BACKGROUND
the insect head is composed of several segments and a non-segmental anterior region.
however, the exact segmental composition of the insect head has long been a matter for debate  <cit> .
the posterior gnathocephalon is made up of the mandibular, maxillary and labial segments that each bear a pair of appendages specialized for feeding.
the anterior procephalon consists of anterior non-segmental parts and an antennal segment, which is separated from the mandibular segment by an appendage-free segment , whose development in insects is significantly delayed, as well as reduced in size.
the genetic mechanisms of head segmentation were first examined in the dipteran fruit fly drosophila melanogaster <cit> .
its gnathal segments are patterned by the trunk segmentation gene cascade, involving maternal, gap, pair-rule and segment polarity genes  <cit> , while the patterning of the procephalic segments follows a different paradigm  <cit> .
whilst segment polarity genes  are involved in establishing these segments, pair-rule genes are not  <cit> .
four drosophila head gap genes, orthodenticle , empty spiracles , buttonhead  and sloppy paired are expressed in broad overlapping domains in the developing anterior head  <cit> .
mutation of these genes leads to classic ‘gap phenotypes’ - the loss of all the adjacent segments covered by their domains of expression  <cit> .
however, mis-expression studies have shown that only otd affects segment polarity gene expression when expressed in ectopic domains, and only ems, with the help of btd, appears to confer identity to head segments  <cit> .
indeed, second order regulators have been identified that operate at levels in between the head gap genes and segment polarity genes: i.e.
collier and cap ‘n’ collar <cit> .
drosophila exhibits an evolutionary derived mode of head development, in which the larval head is greatly reduced and undergoes ‘head involution’ during which head regions are folded into the body cavity  <cit> .
this situation is far from typical for insects and moreover, the reduced and experimentally inaccessible drosophila larval head has limited the comprehensive identification and analysis of insect head development genes for technical reasons  <cit> .
in recent years the red flour beetle tribolium castaneum has emerged as a powerful genetic insect model system  <cit>  and offers an opportunity to study the genetic and cellular mechanisms underlying the development of a more insect-typical head  <cit> .
as in drosophila, the tribolium gnathal segments appear to be patterned using similar mechanisms to those operating in the trunk, including a central role for pair-rule gene homologues  <cit> .
in the anterior head, second order regulators and the segment polarity genes might be relatively well conserved between drosophila and tribolium <cit> .
however, clear differences have been identified at the level of the head gap genes, and the maternally provided anterior protein gradients that establish their expression domains  <cit> .
for example, while the tribolium homologue of orthodenticle  apparently plays a broadly conserved role as a gap gene during head segmentation in tribolium, it appears to be much more involved in axis formation than its drosophila orthologue  <cit> .
the expression of the tribolium homologues of empty spiracles and buttonhead  is limited to single segment wide domains instead of large overlapping domains in the blastodermal head anlagen.
tc-ems is required to form parts of the antennal segment only and knockdown of tc-btd does not lead to a head cuticle phenotype at all  <cit> .
this raised the question of what genes might control development of these head regions in tribolium.
work by cerny et al.
<cit>  suggests that the answer to this question is, at least in part, the single tribolium homologue of the drosophila genes knirps and knirps-related.
the drosophila genes knirps and knirps-related encode steroid hormone receptor-like transcription factors  <cit> .
ancestrally, the insect knirps family consisted of two genes, eagle and knirps-related, while knirps arose via a recent gene duplication of the knirps-related gene in the higher diptera  <cit> .
at the blastoderm stage, knirps and knirps-related are expressed in almost identical anterior and posterior domains  <cit> .
drosophila knirps acts as a canonical gap gene during trunk segmentation  <cit> .
in contrast, the anterior mandibular expression domain is not required for head segmentation, since segment polarity gene  expression in the head is not affected in embryos that lack both paralogues  <cit>  while a loss of the stomatogastric nervous system is observed  <cit> .
cerny et al.
<cit>  have shown that tc-knirps , the single tribolium homologue of the drosophila knirps-family paralogues, is also expressed in anterior and posterior domains during early development  <cit> .
however, the tc-kni posterior domain is shifted anteriorly relative to its position in drosophila, and knockdown of tc-kni does not lead to a canonical gap phenotype in the trunk, but rather minor defects in the posterior abdomen.
the anterior expression domain of tc-kni is largely conserved.
in contrast to drosophila, the anterior domain does play an essential role in head patterning: knockdown of tc-kni leads to loss of both antennae and mandibles  <cit> .
cerny et al.
<cit>  found early loss of tc-wg expression in the antennal segment in tc-kni rnai while the mandibular domain of tc-wg expression disappeared at a later stage.
further, they showed that tc-kni is not needed for correct tc-wg expression in the intercalary segment.
finally, light abnormalities in the maturation and maintenance of the first pair-rule stripe of tc-eve expression were observed, where the distance between the first segmental tc-eve stripe  and the ocular tc-wg expression domain was reduced in tc-kni rnai blastoderms  <cit> .
it has remained unclear, however, whether antennal and mandibular segments are deleted completely and whether the intercalary segment is affected.
in this study we examined the effect of knocking down tc-kni on a comprehensive set of genes that mark sub regions of the antennal, intercalary and/or mandibular segments.
we show that tc-kni is required for correctly specifying only posterior regions of antennal and mandibular head segments .
interestingly, tc-kni is essential for the initial specification of the antennal parasegmental boundary, while it is required only for the maintenance of the mandibular parasegmental boundary.
the intercalary segment does not appear to be affected.
unexpectedly, we find that the trunk pair-rule gene tc-even-skipped is required to set the posterior boundary of the mandibular tc-kni expression.
unlike most rnai studies, we have good evidence that we were investigating a null situation due to our finding that antennaless, a tribolium mutant arising from an ems screen  <cit> , is a likely regulatory null-mutation of tc-kni.
taken together, we show that tc-kni is not a head gap gene, since its mutation does not lead to the complete deletion of several adjacent segments.
further, we provide a model, for how head and trunk patterning mechanisms cooperate to pattern the mandibular segment in tribolium, and how these interactions differ between drosophila and tribolium.
RESULTS
early tc-kni expression prefigures the appearance of the anterior head anlagen
first, we studied in more detail the dynamics of anterior tc-kni expression because previous studies had focused on posterior aspects  <cit> .
zygotic tc-kni expression in the syncytial blastoderm begins as a broad domain covering most of the anterior half of the blastoderm, only absent from a small region around the anterior pole .
tc-kni expression then retreats from the anterior pole, but much more so on the dorsal side of the egg, clearing from regions that will later become the extra-embryonic serosa   <cit> .
tc-kni expression is maintained in the anterior/ventral regions that will become head tissue  <cit>  .
these early shifts in expression occur before embryonic and extra-embryonic tissue can be distinguished at the level of nuclear morphology .
when this distinction becomes apparent , the uniform anterior tc-kni expression domain splits into two anterior lateral domains at the boundary between each head lobe and the abutting extra-embryonic tissue , and a more posterior stripe marking the future anterior compartment of the mandibular segment  .
these domains are maintained through subsequent blastoderm  and early germband  stages, before fading  and then disappearing completely during mid-germband elongation stages.
hence, the early uniform domain of tc-kni expression  encompasses the entire future anterior head anlagen; i.e.
everything anterior to the parasegment 0/ <dig> boundary.
antennaless is likely a regulatory mutation at the tc-kni locus
in most rnai experiments outside drosophila, it remains unclear to what extent a genetic null-situation is phenocopied.
we aimed at determining the null situation by studying a genetic mutant.
antennaless is a homozygous lethal mutation that was recovered from an ems mutagenesis screen  <cit> .
the cuticular phenotypes of antennaless mutant larvae are highly reminiscent of tc-kni rnai phenotypes   <cit> .
larvae of the antennaless mutant lack antennae and mandibles, and occasionally also display minor abdominal defects, very similar to those previously reported for weak tc-knirnai larvae .
in order to check for defects in dorsal and lateral head tissues which are derived from pre-antennal and intercalary regions, respectively  <cit> , we scored both sides of  <dig> antennaless larval cuticles for the head bristle pattern.
all bristles were present, albeit shifted somewhat compared to the wildtype condition in these regions .
this is consistent with pre-antennal regions and much of the intercalary segment not being affected in the mutant  <cit> .
the development of both mandibles and antennae was more severely affected at a higher temperature in antennaless mutants.
a larval cuticle was scored as an antennaless mutant if either the mandibles or antennae, or both, were reduced or absent.
most of the antennaless mutant larvae  lacked both the antennae and mandibles at 25°c.
this increased to 75%  at 32°c.
in a few cases, rudiments of mandibles were found .
in other cases, antennae or antennal rudiments were found .
in contrast, and against the general observation that phenotypes tend to be more penetrant at higher temperatures, the frequency of abdominal defects was found to be higher at 25°c than at 32°c.
at 25°c,  <dig> %  of phenotypic larvae displayed defects within segments a5-a <dig>  compared to only 16%  at 32°c.
an inverse sensitivity with respect to temperature was also observed for defects in the urogomphi, dorsal outgrowths of the ninth segment.
at 25°c this structure was defective in 79% of the phenotypic larvae examined , compared to 44% at 32°c .
in offspring from nine independent pairs of heterozygous parents, the antennaless cuticle phenotype is found in 25%  and 27%  of larvae at 25°c and 32°c respectively, levels consistent with a highly penetrant homozygous lethal mutation.
a significant proportion of mutant larvae were able to hatch despite their lack of antennae and mandibles; 45% at 25°c  compared to only 17% at 32°c .
the vast majority of these larvae died at the l <dig> stage.
only very rarely did individuals reach the l <dig> stage.
when corrected for a background reduction in hatching rate associated with this shift in temperature , these data reveal a 19% increase in sensitivity to temperature with regards to hatching in the antennaless mutant background.
in our tc-kni rnai experiments head defects were also found to be more severe at 32°c compared to 25°c .
the severity of head defects decreased over time post-injection as expected for parental rnai experiments  <cit>  .
however, as for antennaless, we found inverse temperature sensitivity with respect to defects in segments a5-a <dig> following tc-knirnai .
the frequency of abdominal defects also increased with time post-injection from about 20%  at day eight post-injection, to 37%  eleven days post-injection, and to 56%  thirty-one days post-injection, before dropping again .
this is not in line with the usual observation that in rnai experiments phenotypic strength decreases over time and indicates a complex relationship between knockdown and phenotype, which we do not fully understand.
since the antennaless cuticle defects described above are almost identical to the tc-knirnai phenotypes both in terms of physical phenotype and sensitivity to temperature, we suspected tc-kni as the gene affected by the mutation.
we therefore independently sequenced the three tc-kni exons from the genomic dna of two first instar larvae that had been identified as homozygous mutant by their cuticle phenotype.
we found that the coding sequence of tc-kni is not altered in mutant beetles .
in order to test the hypothesis that antennaless is a regulatory mutation at the tc-kni locus, we carried out in situ hybridization on embryos from heterozygous mutant parents with a mix of probes targeting tc-kni and tc-caudal as positive control.
we failed to detect tc-kni expression in 15% of the offspring examined , whereas tc-caudal was well stained in the same colour reaction in all cases .
this is consistent with embryos homozygous for the antennaless allele not expressing tc-kni at levels detectable via in situ hybridization.
taken together, our results are consistent with the hypothesis that antennaless is a regulatory mutation of tc-kni, and as such we now refer to it as tc-kniatl.
however, we cannot rule out that antennaless is a mutation in a gene that acts rather exclusively upstream of, or is a required interaction partner of, tc-kni.
further studies will be necessary to confirm our hypothesis by identifying the cis-regulatory regions that are affected by the mutation.
the tc-kni null phenotype is revealed by both tc-kniatl and tc-knirnai
the lack of tc-kni expression in homozygous tc-kniatl embryos implies that tc-kni function is strongly reduced in the mutant.
however, it remains possible that some residual function remains.
in order to test whether tc-kniatl constitutes a complete null phenotype for tc-kni, we performed tc-kni rnai in the tc-kniatl background, assuming that the added effects should not lead to stronger phenotypes if tc-kniatl is a null mutant.
the frequency of the tc-kni phenotype in the pooled offspring from  <dig> independent pairs of tc-kniatl heterozygous animals was 11%  at 25°c and 17%  at 32°c.
as expected, after injecting the same females with tc-kni dsrna, the frequency of the tc-kni phenotype strongly increased, to 97%  and 81% .
crucially, we did not find evidence for an increase in the severity of the cuticle phenotype: i.e.
there were no larvae with phenotypes more severe than those seen in tc-kniatl or tc-knirnai alone.
hence, unlike in most rnai experiments, we can be rather confident that the most severe rnai phenotypes we observe fully phenocopy a null situation.
therefore, we use results from both mutant and rnai embryos for this work.
tc-kni is differentially required for specifying antennal, intercalary and mandibular parasegment boundaries
the cuticle phenotypes reported by cerny et al.
<cit>  raised the question of whether entire segments are deleted in tc-kni depleted embryos, or only the posterior parts that give rise to the appendages.
these depend on parasegmental boundary formation via the segment polarity genes tc-engrailed , tc-hedgehog  and tc-wingless   <cit> .
therefore, we stained tc-en and tc-hh in tc-knirnai embryos as markers for the posterior segment compartment.
in the case of tc-en, embryos were co-stained for tc-dfd to help distinguish disrupted mandibular tc-en expression domains , from intercalary tc-en expression domains  .
we find that both tc-en  and tc-hh  expression is absent in the antennal segment in tc-knirnai embryos throughout embryogenesis .
occasionally, tc-en and tc-hh expression associated with structures at the base of the antenna remains in later germband embryos, despite the clear failure of the antenna to form .
the mandibular stripe of tc-en expression is initiated in early tc-knirnai germband embryos, albeit abnormally; the stripe of expression is broken and in extreme cases only patches of expression are seen .
in older tc-knirnai germband embryos, tc-en expression associated with the mandibular segment is often missing completely , suggesting a failure to properly maintain tc-en expression in this segment in strong knockdowns of tc-kni expression.
similarly, tc-hh expression in the mandibular segment is greatly reduced in young tc-knirnai embryos, but a thin broken stripe is still detected medially in early germbands .
later, this tc-hh expression disappears in contrast to wildtype .
note that tc-en mandibular expression appears to be more sensitive to the loss of tc-kni expression in lateral regions of the germband .
tc-en and tc-wg expression in the intercalary segment appears relatively late and at a quite variable time point during mid germband elongation in wildtype embryos.
whilst we cannot completely rule out minor disruptions of intercalary tc-en/tc-hh expression initiation and/or maintenance in tc-knirnai embryos, we do detect wildtype expression of these genes in late elongation  and early fully elongated tc-knirnai embryos .
in conclusion, the disruption of tc-kni function leads to the complete failure to initiate segment polarity gene expression in the antennal segment, failure to properly maintain segment polarity gene expression in the mandibular segment, and most likely has no effect on segment polarity gene expression in the intercalary segment  <cit> .
molecular markers show that tc-kni is not a canonical head gap gene
segment polarity genes mark a posterior portion of each segment.
therefore, we also asked whether the anterior parts of the segments are miss-specified and/or missing in tc-knirnai embryos by analyzing markers for the anterior regions of head segments.
tc-goosecoid  expression  <cit>  initially partially overlaps the ocular tc-wg domain and extends posterior to it .
later, this domain widens in lateral regions, forming a wedge shape domain that abuts the antennal tc-wg stripe as it appears  but later retracts from it .
thus, tc-gsc is a marker for posterior ocular and anterior antennal regions in early embryos but later predominantly marks ocular tissue.
in early tc-knirnai germband embryos, tc-gsc expression is down-regulated in posterior-lateral regions of the wedge shaped expression domain .
in older tc-knirnai embryos, the tc-gsc expression domain again closely resembles the wedge shape seen in wild-type embryos at these stages .
this indicates that the ocular parasegment boundary and anterior parts of the antennal segment are not greatly affected by loss of tc-kni, apart from some degree of lateral down-regulation of tc-gsc in early germ bands.
tc-lim <dig> is a similar marker for the posterior of the ocular segment and anterior compartment of the antennal segment  <cit> , albeit shifted slightly posteriorly relative to tc-gsc.
in early embryos, expression is partially overlapping the ocular tc-wg domain and extending posterior to it .
in elongating germbands, tc-lim <dig> expression forms a wedge shaped domain that covers all cells between the ocular and antennal tc-wg stripes .
in the tc-knirnai background, the posterior boundary of this domain is irregular and the domain is somewhat narrower indicating posterior reduction of the tc-lim marked tissues .
in wild-type elongating germbands, additional tc-lim <dig> segmental expression arises that laterally overlaps the segmental tc-wg domains in gnathal and thoracic segments .
at these stages, the ocular-antennal domain of tc-lim <dig> expression splits into a posterior domain that overlaps the antennal tc-wg stripe, and an anterior domain positioned between the ocular and the antennal tc-wg stripes .
expression in the ocular-antennal region remains also in tc-knirnai germbands, but the domain is not split as in wildtype  and appears to be fused with the mandibular domain of lateral tc-lim <dig> expression .
in extreme cases this domain is also fused to the maxillary tc-lim <dig> expression domain .
this confirms that anterior antennal tissue is properly specified while the parasegmental boundaries of the antennal and mandibular segments are not formed correctly.
tc-empty-spiracles  is expressed in a segmentally reiterated pattern during tribolium development  <cit>  in mediolaterally-restricted domains that lie anterior to the tc-wg stripe in each segment  <cit> .
tc-ems expression therefore predominantly marks the anterior portion of each segment.
in contrast to wild-type expression, the antennal and mandibular tc-ems segmental domains are fused in tc-knirnai embryos , as seen for tc-lim <dig>  this is consistent with defects being restricted to the posterior compartment of the antennal segment leading to the fusion of the antennal and mandibular tc-ems expression domains.
tc-sloppy-paired- <dig>  is also expressed in a segmentally reiterated pattern in the developing tribolium head .
tc-slp- <dig> expression domains overlap the tc-wg stripe in each head segment, but also extend further into the anterior compartment, as well as a little across the parasegmental boundary into the posterior compartment  <cit> .
tc-slp- <dig> expression therefore predominantly marks a posterior portion of the anterior segment compartment.
tc-slp- <dig> is expressed in a stripe in the antennal and each gnathal head segment in both tc-knirnai and tc-kniatl embryos , as in wild-type embryos .
however, the antennal stripe often appears to broaden , and the distance between the mandibular and the maxillary tc-slp- <dig> stripes is often decreased, completely fusing in lateral regions .
these data indicate that overall the tc-slp- <dig> stripes are not dramatically affected by tc-kni rnai.
however, the decreased distance between the mandibular and maxillary stripes could indicate incorrectly specified tissue in the intervening posterior compartment of the mandibular segment.
a normal complement of bristles and setae in lateral regions of tc-kniatl larval heads implies the retention of intercalary segment derived cuticle in tc-kni knockdown embryos .
since tc-labial  is expressed during embryogenesis throughout the presumptive intercalary segment  <cit> , and is required for its formation  <cit> , we used it as a molecular marker for the presence/absence of the intercalary segment.
in tc-kni rnai embryos , tc-lab expression appears prematurely but with similar dynamics as in wildtype .
the premature tc-lab expression domain first appears medially as a pair of dots on either side of the median mesoderm .
tc-lab expression then extends into more lateral and medial regions.
expression is not entirely wildtype, as it may form an unusual regular stripe without the typical median expansion , or it may be laterally reduced .
however, in all instances, the stripe was present indicating that the intercalary segment is present albeit its morphology appears not to be entirely unaffected by adjacent defects.
we also examined the expression of another marker for the intercalary segment in tc-knirnai embryos, the second order regulator tc-collier , also called tc-knot.
tc-col acts downstream of tc-lab and is required for wildtype expression of tc-en in the intercalary segment  <cit> .
aside from a slight delay in the initial appearance of the tc-col expression domain in early embryos  we found no evidence of strong disruption of tc-col expression in a tc-knirnai background .
besides, this staining confirmed the identity, and wildtype appearance, of the intercalary spots of tc-en in late elongating and early fully elongated embryos .
the drosophila head gap genes lead to the loss of entire adjacent segments but their tribolium orthologues do not fit this definition  <cit> .
similarly, taken together, our data  show that tc-kni cannot be considered a canonical head gap gene, because tc-kni function is only required for the posterior parts of the antennal and mandibular segments .
moreover, tc-kni is not required for the formation of the intercalary segment, which lies between the affected antennal and mandibular segments.
intercalary segment polarity gene expression is instead likely dependent on a conserved pathway involving tc-lab and tc-col <cit> .
overall, our data confirm that the regulatory networks underlying the establishment of the anterior head segments have diverged significantly between tribolium and drosophila, involving changing roles for knirps, buttonhead and empty spiracles homologues  <cit> .
the tc-kni phenotype is due to a failure to correctly specify cell fates
we decided to check whether missing head regions in tc-kni knockdown and mutant embryos were lost due to a failure to maintain already specified tissue - which might be indicated by high levels of cell death - or through the failure to specify cells to their correct developmental fate.
using tunel staining, we did not detect any apoptosis in either the blastoderm  or early germband stages  in tc-knirnai embryos.
in later germband stages there were a few apoptotic cells detectable in the head , but no more than in wild-type embryos, and there was no specific pattern of apoptosis that would indicate the loss of the tissues in question, namely the posterior portions of the antennal and mandibular segments .
thus, the lack of antennae and mandibles in tc-knirnai and tc-kniatl larvae is not due to tissue degeneration, but rather the failure to specify the respective segmental regions.
the posterior border of the anterior tc-kni expression domain is regulated by tc-even-skipped
in the tribolium blastoderm, expression of tc-kni and the pair rule genes occurs at the same time, opening the possibility tc-kni could be regulated by pair rule genes.
indeed, in tc-evernai blastoderm embryos, we found that the anterior domain of tc-kni expression expanded into more posterior regions .
in contrast, expression defects in this tc-kni mandibular domain were not seen in tc-oddrnai or tc-runrnai germbands .
this is consistent with the first pair-rule and/or segmental stripe of tc-eve expression being required to set the posterior boundary of mandibular tc-kni expression.
head and trunk segmentation gene networks cooperate to establish the mandibular segment in a different way in tribolium and drosophila
in drosophila, the anterior head gene regulatory network  and the gnathal/trunk regulatory gene network  cooperate to pattern aspects of the mandibular segment .
in this study we provide evidence that this is also true in tribolium.
both tc-eve and tc-kni rnai lead to the failure to properly maintain mandibular tc-en stripe expression and as a result to loss of the mandible itself .
in our model  tc-kni contributes to the activation of mandibular tc-wg expression.
this interaction could be direct because the respective expression patterns overlap.
tc-eve in turn could be directly involved in activating tc-hh and tc-en within its anteriormost expression domain.
subsequently, the interactions of the wnt and hh pathways ensure maintenance of the parasegement boundary.
in the absence of tc-kni expression, tc-eve still partially initiates tc-en/tc-hh expression in the posterior compartment, which leads to partial initiation of tc-wg in the anterior adjacent cells .
however, absence of tc-kni leads to failure to maintain tc-wg expression, and as a result failure to maintain tc-en expression in posterior adjacent cells .
interestingly, in drosophila, the maintenance of en and wg expression in the mandibular segment appears more interdependent in dorsal regions  <cit> .
the same might also be true in tribolium, since both tc-en and tc-wg expression is maintained more often in medial  regions in tc-kni mutant and knockdown embryos.
taken together, it appears to be a conserved feature of insects that two systems cooperate in patterning the mandible, but the molecular details of this cooperation have diverged significantly between drosophila and tribolium .
CONCLUSIONS
in this study we show that anterior regions of the antennal and mandibular segment, as well as the intervening intercalary segment, are correctly specified in the absence of tc-kni function.
moreover, we identify key differences in the role of tc-kni in setting up the non-adjacent antennal and mandibular parasegment boundaries.
we identify the pair-rule gene tc-even-skipped as a potential positive regulatory input that enables the initial specification  of the mandibular parasegmental boundary in the absence of tc-kni function.
we also present the first evidence that, as in drosophila, head and trunk gene regulatory networks cooperate to specify the mandibular segment.
however, our data, and those of others, point to significant divergence in the molecular interactions involved in mandibular patterning between drosophila and tribolium.
a recent study of the expression and function of head patterning genes in the hemimetabolous insect oncopeltus fasciatus suggests that the developmental gene networks operating in tribolium castaneum more closely resemble the ancestral insect condition, perhaps not surprisingly given the highly derived drosophila larval head  <cit> .
indeed, recent data from a myriapod suggest that the developmental genetic basis of tribolium larval head development might even closely resemble the ancestral arthropod condition  <cit> .
it will be interesting to see whether studies on other arthropods reveal an ancestral role for knirps-family homologues in head segmentation, which would imply ems and btd have usurped the role of knirps, and potentially other genes, in the lineage leading to drosophila.

 the insect head is composed of several segments and a non-segmental anterior region. however, the exact segmental composition of the insect head has long been a matter for debate. the posterior gnathocephalon is made up of the mandibular, maxillary and labial segments that each bear a pair of appendages specialized for feeding. the anterior procephalon consists of anterior non-segmental parts and an antennal segment, which is separated from the mandibular segment by an appendage-free segment, whose development in insects is significantly delayed, as well as reduced in size. the genetic mechanisms of head segmentation were first examined in the dipteran fruit fly drosophila melanogaster. its gnathal segments are patterned by the trunk segmentation gene cascade, involving maternal, gap, pair-rule and segment polarity genes, while the patterning of the procephalic segments follows a different paradigm. whilst segment polarity genes are involved in establishing these segments, pair-rule genes are not. four drosophila head gap genes, orthodenticle, empty spiracles, buttonhead and sloppy paired are expressed in broad overlapping domains in the developing anterior head. mutation of these genes leads to classic ‘gap phenotypes’ - the loss of all the adjacent segments covered by their domains of expression. however, mis-expression studies have shown that only otd affects segment polarity gene expression when expressed in ectopic domains, and only ems, with the help of btd, appears to confer identity to head segments. indeed, second order regulators have been identified that operate at levels in between the head gap genes and segment polarity genes: i.e. collier and cap ‘n’ collar. drosophila exhibits an evolutionary derived mode of head development, in which the larval head is greatly reduced and undergoes ‘head involution’ during which head regions are folded into the body cavity. this situation is far from typical for insects and moreover, the reduced and experimentally inaccessible drosophila larval head has limited the comprehensive identification and analysis of insect head development genes for technical reasons. in recent years the red flour beetle tribolium castaneum has emerged as a powerful genetic insect model system and offers an opportunity to study the genetic and cellular mechanisms underlying the development of a more insect-typical head. as in drosophila, the tribolium gnathal segments appear to be patterned using similar mechanisms to those operating in the trunk, including a central role for pair-rule gene homologues. in the anterior head, second order regulators and the segment polarity genes might be relatively well conserved between drosophila and tribolium. however, clear differences have been identified at the level of the head gap genes, and the maternally provided anterior protein gradients that establish their expression domains. for example, while the tribolium homologue of orthodenticle apparently plays a broadly conserved role as a gap gene during head segmentation in tribolium, it appears to be much more involved in axis formation than its drosophila orthologue. the expression of the tribolium homologues of empty spiracles and buttonhead is limited to single segment wide domains instead of large overlapping domains in the blastodermal head anlagen. tc-ems is required to form parts of the antennal segment only and knockdown of tc-btd does not lead to a head cuticle phenotype at all. this raised the question of what genes might control development of these head regions in tribolium. work by cerny et al. suggests that the answer to this question is, at least in part, the single tribolium homologue of the drosophila genes knirps and knirps-related. the drosophila genes knirps and knirps-related encode steroid hormone receptor-like transcription factors. ancestrally, the insect knirps family consisted of two genes, eagle and knirps-related, while knirps arose via a recent gene duplication of the knirps-related gene in the higher diptera. at the blastoderm stage, knirps and knirps-related are expressed in almost identical anterior and posterior domains. drosophila knirps acts as a canonical gap gene during trunk segmentation. in contrast, the anterior mandibular expression domain is not required for head segmentation, since segment polarity gene expression in the head is not affected in embryos that lack both paralogues while a loss of the stomatogastric nervous system is observed. cerny et al. have shown that tc-knirps, the single tribolium homologue of the drosophila knirps-family paralogues, is also expressed in anterior and posterior domains during early development. however, the tc-kni posterior domain is shifted anteriorly relative to its position in drosophila, and knockdown of tc-kni does not lead to a canonical gap phenotype in the trunk, but rather minor defects in the posterior abdomen. the anterior expression domain of tc-kni is largely conserved. in contrast to drosophila, the anterior domain does play an essential role in head patterning: knockdown of tc-kni leads to loss of both antennae and mandibles. cerny et al. found early loss of tc-wg expression in the antennal segment in tc-kni rnai while the mandibular domain of tc-wg expression disappeared at a later stage. further, they showed that tc-kni is not needed for correct tc-wg expression in the intercalary segment. finally, light abnormalities in the maturation and maintenance of the first pair-rule stripe of tc-eve expression were observed, where the distance between the first segmental tc-eve stripe and the ocular tc-wg expression domain was reduced in tc-kni rnai blastoderms. it has remained unclear, however, whether antennal and mandibular segments are deleted completely and whether the intercalary segment is affected. in this study we examined the effect of knocking down tc-kni on a comprehensive set of genes that mark sub regions of the antennal, intercalary and/or mandibular segments. we show that tc-kni is required for correctly specifying only posterior regions of antennal and mandibular head segments. interestingly, tc-kni is essential for the initial specification of the antennal parasegmental boundary, while it is required only for the maintenance of the mandibular parasegmental boundary. the intercalary segment does not appear to be affected. unexpectedly, we find that the trunk pair-rule gene tc-even-skipped is required to set the posterior boundary of the mandibular tc-kni expression. unlike most rnai studies, we have good evidence that we were investigating a null situation due to our finding that antennaless, a tribolium mutant arising from an ems screen, is a likely regulatory null-mutation of tc-kni. taken together, we show that tc-kni is not a head gap gene, since its mutation does not lead to the complete deletion of several adjacent segments. further, we provide a model, for how head and trunk patterning mechanisms cooperate to pattern the mandibular segment in tribolium, and how these interactions differ between drosophila and tribolium.  early tc-kni expression prefigures the appearance of the anterior head anlagen first, we studied in more detail the dynamics of anterior tc-kni expression because previous studies had focused on posterior aspects. zygotic tc-kni expression in the syncytial blastoderm begins as a broad domain covering most of the anterior half of the blastoderm, only absent from a small region around the anterior pole. tc-kni expression then retreats from the anterior pole, but much more so on the dorsal side of the egg, clearing from regions that will later become the extra-embryonic serosa. tc-kni expression is maintained in the anterior/ventral regions that will become head tissue. these early shifts in expression occur before embryonic and extra-embryonic tissue can be distinguished at the level of nuclear morphology. when this distinction becomes apparent, the uniform anterior tc-kni expression domain splits into two anterior lateral domains at the boundary between each head lobe and the abutting extra-embryonic tissue, and a more posterior stripe marking the future anterior compartment of the mandibular segment. these domains are maintained through subsequent blastoderm and early germband stages, before fading and then disappearing completely during mid-germband elongation stages. hence, the early uniform domain of tc-kni expression encompasses the entire future anterior head anlagen; i.e. everything anterior to the parasegment 0/ boundary. antennaless is likely a regulatory mutation at the tc-kni locus in most rnai experiments outside drosophila, it remains unclear to what extent a genetic null-situation is phenocopied. we aimed at determining the null situation by studying a genetic mutant. antennaless is a homozygous lethal mutation that was recovered from an ems mutagenesis screen. the cuticular phenotypes of antennaless mutant larvae are highly reminiscent of tc-kni rnai phenotypes. larvae of the antennaless mutant lack antennae and mandibles, and occasionally also display minor abdominal defects, very similar to those previously reported for weak tc-knirnai larvae. in order to check for defects in dorsal and lateral head tissues which are derived from pre-antennal and intercalary regions, respectively, we scored both sides of antennaless larval cuticles for the head bristle pattern. all bristles were present, albeit shifted somewhat compared to the wildtype condition in these regions. this is consistent with pre-antennal regions and much of the intercalary segment not being affected in the mutant. the development of both mandibles and antennae was more severely affected at a higher temperature in antennaless mutants. a larval cuticle was scored as an antennaless mutant if either the mandibles or antennae, or both, were reduced or absent. most of the antennaless mutant larvae lacked both the antennae and mandibles at 25°c. this increased to 75% at 32°c. in a few cases, rudiments of mandibles were found. in other cases, antennae or antennal rudiments were found. in contrast, and against the general observation that phenotypes tend to be more penetrant at higher temperatures, the frequency of abdominal defects was found to be higher at 25°c than at 32°c. at 25°c, % of phenotypic larvae displayed defects within segments a5-a compared to only 16% at 32°c. an inverse sensitivity with respect to temperature was also observed for defects in the urogomphi, dorsal outgrowths of the ninth segment. at 25°c this structure was defective in 79% of the phenotypic larvae examined, compared to 44% at 32°c. in offspring from nine independent pairs of heterozygous parents, the antennaless cuticle phenotype is found in 25% and 27% of larvae at 25°c and 32°c respectively, levels consistent with a highly penetrant homozygous lethal mutation. a significant proportion of mutant larvae were able to hatch despite their lack of antennae and mandibles; 45% at 25°c compared to only 17% at 32°c. the vast majority of these larvae died at the l stage. only very rarely did individuals reach the l stage. when corrected for a background reduction in hatching rate associated with this shift in temperature, these data reveal a 19% increase in sensitivity to temperature with regards to hatching in the antennaless mutant background. in our tc-kni rnai experiments head defects were also found to be more severe at 32°c compared to 25°c. the severity of head defects decreased over time post-injection as expected for parental rnai experiments. however, as for antennaless, we found inverse temperature sensitivity with respect to defects in segments a5-a following tc-knirnai. the frequency of abdominal defects also increased with time post-injection from about 20% at day eight post-injection, to 37% eleven days post-injection, and to 56% thirty-one days post-injection, before dropping again. this is not in line with the usual observation that in rnai experiments phenotypic strength decreases over time and indicates a complex relationship between knockdown and phenotype, which we do not fully understand. since the antennaless cuticle defects described above are almost identical to the tc-knirnai phenotypes both in terms of physical phenotype and sensitivity to temperature, we suspected tc-kni as the gene affected by the mutation. we therefore independently sequenced the three tc-kni exons from the genomic dna of two first instar larvae that had been identified as homozygous mutant by their cuticle phenotype. we found that the coding sequence of tc-kni is not altered in mutant beetles. in order to test the hypothesis that antennaless is a regulatory mutation at the tc-kni locus, we carried out in situ hybridization on embryos from heterozygous mutant parents with a mix of probes targeting tc-kni and tc-caudal as positive control. we failed to detect tc-kni expression in 15% of the offspring examined, whereas tc-caudal was well stained in the same colour reaction in all cases. this is consistent with embryos homozygous for the antennaless allele not expressing tc-kni at levels detectable via in situ hybridization. taken together, our results are consistent with the hypothesis that antennaless is a regulatory mutation of tc-kni, and as such we now refer to it as tc-kniatl. however, we cannot rule out that antennaless is a mutation in a gene that acts rather exclusively upstream of, or is a required interaction partner of, tc-kni. further studies will be necessary to confirm our hypothesis by identifying the cis-regulatory regions that are affected by the mutation. the tc-kni null phenotype is revealed by both tc-kniatl and tc-knirnai the lack of tc-kni expression in homozygous tc-kniatl embryos implies that tc-kni function is strongly reduced in the mutant. however, it remains possible that some residual function remains. in order to test whether tc-kniatl constitutes a complete null phenotype for tc-kni, we performed tc-kni rnai in the tc-kniatl background, assuming that the added effects should not lead to stronger phenotypes if tc-kniatl is a null mutant. the frequency of the tc-kni phenotype in the pooled offspring from independent pairs of tc-kniatl heterozygous animals was 11% at 25°c and 17% at 32°c. as expected, after injecting the same females with tc-kni dsrna, the frequency of the tc-kni phenotype strongly increased, to 97% and 81%. crucially, we did not find evidence for an increase in the severity of the cuticle phenotype: i.e. there were no larvae with phenotypes more severe than those seen in tc-kniatl or tc-knirnai alone. hence, unlike in most rnai experiments, we can be rather confident that the most severe rnai phenotypes we observe fully phenocopy a null situation. therefore, we use results from both mutant and rnai embryos for this work. tc-kni is differentially required for specifying antennal, intercalary and mandibular parasegment boundaries the cuticle phenotypes reported by cerny et al. raised the question of whether entire segments are deleted in tc-kni depleted embryos, or only the posterior parts that give rise to the appendages. these depend on parasegmental boundary formation via the segment polarity genes tc-engrailed, tc-hedgehog and tc-wingless. therefore, we stained tc-en and tc-hh in tc-knirnai embryos as markers for the posterior segment compartment. in the case of tc-en, embryos were co-stained for tc-dfd to help distinguish disrupted mandibular tc-en expression domains, from intercalary tc-en expression domains. we find that both tc-en and tc-hh expression is absent in the antennal segment in tc-knirnai embryos throughout embryogenesis. occasionally, tc-en and tc-hh expression associated with structures at the base of the antenna remains in later germband embryos, despite the clear failure of the antenna to form. the mandibular stripe of tc-en expression is initiated in early tc-knirnai germband embryos, albeit abnormally; the stripe of expression is broken and in extreme cases only patches of expression are seen. in older tc-knirnai germband embryos, tc-en expression associated with the mandibular segment is often missing completely, suggesting a failure to properly maintain tc-en expression in this segment in strong knockdowns of tc-kni expression. similarly, tc-hh expression in the mandibular segment is greatly reduced in young tc-knirnai embryos, but a thin broken stripe is still detected medially in early germbands. later, this tc-hh expression disappears in contrast to wildtype. note that tc-en mandibular expression appears to be more sensitive to the loss of tc-kni expression in lateral regions of the germband. tc-en and tc-wg expression in the intercalary segment appears relatively late and at a quite variable time point during mid germband elongation in wildtype embryos. whilst we cannot completely rule out minor disruptions of intercalary tc-en/tc-hh expression initiation and/or maintenance in tc-knirnai embryos, we do detect wildtype expression of these genes in late elongation and early fully elongated tc-knirnai embryos. in conclusion, the disruption of tc-kni function leads to the complete failure to initiate segment polarity gene expression in the antennal segment, failure to properly maintain segment polarity gene expression in the mandibular segment, and most likely has no effect on segment polarity gene expression in the intercalary segment. molecular markers show that tc-kni is not a canonical head gap gene segment polarity genes mark a posterior portion of each segment. therefore, we also asked whether the anterior parts of the segments are miss-specified and/or missing in tc-knirnai embryos by analyzing markers for the anterior regions of head segments. tc-goosecoid expression initially partially overlaps the ocular tc-wg domain and extends posterior to it. later, this domain widens in lateral regions, forming a wedge shape domain that abuts the antennal tc-wg stripe as it appears but later retracts from it. thus, tc-gsc is a marker for posterior ocular and anterior antennal regions in early embryos but later predominantly marks ocular tissue. in early tc-knirnai germband embryos, tc-gsc expression is down-regulated in posterior-lateral regions of the wedge shaped expression domain. in older tc-knirnai embryos, the tc-gsc expression domain again closely resembles the wedge shape seen in wild-type embryos at these stages. this indicates that the ocular parasegment boundary and anterior parts of the antennal segment are not greatly affected by loss of tc-kni, apart from some degree of lateral down-regulation of tc-gsc in early germ bands. tc-lim is a similar marker for the posterior of the ocular segment and anterior compartment of the antennal segment, albeit shifted slightly posteriorly relative to tc-gsc. in early embryos, expression is partially overlapping the ocular tc-wg domain and extending posterior to it. in elongating germbands, tc-lim expression forms a wedge shaped domain that covers all cells between the ocular and antennal tc-wg stripes. in the tc-knirnai background, the posterior boundary of this domain is irregular and the domain is somewhat narrower indicating posterior reduction of the tc-lim marked tissues. in wild-type elongating germbands, additional tc-lim segmental expression arises that laterally overlaps the segmental tc-wg domains in gnathal and thoracic segments. at these stages, the ocular-antennal domain of tc-lim expression splits into a posterior domain that overlaps the antennal tc-wg stripe, and an anterior domain positioned between the ocular and the antennal tc-wg stripes. expression in the ocular-antennal region remains also in tc-knirnai germbands, but the domain is not split as in wildtype and appears to be fused with the mandibular domain of lateral tc-lim expression. in extreme cases this domain is also fused to the maxillary tc-lim expression domain. this confirms that anterior antennal tissue is properly specified while the parasegmental boundaries of the antennal and mandibular segments are not formed correctly. tc-empty-spiracles is expressed in a segmentally reiterated pattern during tribolium development in mediolaterally-restricted domains that lie anterior to the tc-wg stripe in each segment. tc-ems expression therefore predominantly marks the anterior portion of each segment. in contrast to wild-type expression, the antennal and mandibular tc-ems segmental domains are fused in tc-knirnai embryos, as seen for tc-lim this is consistent with defects being restricted to the posterior compartment of the antennal segment leading to the fusion of the antennal and mandibular tc-ems expression domains. tc-sloppy-paired- is also expressed in a segmentally reiterated pattern in the developing tribolium head. tc-slp- expression domains overlap the tc-wg stripe in each head segment, but also extend further into the anterior compartment, as well as a little across the parasegmental boundary into the posterior compartment. tc-slp- expression therefore predominantly marks a posterior portion of the anterior segment compartment. tc-slp- is expressed in a stripe in the antennal and each gnathal head segment in both tc-knirnai and tc-kniatl embryos, as in wild-type embryos. however, the antennal stripe often appears to broaden, and the distance between the mandibular and the maxillary tc-slp- stripes is often decreased, completely fusing in lateral regions. these data indicate that overall the tc-slp- stripes are not dramatically affected by tc-kni rnai. however, the decreased distance between the mandibular and maxillary stripes could indicate incorrectly specified tissue in the intervening posterior compartment of the mandibular segment. a normal complement of bristles and setae in lateral regions of tc-kniatl larval heads implies the retention of intercalary segment derived cuticle in tc-kni knockdown embryos. since tc-labial is expressed during embryogenesis throughout the presumptive intercalary segment, and is required for its formation, we used it as a molecular marker for the presence/absence of the intercalary segment. in tc-kni rnai embryos, tc-lab expression appears prematurely but with similar dynamics as in wildtype. the premature tc-lab expression domain first appears medially as a pair of dots on either side of the median mesoderm. tc-lab expression then extends into more lateral and medial regions. expression is not entirely wildtype, as it may form an unusual regular stripe without the typical median expansion, or it may be laterally reduced. however, in all instances, the stripe was present indicating that the intercalary segment is present albeit its morphology appears not to be entirely unaffected by adjacent defects. we also examined the expression of another marker for the intercalary segment in tc-knirnai embryos, the second order regulator tc-collier, also called tc-knot. tc-col acts downstream of tc-lab and is required for wildtype expression of tc-en in the intercalary segment. aside from a slight delay in the initial appearance of the tc-col expression domain in early embryos we found no evidence of strong disruption of tc-col expression in a tc-knirnai background. besides, this staining confirmed the identity, and wildtype appearance, of the intercalary spots of tc-en in late elongating and early fully elongated embryos. the drosophila head gap genes lead to the loss of entire adjacent segments but their tribolium orthologues do not fit this definition. similarly, taken together, our data show that tc-kni cannot be considered a canonical head gap gene, because tc-kni function is only required for the posterior parts of the antennal and mandibular segments. moreover, tc-kni is not required for the formation of the intercalary segment, which lies between the affected antennal and mandibular segments. intercalary segment polarity gene expression is instead likely dependent on a conserved pathway involving tc-lab and tc-col. overall, our data confirm that the regulatory networks underlying the establishment of the anterior head segments have diverged significantly between tribolium and drosophila, involving changing roles for knirps, buttonhead and empty spiracles homologues. the tc-kni phenotype is due to a failure to correctly specify cell fates we decided to check whether missing head regions in tc-kni knockdown and mutant embryos were lost due to a failure to maintain already specified tissue - which might be indicated by high levels of cell death - or through the failure to specify cells to their correct developmental fate. using tunel staining, we did not detect any apoptosis in either the blastoderm or early germband stages in tc-knirnai embryos. in later germband stages there were a few apoptotic cells detectable in the head, but no more than in wild-type embryos, and there was no specific pattern of apoptosis that would indicate the loss of the tissues in question, namely the posterior portions of the antennal and mandibular segments. thus, the lack of antennae and mandibles in tc-knirnai and tc-kniatl larvae is not due to tissue degeneration, but rather the failure to specify the respective segmental regions. the posterior border of the anterior tc-kni expression domain is regulated by tc-even-skipped in the tribolium blastoderm, expression of tc-kni and the pair rule genes occurs at the same time, opening the possibility tc-kni could be regulated by pair rule genes. indeed, in tc-evernai blastoderm embryos, we found that the anterior domain of tc-kni expression expanded into more posterior regions. in contrast, expression defects in this tc-kni mandibular domain were not seen in tc-oddrnai or tc-runrnai germbands. this is consistent with the first pair-rule and/or segmental stripe of tc-eve expression being required to set the posterior boundary of mandibular tc-kni expression. head and trunk segmentation gene networks cooperate to establish the mandibular segment in a different way in tribolium and drosophila in drosophila, the anterior head gene regulatory network and the gnathal/trunk regulatory gene network cooperate to pattern aspects of the mandibular segment. in this study we provide evidence that this is also true in tribolium. both tc-eve and tc-kni rnai lead to the failure to properly maintain mandibular tc-en stripe expression and as a result to loss of the mandible itself. in our model tc-kni contributes to the activation of mandibular tc-wg expression. this interaction could be direct because the respective expression patterns overlap. tc-eve in turn could be directly involved in activating tc-hh and tc-en within its anteriormost expression domain. subsequently, the interactions of the wnt and hh pathways ensure maintenance of the parasegement boundary. in the absence of tc-kni expression, tc-eve still partially initiates tc-en/tc-hh expression in the posterior compartment, which leads to partial initiation of tc-wg in the anterior adjacent cells. however, absence of tc-kni leads to failure to maintain tc-wg expression, and as a result failure to maintain tc-en expression in posterior adjacent cells. interestingly, in drosophila, the maintenance of en and wg expression in the mandibular segment appears more interdependent in dorsal regions. the same might also be true in tribolium, since both tc-en and tc-wg expression is maintained more often in medial regions in tc-kni mutant and knockdown embryos. taken together, it appears to be a conserved feature of insects that two systems cooperate in patterning the mandible, but the molecular details of this cooperation have diverged significantly between drosophila and tribolium.  in this study we show that anterior regions of the antennal and mandibular segment, as well as the intervening intercalary segment, are correctly specified in the absence of tc-kni function. moreover, we identify key differences in the role of tc-kni in setting up the non-adjacent antennal and mandibular parasegment boundaries. we identify the pair-rule gene tc-even-skipped as a potential positive regulatory input that enables the initial specification of the mandibular parasegmental boundary in the absence of tc-kni function. we also present the first evidence that, as in drosophila, head and trunk gene regulatory networks cooperate to specify the mandibular segment. however, our data, and those of others, point to significant divergence in the molecular interactions involved in mandibular patterning between drosophila and tribolium. a recent study of the expression and function of head patterning genes in the hemimetabolous insect oncopeltus fasciatus suggests that the developmental gene networks operating in tribolium castaneum more closely resemble the ancestral insect condition, perhaps not surprisingly given the highly derived drosophila larval head. indeed, recent data from a myriapod suggest that the developmental genetic basis of tribolium larval head development might even closely resemble the ancestral arthropod condition. it will be interesting to see whether studies on other arthropods reveal an ancestral role for knirps-family homologues in head segmentation, which would imply ems and btd have usurped the role of knirps, and potentially other genes, in the lineage leading to drosophila. 
using the mutant antennaless, a likely regulatory null mutation at the tc-kni locus, we provide evidence that our rnai studies represent a null situation. however, published tc-kni data are restricted to cuticle phenotypes and tc-even-skipped and tc-wingless stainings in knockdown embryos. hence, it has remained unclear whether the entire antennal and mandibular segments depend on tc-kni function, and whether the intervening intercalary segment is formed completely. we address these questions with a detailed examination of tc-kni function. surprisingly, tc-kni controls the timing of expression of the hox gene tc-labial in the intercalary segment, although this segment does form in the absence of tc-kni function. in tribolium, as in drosophila, head and trunk segmentation gene networks cooperate to pattern the mandibular segment, albeit involving tc-kni as novel component. the drosophila larval head is evolutionarily derived at the genetic and morphological level. in the beetle tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. by examining the expression of marker genes in rnai embryos, we show that tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments. unexpectedly, we find that the pair-rule gene tc-even-skipped helps set the posterior boundary of tc-kni expression in the mandible. tc-kni is not required for specification of the anterior regions of these segments, nor the intervening intercalary segment, confirming that tc-kni is not a canonical ‘gap-gene’. moreover, we find that the role of tc-kni is distinct in these segments: tc-kni is required for the initiation of the antennal parasegment boundary, but only for the maintenance of the mandibular parasegmental boundary.
 the drosophila larval head is evolutionarily derived at the genetic and morphological level. in the beetle tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. unlike in drosophila, a knirps homologue is required for development of the antennae and mandibles. however, published tc-kni data are restricted to cuticle phenotypes and tc-even-skipped and tc-wingless stainings in knockdown embryos. hence, it has remained unclear whether the entire antennal and mandibular segments depend on tc-kni function, and whether the intervening intercalary segment is formed completely. we address these questions with a detailed examination of tc-kni function.  by examining the expression of marker genes in rnai embryos, we show that tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments. moreover, we find that the role of tc-kni is distinct in these segments: tc-kni is required for the initiation of the antennal parasegment boundary, but only for the maintenance of the mandibular parasegmental boundary. surprisingly, tc-kni controls the timing of expression of the hox gene tc-labial in the intercalary segment, although this segment does form in the absence of tc-kni function. unexpectedly, we find that the pair-rule gene tc-even-skipped helps set the posterior boundary of tc-kni expression in the mandible. using the mutant antennaless, a likely regulatory null mutation at the tc-kni locus, we provide evidence that our rnai studies represent a null situation.  tc-kni is required for the initiation of the antennal and the maintenance of the mandibular parasegmental boundaries. tc-kni is not required for specification of the anterior regions of these segments, nor the intervening intercalary segment, confirming that tc-kni is not a canonical ‘gap-gene’. our finding that a gap gene orthologue is regulated by a pair rule gene adds to the view that the segmentation gene hierarchies differ between tribolium and drosophila upstream of the pair rule gene level. in tribolium, as in drosophila, head and trunk segmentation gene networks cooperate to pattern the mandibular segment, albeit involving tc-kni as novel component. knirpshead gap genetriboliumantennamandibleintercalary segment 