in embryos from an mxp^StmR1/A^Es self-cross, and half of the embryos show no mxp expression. A^Es homozygotes (one-quarter of the progeny) die early in embryogenesis before mxp is expressed. It seems likely that the mxp^StmR1 lesion eliminates mxp expression and that the remaining unstained embryos are mxp^StmR1 homozygotes.

mxp alleles not associated with changes in embryonic mxp expression:
A number of mxp alleles for which mxp expression was assayed showed no changes in expression pattern. The GOF/LOF alleles mxp^Stb, mxp^Ng, and mxp^Dch-1, as well as the putative GOF allele, Apl, showed no abnormalities in mxp expression in either heterozygotes or homozygotes. The lack of ectopic embryonic expression from these alleles is not particularly surprising since their dominant effects are limited to adult structures. Most of these alleles, however, are also associated with embryonic LOF effects (see above). mxp^Stb and mxp^Dch-1 cause only partial mouthpart to leg transformations, so levels of mxp expression in the normal domain may be only moderately reduced. mxp^Ng apparently lacks all normal mxp function, but is capable of causing dominant head capsule abnormalities. It is feasible that a nonfunctional protein is produced both in the normal domain and ectopically (in the adult). Such a protein might interfere with the action of endogenous proteins, in a manner analogous to the reported dominant negative effect of nonfunctional Pb on Sex combs reduced (PERCIVAL-SMITH et al. 1997 ). The null allele mxp¹⁹, which is predicted to encode a truncated, nonfunctional Mxp protein (SHIPPY et al. 2000 ), appears to retain normal mxp expression, since virtually all offspring from a self-cross display wild-type expression (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have classified available mxp alleles by classical genetic methods. Null mxp alleles cause larval lethality with strong transformation of maxillary and labial palps to legs, consistent with the hypothesis that an ancestral pb-like gene had an embryonic function. Hypomorphic mxp alleles cause incomplete transformations of larval (and sometimes adult) mouthparts to leg. In contrast, the effects of hypomorphic pb alleles are visible only in adult flies, where they cause transformation of the distal portion of the labial palps to antennal aristae. The weakest mxp phenotype, seen in many null heterozygotes and in some homozygous hypomorphs, is the presence of an abnormally long spike on an otherwise normal maxillary palp. The significance of this spike is unclear, but we have seen similar structures on the maxillary and labial palps of embryos lacking TcDfd (S. J. BROWN, unpublished results). Since TcDfd is not expressed in the labial palps, it is possible that perturbation of normal head development results in an unspecialized sensory structure.

The mxp LOF phenotypes are consistent with the wild-type mxp expression pattern, since the epidermal sites of embryonic mxp expression are the maxillary and labial appendages. Although mxp is expressed in both the telopodite and endite of the maxillary appendages, the maxillary endites are unaffected in mxp mutants (see Fig 1B). In contrast, the maxillary appendages of embryos homozygous for a null allele of TcDfd lack endites, but have relatively normal telopodites (BROWN et al. 1999 ). Thus, mxp and TcDfd apparently function in distinct domains of the embryonic maxillary palps. A strictly additive model based on these data predicts that the maxillary appendages of a Tribolium embryo lacking both mxp and TcDfd would be transformed toward legs (as in mxp nulls) but would lack endites. However, according to the current paradigm, homeotic gene function is necessary in appendages to repress homothorax and thus prevent antennal identity. Embryos lacking both mxp and TcDfd would have no homeotic genes expressed in the maxillary appendages, and thus the maxillary palps might be transformed to antennae. We are currently performing RNAi experiments to test these predictions.

In Drosophila embryos, the maxillary segment has no true appendage, but the maxillary sense organ is believed to be a vestige of the telopodite (JURGENS et al. 1986 ). Interestingly, the maxillary sense organ is present in Dfd null mutants (MERRILL et al. 1987 ; REGULSKI et al. 1987 ), suggesting that the Dfd-independent development of the maxillary telopodite evolved in a common ancestor to beetles and flies. It seems probable that, in that same common ancestor, a pb-like gene specified the identity of the maxillary telopodite. However, all larval structures, including the maxillary sense organ, are apparently normal in pb null Drosophila larvae.

It is intriguing that the embryonic expression patterns of mxp and Pb are so similar, since pb has no apparent function in Drosophila embryos. The similar expression patterns could be interpreted to suggest that the two genes share a common regulatory mechanism. Studies of pb regulation (KAPOUN and KAUFMAN 1995 ) using a pb minigene and a lacZ reporter gene have revealed the existence of both positive and negative regulatory elements. Region-specific enhancers of pb expression are located within its second intron. The region upstream of pb is not required for its normal spatial expression pattern. However, removal of this region from a pb minigene driven by the pb basal promoter results in ectopic expression in the eye-antennal and leg discs. The only observable effect of this ectopic expression is a thickening of the antennal aristae. In contrast, when the pb minigene is driven with the Hsp70 promoter (without heat induction) the adult antennae are transformed toward maxillae (CRIBBS et al. 1995 ). Similarly, pb expression driven by the decapentaplegic promoter was shown to transform the adult legs toward a labial identity (APLIN and KAUFMAN 1997 ).

GOF alleles of mxp, like ectopic expression of pb driven by a foreign promoter, transform adult legs and/or antennae toward palps (BEEMAN et al. 1989 ). Somewhat surprisingly, since most of the GOF effects are limited to adult structures, we have, in some cases, correlated transformations with ectopic embryonic (and presumably adult) mxp expression. Of these mutations, only mxp^Dch-3 (which produces very strong T1 expression and a corresponding shortening of the embryonic prothoracic legs) is associated with embryonic GOF effects, suggesting that embryonic limbs may be less sensitive to perturbation by ectopic mxp expression. The frequent occurrence of mxp GOF alleles affecting the antennae and legs suggests that mxp expression, like that of pb, is normally negatively regulated by upstream antennal and thoracic silencers. Why are GOF mutations, apparently resulting from separation of these elements from the transcription unit, so common for mxp when they are never found for pb? Since a pb minigene lacking the upstream silencer-containing region can, under control of certain promoters, cause transformation of antennae and legs toward palps, the simplest explanation is that the level of pb expression driven by the endogenous pb promoter is insufficient to produce recognizable transformations. It is also possible that GOF mutations of pb are usually embryonic lethal, since heat-shock-induced ubiquitous expression of pb during early embryogenesis inhibited germband retraction and head involution (PERCIVAL-SMITH et al. 1997 ).

It is interesting to note that while some mutations associated with adult GOF effects cause ectopic embryonic mxp expression, other mutations with similar adult phenotypes do not. Perhaps independent silencer elements exist for control of embryonic and adult expression. Alternatively, one class of mutants may result from juxtaposition of novel enhancers.

A precise model of mxp regulation cannot yet be built. Numerous potential explanations exist for the modified expression patterns observed in mxp mutants. Since the molecular lesions associated with these mutations have not yet been characterized, it is possible that complex rearrangements have occurred. Furthermore, it is not clear whether loss of mxp expression in portions of the normal domain is caused by introducing upstream silencers or removing endogenous enhancers. Likewise, ectopic expression might result from either removal of silencers or juxtaposition of novel enhancers. For example, the mxp expression pattern in mxp^Dch-3 homozygotes is reminiscent of the expression pattern of the Tribolium Sex combs reduced ortholog Cephalothorax (Cx; our unpublished results), raising the possibility that Cx regulatory elements have been juxtaposed with the mxp transcription unit. In mxp^Dch-4 homozygotes, where expression is seen only in the tips of antennal, gnathal, and trunk appendages, the expression pattern may represent a completely novel pattern. Conversely, this pattern could result from removal of silencers, and a reduction in the level of expression in the normal domain. Consistent with the latter scenario, KAPOUN and KAUFMAN 1995 proposed that the region upstream of pb contains sequences that upregulate pb expression throughout its normal domain.

Our analysis of mxp mutant alleles has raised numerous questions about mxp regulation. Answering these questions will require several approaches. Identification of molecular lesions associated with particular mxp alleles may be useful in broadly defining important regulatory regions. Reporter gene assays (similar to those performed for pb) should determine the location of particular types of regulatory elements. With the recent development of a system for transforming Tribolium (BERGHAMMER et al. 1999 ), these experiments are now feasible. Once regulatory regions are identified, sequence comparison between mxp and its orthologs might reveal conserved sequences, the functional significance of which can be tested by targeted mutagenesis.

	FOOTNOTES

¹ Present address: Department of Pediatrics-Hematology/Oncology, Washington University, St. Louis, MO 63130.

	ACKNOWLEDGMENTS

We thank Marcé Lorenzen, Kathryn Hummels, and Sandra Koo for technical assistance, and Mark DeCamillis for allowing the Lucifer Revertant 1 mutation to be used prior to publication. This is contribution number 00-286J from the Kansas Agricultural Experiment Station. This work was supported by National Science Foundation grant MCB-9630179 and National Institutes of Health grant HD29594 to R.E.D., R.W.B., and S.J.B. T.D.S. was supported by a Virology and Tumor Biology predoctoral training grant from the National Institutes of Health.

Manuscript received December 13, 1999; Accepted for publication February 22, 2000.

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