Do teashirt family genes specify trunk identity? Insights from the single tiptop/teashirt homolog of Tribolium castaneum

The Drosophila teashirt gene acts in concert with the homeotic selector (Hox) genes to specify trunk (thorax and abdomen) identity. There has been speculation that this trunk-specifying function might be very ancient, dating back to the common ancestor of insects and vertebrates. However, other evidence suggests that the role of teashirt in trunk identity is not well conserved even within the Insecta. To address this issue, we have analyzed the function of Tc-tiotsh, the lone teashirt family member in the red flour beetle, Tribolium castaneum. Although Tc-tiotsh is important for aspects of both embryonic and imaginal development including some trunk features, we find no evidence that it acts as a trunk identity gene. We discuss this finding in the context of recent insights into the evolution and function of the Drosophila teashirt family genes.
Introduction
Homeotic genes are responsible for defining regional identity, and mutations in these genes often transform one body part into another. The best characterized homeotic genes are the Hox genes, which have been identified in representatives of most animal phyla (reviewed by Martindale 2005) and have been extensively studied in several organisms. However, some non-Hox genes have also been classified as homeotic genes based on their mutant phenotypes. One such gene encodes the zinc finger transcription factor Teashirt (Fasano et al. 1991). Loss-of-function (LOF) teashirt (tsh) mutations are reported to cause partial transformation of the larval prothorax to a labial identity due to ectopic expression of the Hox gene Sex combs reduced (Fasano et al. 1991; Roder et al. 1992). The trunk (thoracic and abdominal) segments of tsh− larvae are shorter than normal and the denticle belts (a trunk-specific feature) are severely disorganized. Moreover, patches of sclerotic cuticle, which have been interpreted as head structures, form between the remaining denticle belts (Fasano et al. 1991). Conversely, ectopic expression of tsh results in the development of denticle belts in the head (de Zulueta et al. 1994). On the basis of these phenotypes, tsh has been suggested to function as a specifier of trunk vs head identity (Roder et al. 1992).
tsh is believed to act in parallel with the trunk Hox genes (Scr, Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)) to determine segmental identity (de Zulueta et al. 1994; Roder et al. 1992). When all of these Hox genes are inactivated, trunk segments develop with what has been interpreted as a mixed head/trunk identity. That is, sclerotized tissue develops in the posterior compartment of each trunk segment, whereas denticle belts (a trunk-specific feature) still develop in the anterior compartment (Sato et al. 1985; Struhl 1983). When tsh function is also eliminated, the trunk segments are more severely affected in that denticle belts disappear and additional head-like cuticle is observed (Roder et al. 1992).
Recent data suggests that tsh may play an even greater role in trunk development than was previously recognized. In the absence of tsh function, its paralog tiptop (tio) is ectopically expressed in the trunk region (Laugier et al. 2005). When tio and tsh functions are both absent, the trunk denticles are even more severely affected than in mutants that lack only tsh, suggesting that tio can partially substitute for tsh and reduce the effects of tsh mutations.
Robertson and coworkers (2004) proposed the existence of a system of zinc-finger proteins that divides the Drosophila embryo into sections and limits the functional domains of Hox genes. They suggest that within this system tsh specifies the trunk region, while disconnected (disco) specifies the gnathal region. In the absence of tsh, disco (as well as its paralog disco-related) is ectopically expressed in the trunk, and there is some evidence that ectopic disco expression is responsible for at least some features of tsh mutants (e.g., loss of denticles and abnormal sensory neurons) that are thought to represent a switch to head/gnathal identity (Robertson et al. 2004).
Manfroid et al. (2004) speculated that the trunk-specifying function of tsh family members might be conserved in vertebrates, as tsh genes from mice are expressed in the trunk region and can also rescue the phenotype of Drosophila tsh mutants when ectopically expressed. However, such conjecture requires a rather large phylogenetic leap, as it is not clear that the role of tsh in trunk specification is conserved even in other insects. tsh family genes of the firebrat, Thermobia domestica, and the milkweed bug, Oncopeltus fasciatus, are expressed predominantly in the gnathos and thorax, arguing against a function in trunk specification (Herke et al. 2005; Peterson et al. 1999). Furthermore, when Herke et al. (2005) knocked down the function of the Oncopeltus tsh family gene by RNA interference (RNAi), they observed transformations of distal leg to antenna, but did not report any changes in trunk identity. Finally, our previous studies suggest that genetic specification of trunk identity in the red flour beetle, Tribolium castaneum, differs significantly from that in Drosophila. Removal of trunk Hox genes alone is sufficient to produce dramatic transformation of trunk to head identity (Brown et al. 2002). Antennae develop on all gnathal and trunk segments of embryos homozygous for a deficiency that removes the Tribolium orthologs of Deformed (Dfd), Scr, Antp, Ubx, and abd-A (Stuart et al. 1991). Thus, there is apparently no need to invoke the function of another gene for trunk identity. Peterson et al. (1999) predicted that either the Tribolium tsh gene has no function in trunk specification or that the expression of tsh is controlled by Hox genes. Taken together, these observations suggest that the trunk-specifying role of tsh observed in Drosophila might not be conserved in other insects.
To better understand the evolution of tsh family gene function, we cloned and characterized a tsh homolog from Tribolium. This gene, Tc-tiotsh, is the sole member of this family in the Tribolium genome. It has a broad embryonic expression domain, encompassing not only the trunk region but much of the head as well. RNAi experiments revealed that Tc-tiotsh functions in several embryonic and imaginal tissues, but they did not suggest a conserved function in trunk specification. We discuss these results and reexamine the classification of tsh as a trunk-specifying gene in Drosophila.
Materials and methods
cDNA cloning
A genomic fragment of the Tc-tiotsh gene was amplified using degenerate primers based on the conserved amino acid motifs VWLGKG and CKVCDK (first round) and ILKCMW and CKVCDK (second round). Additional, overlapping fragments were identified by RACE, using the 5′ and 3′ RACE Systems (GibcoBRL), by polymerase chain reaction (PCR) screening a Tribolium castaneum cDNA library as described previously (Nie et al. 2001), or by inverse PCR (Ochman et al. 1988). To confirm that these overlapping fragments represent a contiguous sequence, primers were designed near the ends of the putative contig, and reverse transcriptase (RT)-PCR was performed using total embryonic RNA (0–30 h of development) as template. The resulting PCR product was cloned and sequenced (GenBank accession number AF356647).
Sequence analysis
The nucleotide contig for Tc-tiotsh was assembled using the Seqman module of Lasergene (DNASTAR). To identify putative tsh family members in other genomes, we performed tBLASTn searches using Drosophila melanogaster Tsh and Tio as query sequences. Multiple alignments were created with the AlignX module of Vector NTI 8 (InforMax). Minor manual adjustments were performed to maximize the alignments. The final output of the multiple alignment was created using BOXSHADE (available at http://www.ch.embnet.org/software/BOX_form.html). Phylogenetic analyses were performed using MEGA version 3.1 (Kumar et al. 2004) or TreePuzzle (http://bioweb.pasteur.fr/seqanal/interfaces/Puzzle.html), and TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) was used to visualize the output.
In situ hybridization and immunostaining of embryos
A digoxigenin-labeled fragment of Tc-tiotsh was hybridized to whole-mount wild-type (Georgia-1) embryos as previously described (Brown et al. 1994b). Embryos were secondarily immunostained with either α-Invected antibody (4D9) (Patel et al. 1989) or with α-Even-skipped antibody (2B8) (Patel et al. 1994), which cross-react with Tc-Engrailed and Tc-Even-skipped, respectively. Tc-spalt (Tc-sal) expression was assayed using the probe described by Tomoyasu et al. (2005).
RNA interference (RNAi)
dsRNA of two different, non-overlapping fragments of Tc-tiotsh was synthesized using the PCR template method described by Kennerdell and Carthew (2000). Briefly, primers were chosen to amplify a fragment of Tc-tiotsh (bases 1358–1786 or 1848–2581 of GenBank sequence AF356647) and the T7 promoter sequence (TAATACGACTCACTATAGG) was added to the 5′ end of each primer. The resulting primers were used to amplify template for RNA synthesis. The resulting product was purified and 1 μg used as template for RNA synthesis using the Megascript T7 High Yield Transcription kit (Ambion).
For embryonic RNAi, 0–2 h eggs were collected from wild-type beetles (GA-1) and injected with dsRNA (2–4 μg/μl) as previously described by Brown et al. (1999). After several days, hatched larvae and unhatched eggs were treated with lactic acid/ethanol. Additional larvae were documented by SEM as described by Curtis et al. (2001).
Injection into non-virgin adult females was performed by manually opening the elytra and wings of ether-anesthetized beetles and injecting 2 μg/μl dsRNA into the dorsal abdomen. Larval RNAi was performed as described by Tomoyasu and Denell (2004).
Results
Tribolium has a single teashirt family gene
We cloned a member of the tsh gene family from the red flour beetle, Tribolium castaneum (see Materials and methods for details). Comparison of the cDNA sequence to the recently completed Tribolium genome sequence revealed three exons distributed over about 90 kb (Fig. 1). Most of this distance can be attributed to an approximately 86-kb intron between the first and second exons.
Fig. 1Gene structure of the Tc-tiotsh gene and its Drosophila homologs. Exons are denoted by boxes and introns by thin lines. The coding region is shaded gray and zinc finger-encoding motifs are shown in black
Only three full-length Tsh family proteins have been described from insects: the Drosophila melanogaster paralogs Tsh and Tio, and the single member of the Tsh family in Anopheles gambiae, Ag-TioTsh (Fasano et al. 1991; Laugier et al. 2005). In addition, small fragments of tsh family genes have been amplified from several other insects (Herke et al. 2005; Peterson et al. 1999). Two conclusions have been drawn from these limited data. First, the two Drosophila genes seem to be the result of a recent duplication in the Drosophila lineage. Second, all of the Tsh family members from other insects appear to be more similar in sequence to Tio than to Tsh. In this paper, we present data that support and greatly strengthen these conclusions.
We performed a tBLASTn search of several recently sequenced insect genomes for tsh family members (Table 1). The Tribolium castaneum gene that we cloned is the only member of the tsh family present in the genome sequence of this insect. Likewise, the Apis mellifera (honeybee) genome contains only a single member of this gene family. In contrast, all of the additional Drosophila species we searched (D. pseudoobscura, D. simulans, D. yakuba and D. virilis) have orthologs of both tio and tsh. Consistent with the situation in D. melanogaster (Laugier et al 2005), tio and tsh are closely linked (less than 250 kb apart) in all of the Drosophila species examined, suggesting that these genes likely arose from a relatively recent tandem duplication event.
Table 1Teashirt family genes in sequenced insect genomesOrganismOrdertsh family genesGenome ReferenceTribolium castaneumColeoptera1The Tribolium Genome Sequencing Consortium 2008Apis melliferaHymenoptera1The Honeybee Genome Sequencing Consortium 2006Anopheles gambiaeDiptera1Holt et al. 2002Drosophila simulansDiptera2 (tio and tsh)Drosophila 12 Genomes Consortium 2007Drosophila yakubaDiptera2 (tio and tsh)Drosophila 12 Genomes Consortium 2007Drosophila pseudoobscuraDiptera2 (tio and tsh)Richards et al. 2005Drosophila virilisDiptera2 (tio and tsh)Drosophila 12 Genomes Consortium 2007tBLASTn searches were used to determine the number of teashirt (tsh) family genes in several assembled insect genomes. All Drosophila species examined had orthologs of both tsh and tiptop (tio), while the non-drosophilid species each contained only one tsh family member.
As the Tribolium gene is related to both tio and tsh, we have named it Tc-tiotsh, after the convention established by Laugier et al. (2005) for the single Anopheles gene. We compared the predicted Tc-TioTsh protein to the other insect Tsh family members for which full-length predicted proteins are available. (Although, we were able to identify a tsh family gene in Apis, the 5′ portion of the transcription unit was apparently missing from the genome sequence.) Like tio and Ag-tiotsh (Laugier et al. 2005), Tc-tiotsh encodes four zinc fingers (Fig. 1), three of which are clustered near the middle of the protein (Fig. 2a), and one that is located near the C terminus (Fig. 2b). Besides the zinc finger regions, several additional short regions of sequence identity (shaded in Fig. 2 and data not shown) are present in all of the full-length predicted proteins. The three dipteran proteins are highly similar at their N termini, while Tio, Ag-TioTsh, and Tc-TioTsh share a C-terminal motif (in addition to the fourth zinc finger) that is absent in Tsh. Regions shared between the beetle and dipteran proteins have been conserved over a long phylogenetic distance, suggesting that they likely have important functions.
Fig. 2Multiple alignments of Teashirt family members. Amino acids identical or similar in at least two of the proteins are shaded black or gray, respectively. Tc Tribolium castaneum; Ag Anopheles gambiae, Dm Drosophila melanogaster; Mm Mus musculus; Dr Danio rerio. a Multiple alignment of the three clustered zinc fingers. Zinc fingers (ZF) 1–3 are indicated above the sequence. Regions underscored with asterisks were concatenated and subjected to phylogenetic analysis. b Multiple alignment of the C-terminal zinc finger region. Zinc finger 4 is delimited above the alignment. Tsh lacks this zinc finger. c Distinct N-terminal motifs in dipteran Tsh family members (upper) and in Tribolium and vertebrates (lower)
To better understand the evolutionary history of the tsh gene family, we have performed phylogenetic analyses using conserved portions of the zinc-finger region (underlined in Fig. 2) from the full-length insect Tsh family proteins that are currently available (Drosophila, Tribolium, and Anopheles). Both maximum likelihood (Fig. 3) and neighbor-joining (data not shown) methods produce trees in which the two Drosophila proteins cluster together, suggesting these genes were produced by duplication after the divergence of the mosquito and fly lineages. The branch lengths of the Drosophila proteins are much longer than those of Anopheles and Tribolium, suggesting rapid divergence of both genes after duplication.
Fig. 3Phylogenetic analysis of insect Teashirt family members. The two Drosophila paralogs cluster together in an unrooted tree produced by maximum likelihood analysis, suggesting that these genes resulted from a recent duplication in the Drosophila lineage. The bootstrap value of 100 indicates that the tree is very robust
The mouse and human genomes each have three tsh family members (Caubit et al. 2000; Manfroid et al. 2004), which are much more similar to one another than they are to any of the insect proteins. Interestingly, vertebrate Tsh family proteins share an N-terminal motif (MPRRKQ) with the Tribolium protein (Fig. 2c), which is distinct from the N-terminal motif shared by the dipteran family members (Laugier et al. 2005). The conservation of this sequence in an insect and vertebrates suggests it has an important function and that it was probably present in the common ancestor of protostomes and deuterostomes.
Taken together, these comparisons of tsh family members indicate that the ancestor of protostomes and deuterostomes had a single tsh family gene. We were unable to identify any tsh family members in the genomes of two non-bilaterian metazoans: the placozoan Trichoplax adhaerens (http://genome.jgi-psf.org/Triad1/Triad1.home.html) and the cnidarian Nematostella vectensis (Putnam et al. 2007), suggesting this gene family may have arisen after the bilaterian/non-bilaterian split. Within the vertebrate lineage, duplication events (perhaps in association with whole genome duplications) produced at least three paralogs. A duplication in the Drosophila lineage (after divergence from the mosquito lineage) resulted in the tsh and tio paralogs.
Tc-tiotsh embryonic expression is not limited to the trunk
We examined the embryonic expression pattern of Tc-tiotsh (Fig. 4) by in situ hybridization, using the posterior compartment marker Engrailed (Brown et al. 1994a; Patel et al. 1989) to determine the stage of embryonic development (segments are added sequentially) and provide a reference for the register of Tc-tiotsh expression. Tc-tiotsh expression develops in a dynamic, pair rule-like manner (Fig. S1 and S2), but eventually encompasses not only the trunk segments (thorax and abdomen), but also the gnathal segments and the labrum and intercalary segment in the procephalon (Fig. 4a). Tc-tiotsh expression in the thorax and the first abdominal segment appears somewhat stronger than in other regions (Fig. 4b). As appendages develop, Tc-tiotsh is expressed in the proximal portion of each leg (Fig. 4d).
Fig. 4Tc-tiotsh expression in Tribolium embryos. Engrailed expression is shown in brown, while Tc-tiotsh expression is in purple. Embryos are oriented with anterior to the left (except in panel d in which anterior is to the top). a During germband elongation, Tc-tiotsh is expressed in the gnathal and trunk segments as well as two procephalic regions (the intercalary segment [Ic] and the labrum [Lr]). b When germband elongation is completed, Tc-tiotsh expression is strongest in the thoracic (T1–T3) and first abdominal (A1) segments. c Slightly later, strong Tc-tiotsh expression appears in the posterior terminal region of the embryo (arrowhead). dTc-tiotsh is expressed in only the proximal portion (arrow) of the developing legs. e In embryos undergoing germ-band retraction, Tc-tiotsh expression is present in the six Malpighian tubule primordia that evert from the hindgut. f In a Df(HOMC) homozygote lacking most Hox genes, Tc-tiotsh expression in the segmented portion of the germband is greatly reduced. In contrast, expression in the Malpighian tubule primordia remains strong
After germ-band elongation, Tc-tiotsh is expressed in the posterior region of the embryo. Initially, expression is seen in a patch on either side of the ventral midline (Fig. 4c), and later in six buds that extend from the invaginating hindgut (Fig. 4e). These structures are the primordia of the Malpighian tubules; they also express Tc-dachsund (Prpic et al. 2001) and Tc-Cut (data not shown).
spalt does not appear to repress Tc-tiotsh in Tribolium embryos
In Drosophila, the limits of tsh expression are controlled either directly or indirectly by spalt-major (salm). In a salm mutant, the tsh expression domain expands both anteriorly and posteriorly (Roder et al. 1992). To assess whether the broader expression of Tc-tiotsh (compared to tsh) might reflect a difference in Tribolium spalt (Tc-sal) expression, we examined the embryonic expression pattern of the single Tribolium sal gene (Tomoyasu et al. 2005). We find that the Tc-sal expression domain (Fig. S3) significantly overlaps that of Tc-tiotsh, suggesting that Tc-sal does not repress Tc-tiotsh expression. It is possible that Tribolium has lost the regulatory interaction between these genes. Alternatively, regulation of tsh by sal may have evolved more recently, after the divergence of these lineages.
Tc-tiotsh expression is reduced in embryos lacking Hox genes
In Drosophila, some aspects of tsh expression are regulated by the Hox genes (Roder et al. 1992). We have examined expression of Tc-tiotsh in embryos homozygous for Df(HOMC), a deletion that removes most of the Tribolium Hox cluster. Tc-tiotsh expression is severely reduced in the ventral region of these embryos (Fig. 4f). These results suggest that reduction of Tc-tiotsh expression might contribute to the transformations seen in Df(HOMC) embryos. However, our functional studies of Tc-tiotsh (presented below) reveal no evidence for this.
Tc-tiotsh functions in the embryonic labial and thoracic segments
We used RNAi to ascertain the probable LOF phenotype of Tc-tiotsh. Pupae injected with Tc-tiotsh dsRNA produced very few eggs upon reaching adulthood, and none of these progeny showed an abnormal phenotype. To circumvent the apparent effect of Tc-tiotsh on oogenesis, we injected Tc-tiotsh dsRNA either directly into eggs or into the abdomens of non-virgin adult females (egg production from these females quickly tapers off, further indicating an effect on oogenesis). Injection of Tc-tiotsh dsRNA by either technique produced embryos with defects in the labial and thoracic segments (Fig. 5, Table 2, and data not shown). During normal Tribolium embryonic development, the two labial appendages fuse at the ventral midline and migrate anteriorly to a position between the maxillary appendages (Fig. 5a). In moderately affected Tc-tiotsh RNAi larvae (Fig. 5b), the labial appendages are smaller than normal, and in the most severely affected embryos (Fig. 5c) these appendages fail to fuse. In all of these cases, the abnormal labial appendages fail to migrate to their normal position between the maxillary palps. These abnormalities produce secondary effects on the orientation of the other head appendages.
Fig. 5Scanning electron micrographs of Tc-tiotsh RNAi larvae. For all panels, the scale bar represents 50um. a–c Ventral views of larval heads. Maxillary (pink) and labial (green) appendages have been shaded. d–e Lateral views with leg segments labeled as follows: coxa (C), trochanter (Tr), femur (F), tibiotarsus (Tt), and pretarsus (Pt). a Wild-type larval head showing the proper position of the maxillary (Mx) and labial (L) appendages. b Moderately affected Tc-tiotsh RNAi larva in which the labial appendages (especially the proximal region) are highly reduced. c Severely affected Tc-tiotsh RNAi larva in which the labial appendages have failed to fuse. Note that in both b and c, the ventral, posterior region of the head (presumed to derive from the labial segment) is almost completely absent. d Wild-type larva. eTc-tiotsh RNAi larva with short legs. On each leg, the trochanter is presumably fused with the reduced coxaTable 2Tc-tiotsh RNA interference cuticular phenotypesdsRNA fragmentSeverity of EffectNoneMildModerateSevere1358–178612/74 (16.2%)4/74 (5.4%)14/74 (18.9%)44/74 (59.5%)1358–17862/58 (3.4%)14/58 (24.1%)39/58 (67.2%)3/58 (5.2%)1848–25810/24 (0%)4/24 (16.6%)20/24 (83.3%)0/24 (0%)All results were obtained by injection of dsRNA into eggs. dsRNA fragment names refer to the nucleotides of GenBank sequence AF356647 included in the fragment. Severity of effect was classified as follows. Larvae with mild effects had short legs resulting from reduction and fusion of proximal segments. Moderately affected larvae had reductions in the ventral head and labial appendages in addition to short legs. Severely affected larvae were distinguished from those with moderate effects by the failure of the labial appendages to fuse at the ventral midline.
Short legs were the most common feature seen in Tc-tiotsh RNAi larvae, being present in all larvae with labial defects, as well as in some larvae with apparently normal labial appendages (Table 2). The reduction in leg length stems from effects on the proximal leg segments. The coxa and trochanter are reduced and apparently fused, and the femur is slightly shortened (Fig. 5e). This phenotype is similar to that observed in the adult legs of hypomorphic Drosophila tsh mutants (Erkner et al. 1999) and thus may indicate a conserved role for tsh family genes in proximal limb development.
We also noted that Tc-tiotsh RNAi larvae show a pronounced curvature of the head and thoracic segments, such that the head bends forward (Fig. 5e). This phenotype apparently results from reduction in length (anterior to posterior) of the ventral portions of the thoracic segments and posterior head (Fig. 5b,c,f).
Tc-tiotsh is important for proper development of adult trunk segments
We examined the role of Tc-tiotsh in adult development using larval RNAi (Tomoyasu and Denell 2004). The resulting adults display a number of defects in the thoracic and abdominal segments (Fig. 6). Most noticeably, these individuals have abnormalities of the T2 notum that prevent the elytra from closing properly (data not shown). We also observed mild defects in the proximal legs. In addition, adults resulting from Tc-tiotsh larval RNAi show abnormalities in many of the ventral sclerites (sternites) (see El-Kifl 1953 for a description of wild-type structures). In the ventral prothorax (T1) of RNAi-treated individuals, the epimera, which are normally located posterior to the T1 coxae, are missing and the basisternum is reduced in size resulting in the exposure of membranous areas (Fig. 6b,d). In addition, the shape of the sternellum (the process extending between the T1 legs) is abnormal. Ventral T2 is not noticeably affected, but the T3 sternite is missing its anterior process (meso-spinasternum) (Fig. 6d). The anterior abdominal segments are also affected by Tc-tiotsh larval RNAi. In wild-type adults, the first and second abdominal segments (A1-A2) form an internal portion of the T3 leg sockets and are only lightly sclerotized, while A3 is distinguished by a sharply pointed process extending between the T3 legs (Fig. 6e). In Tc-tiotsh RNAi individuals, an additional anterior region (A1 and/or A2) is heavily sclerotized and A3 lacks its anterior process, thus resembling the more posterior abdominal segments (A4–A6) (Fig. 6f).
Fig. 6Adult phenotypes resulting from Tc-tiotsh larval RNAi. All images are ventral views with anterior to the top. a Wild-type adult head and thorax. b Head and thorax of Tc-tiotsh RNAi individual. The T1 basisternum (Bs) is smaller than normal. c Enlarged view of region outlined in A. d Enlarged view of region outlined in B. The T1 epimera (Epm) and the T3 meso-spinasternum (Ss) are reduced or missing. e Wild-type adult abdomen. f Abdomen of Tc-tiotsh RNAi adult. The third abdominal segment (A3) lacks its anterior process, and an additional heavily sclerotized segment is present anterior to A3
Discussion
We have identified and cloned the lone member of the tsh gene family from Tribolium castaneum. Our analysis of this gene provides important insights into the evolution of the tsh gene family at the levels of both sequence and function.
Evolution of tsh family genes
Searches of several non-drosophilid insect genome sequences identified only a single tsh family member in each genome. These results corroborate previous suggestions (based on more limited data) that most insects have only a single tsh-related gene and that this is the ancestral state (Herke et al. 2005; Laugier et al. 2005). A key implication of this model is that the Drosophila genes tio and tsh are the result of a recent duplication event that occurred sometime after the divergence of mosquitoes and flies. Since that duplication, tsh has apparently diverged from the ancestral sequence faster than has tio (although both have diverged more than the Tribolium and Anopheles homologs). More rapid divergence by one member of a recently duplicated gene pair is not unprecedented. For example, the Hox3 paralogs zerknüllt (zen) and bicoid (bcd) are believed to have arisen by duplication late in the dipteran lineage (Brown et al. 2001; Stauber et al. 1999, 2002), but the sequence of Zen is more similar to that of the Hox 3 proteins of other insects than it is to Bcd.
Much of the sequence conservation between Tsh family members is found in the zinc finger regions. However, a few additional conserved motifs are present, particularly at the protein termini. The C-terminal region is well conserved between Tc-TioTsh, Ag-TioTsh, and Tio, but this conserved region is not found in Tsh. The conservation of the N-terminal region is particularly interesting. The Tsh family members for which N-terminal sequence is available fall into two distinct classes (Fig. 2c). Ag-TioTsh and both Drosophila proteins share an extended region of identity that is completely different than the short motif (MPRRKQ) shared by the Tribolium and vertebrate proteins. This pattern of conservation suggests that an ancestral Tsh family member had the MPRRKQ motif, which was replaced with a different motif sometime before the separation of Anopheles and Drosophila within the dipteran lineage. It is not clear what the function of these disparate motifs might be, as the vertebrate Tsh proteins function very similarly to Dm-Tsh when expressed in Drosophila (Manfroid et al. 2004). Perhaps these motifs are important in a particular tissue or at a different stage in development than those assayed. As additional insect Tsh family protein sequences become available, it will be possible to more accurately determine the point at which these N-terminal sequences began to diverge.
Evolution of tsh family gene expression
Although the sequences of other insect tsh family members are most similar to tio, their expression patterns (where known) seem to be more similar to tsh. The embryonic expression patterns of tsh and tio in Drosophila are strikingly different (Laugier et al. 2005). tsh is expressed throughout the ectoderm of trunk segments, while tio is expressed in only a few cells of the trunk ectoderm (as well as in the Malpighian tubules and parts of the CNS and gut). In tsh mutants, however, tio is more broadly expressed in the trunk, indicating that tsh represses tio expression in this region. Likewise, tsh is ectopically expressed in the stomodeal region when tio expression is knocked down by RNAi.
The Tc-tiotsh expression pattern includes similiarities to both the tsh and tio expression patterns. The ectodermal expression in the segmented region of the germband is reminiscent of, although more extensive than, that of tsh. On the other hand, Tc-tiotsh is expressed in the Malpighian tubule primordia, similar to tio. The expression of tio near the stomodeum might also correspond to the expression of Tc-tiotsh in the labrum and intercalary segment, although it is difficult to compare expression patterns in the cephalic regions of these insects due to the derived structure of the Drosophila larval head.
It is interesting that the Tc-tiotsh expression pattern approximates the sum of the tsh and tio patterns. This may indicate that the expression pattern of the ancestral gene that gave rise to tsh and tio had an expression pattern similar to Tc-tiotsh and that parts of its expression pattern (and perhaps its function) have been split between its daughters. Such division of labor resulting from complementary loss of particular expression domains or functions is termed subfunctionalization and has been suggested to play a key role in the maintenance of duplicated genes (Force et al. 1999).
Among other insects, expression patterns of tsh family members have been reported only for the firebrat, Thermobia domestica, and the milkweed bug, Oncopeltus fasciatus (Herke et al. 2005; Peterson et al. 1999). In both cases, these genes are expressed in the gnathal, thoracic, and first abdominal segments, with very weak (if any) expression in the remainder of the abdomen. Both insects also show expression in the posterior tip of the embryo (see below). Oncopeltus has additional expression in the procephalon. The leg expression pattern in firebrats has not been described, but Herke et al. (2005) reported that milkweed bugs show expression throughout the legs, which fades from the distal tips later in development.
Interestingly, all of the insect tsh family genes whose expression has been described are expressed in the post-abdominal region. However, these domains have been interpreted differently. In firebrats, expression in this region is reported to represent the anlagen of the cerci and the median caudal filament (appendages of the 11th abdominal segment) (Peterson et al. 1999). The posterior expression of tsh has been interpreted as the future anal opening (Fasano et al. 1991), and that of Oncopeltus tiptop is described only as the caudal region (Herke et al. 2005). Our results show that Tc-tiotsh is expressed in the Malpighian tubule primordia. Likewise, tio is expressed in the developing Malpighian tubules (Laugier et al. 2005). Thus, Malpighian tubule expression may have been part of the ancestral expression pattern of this gene family. It is possible that the posterior expression domains in other insects will, upon closer examination, be found to represent the developing Malpighian tubules as well.
Evolution of tsh family function
Tc-tiotsh is required for the proper development of the proximal portion of developing larval legs. Drosophila larvae lack external appendages, but tsh is expressed in the proximal regions of leg imaginal discs and hypomorphic tsh alleles cause proximal leg defects (Erkner et al. 1999). These results suggest that proximal leg expression and function were probably a feature of a tsh-like gene in the ancestor of beetles and flies. In contrast, Herke et al. (2005) reported that knockdown of Oncopeltus tiptop caused transformations of the distal legs to antennae. Based on this observation, they concluded that Oncopeltus tiptop acts as a distal leg selector gene, and suggested that tio or tsh might play a similar role in Drosophila. tsh expression is known to be restricted to the portion of the imaginal disc that will form the body wall and proximal leg segments (Gonzalez-Crespo and Morata 1996). Similarly, Tc-tiotsh is expressed in the proximal region of embryonic legs and leg imaginal discs (Fig. 4d and data not shown). We have also examined the expression of tio in Drosophila leg imaginal discs and found that expression is restricted to the body wall and proximal portion of the developing leg. Thus, it is highly unlikely that the “tiptop-like” distal leg-specifying function suggested by Herke et al. (2005) is performed by Tio, either alone or in combination with Tsh.
We cannot rule out additional, internal functions for Tc-TioTsh. In particular, the expression of Tc-tiotsh in the Malpighian tubules is intriguing, as Drosophila tio (and possibly other homologs) show similar expression.
The striking effects of larval Tc-tiotsh RNAi would not have been predicted based on Drosophila experiments, as relatively few adult effects of LOF tsh mutations have been reported. Other than the proximal leg defects that have been discussed above, the only other adult phenotypes reported are defects in wing posture (Soanes et al. 2001) and in structures that develop from the eye-antennal disc, namely, the eyes (Singh et al. 2002) and the maxillary palps (Bhojwani et al. 1997). This dearth of adult phenotypes could be a consequence of the pre-adult lethality of many tsh allele combinations. However, it might also be due to functional redundancy with tio. We examined tio expression in Drosophila imaginal discs and found that it is indeed expressed in a pattern very similar to that of tsh (data not shown). Thus, loss of both tio and tsh during Drosophila imaginal development might produce a more severe phenotype.
The principal question prompting our studies of the Tribolium Tc-tiotsh gene was whether the trunk-specifying function reported for tsh in Drosophila was conserved in other insects. However, insights from our work and other recent reports compel us to first reexamine whether tsh itself is really a trunk-specifying gene. The key observations, which originally led to this conclusion, were the presence of head-like sclerites in embryos lacking tsh, and the positive correlation between expression of tsh and the presence of denticle belts (a trunk-specific feature) (de Zulueta et al. 1994; Fasano et al. 1991; Roder et al. 1992). The later discovery that tsh and disco form mutually exclusive domains in the trunk and head, respectively, seemed to support this view (Robertson et al. 2004). Below we synthesize recent studies that may lead to new interpretations of these observations.
A number of mutant phenotypes in Drosophila, including those of tsh and some Hox genes, feature small patches of sclerotic tissue. The previous interpretation of such patches as ectopic head tissue has been called into question. Mace et al. (2005) showed that the sclerites that form in a segment lacking Hox gene expression are correlated with breaches in epidermal integrity and the resulting activation of a wound response pathway. If in fact, the sclerotized cuticle seen in tsh mutants is scar tissue rather than head cuticle, the interpretation of tsh as a trunk-specifying gene would be less convincing, but still possible.
The case for tsh acting as a trunk specifier was apparently strengthened when Robertson et al. (2004) proposed the attractive hypothesis that a system of zinc finger proteins acted in conjunction with the Hox genes to specify body regions. Within this system, disco specified gnathal segments and tsh specified trunk segments. However, the same laboratory recently reported that the conserved role of disco is to specify the medial portion of appendages, and suggested that the boundary between disco and tsh expression is actually an artifact of maggot morphology (Patel et al. 2007). That is, the reduced Drosophila larval head might lack proximal, tsh-expressing tissue (body wall) while retaining medial appendage tissue, whereas the trunk retains tsh-expressing tissue but has lost most appendage tissue. Taking this scenario a step further, ectopic expression of tsh might “restore” the body wall tissue of the head, creating larger, more complete segments. As the genes controlling segment formation also control denticle belt patterning, these enlarged head segments would have denticle belts as well. This interpretation is consistent with the observation that the additional denticle belts produced by ectopic tsh expression are interspersed with head-specific structures (de Zulueta et al. 1994). When considered in this light, perhaps tsh is no more a trunk identity gene than are other genes involved in segment formation and denticle patterning such as wingless, rhomboid, or shavenbaby. On the other hand, tsh does seem to provide the spatial information that limits full segment formation to the trunk, and thus in Drosophila could be considered a trunk-specifying gene.
In contrast, Tc-tiotsh does not appear to act in any way as a trunk identity gene. This is not to say that Tc-tiotsh has no role in the development of trunk segments, but rather that we find no evidence that it specifies trunk vs head fates. In embryos depleted of Tc-tiotsh the ventral portions of the labial and thoracic segments are reduced, but not visibly transformed. Interestingly, some of the labial abnormalities observed in Tc-tiotsh RNAi larvae are reminiscent of those produced by loss of function alleles of prothoraxless (Tc-Antp) (Shippy, unpublished data). Knockdown of Tc-tiotsh during imaginal development produces adults with patterning defects in the thoracic and abdominal sclerites, including possible transformations of anterior abdominal sclerites to a more posterior identity. These phenotypes, together with the reduction in Tc-tiotsh expression seen in Df(HOMC) embryos, suggest that Tc-tiotsh is regulated to some extent by Hox genes and may carry out a subset of the functions of these genes in both the head and the trunk.
Our studies of Tc-tiotsh indicate that trunk specification is not a universal function of tsh family members. It is possible that this function has been lost in Tribolium. However, expression pattern data from other insects and functional analysis in Oncopeltus argues against this conclusion. A more likely scenario is that tsh family members acquired a role in defining trunk at some point after the divergence of the coleopteran and dipteran lineages. Analysis of tsh function in additional holometabolous insects should help pinpoint when this event occurred.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
Dynamic expression of Tc-tiotsh during germband elongation. Tc-tiotsh expression always appears in an odd-numbered parasegment and the anterior compartment of the adjacent posterior even-numbered parasegment before it is detected in the posterior compartment of the adjacent anterior even-numbered parasegment. An arrowhead marks the mandibular Engrailed stripe in each embryo, while numbers indicate parasegment identity. aTc-tiotsh expression is first observed spanning the mandibular Engrailed stripe. The mesoderm, which has not yet invaginated at the ventral midline, does not show Tc-tiotsh expression. bTc-tiotsh is expressed in PS3 and the anterior compartment of PS4, but not yet in the posterior compartment of PS2. c An embryo with a gap in Tc-tiotsh expression in the posterior compartment of PS10. Tc-tiotsh expression is also visible in the intercalary segment and the developing labrum. d An embryo in which the gap in Tc-tiotsh expression corresponds to the posterior compartment of PS12 (GIF 0 kb)
Fig. S2
Tc-tiotsh and Tc-Even-skipped (Tc-Eve) expression. Tc-Eve expression is shown in brown, while Tc-tiotsh expression is in purple. Embryos are shown in ventral view with anterior to the left. Numbers refer to Tc-Eve stripes. a The first Tc-Eve primary stripe has resolved into two secondary stripes, 1a and 1b. The Tc-tiotsh expression domain at this time covers not only the Tc-Eve first primary stripe region, but also the region anterior to it. The second Tc-Eve primary stripe has formed too, but no Tc-tiotsh expression is present in this region yet. Tc-tiotsh is also expressed in the amnion. b The second Tc-Eve primary stripe is resolving into 2a and 2b, but there is still no Tc-tiotsh expression in this region. The expression of Tc-tiotsh in the amnion is still visible at this stage. c The third Tc-Eve primary stripe has formed and is just beginning to split into secondary stripes. A new stripe of Tc-tiotsh expression covers the region delimited by Tc-Eve stripes 2a and 2b (PS3 and the anterior compartment of PS4). No Tc-tiotsh expression is seen in the posterior compartment of PS2 yet. d A much older embryo than in A, B, and C shows the same relationship between Tc-Eve and Tc-tiotsh expression in the posterior region of the embryo (GIF 0 kb)
Fig. S3
Tc-sal expression. Anterior is to the top. In a wild-type embryo in which the abdominal segments are just beginning to form, Tc-sal is expressed in most of the gnathal and thoracic segments (GIF 0 kb)

teashirt trunk tiptop tribolium homeotic


Clinical review: Treatment of heat stroke: should dantrolene be considered?

Rapid and efficient cooling is the most important therapeutic objective in patients with heat stroke (HS). This article reviews the mechanism of action and rationale for the use of dantrolene as a potential supportive cooling method in the treatment of HS. Relevant studies were included to support discussion of the role of dantrolene for the treatment of HS. In some studies dantrolene was shown to accelerate cooling rate when administered after the development of exertional HS. Dantrolene was also found to be effective in reducing the extent of HS signs when given as pretreatment in an animal model. Accumulated data do not support the routine use of dantrolene as an adjuvant cooling technique in HS, but administration of this drug in severe cases, or in which no improvement is observed, appears rational. Further trials are needed in order to assess the true effectiveness of dantrolene in HS.
Introduction
Hyperthermia is defined as any core temperature rise to above the hypothalamic set-point at which heat-dissipating mechanisms are impaired. Normal core temperature values are in the 36.5–37.5°C range at rest and can rise to 40°C during strenous exercise. Hyperthermia is usually caused by an imbalance between total heat (metabolic and environmental heat) accumulated and total heat lost from the body. Hyperthermia per se may be physiological and can be compensated for (as in the moderate elevation in core temperature that occurs in an exercising and otherwise asymptomatic individual), or it may be pathophysiological but generally well tolerated. It may also be associated with adverse pathophysiological consequences such as inability to continue physical exertion (heat exhaustion). At the extreme, hyperthermia represents a state in which the elevation in core temperature is either already accompanied by organ injury or is sufficient to produce such injury if left untreated.
Several syndromes have been reported to be associated with extreme hyperthermia, including malignant hyperthermia (MH), neuroleptic malignant syndrome (NMS) and heat stroke (HS). The mainstay of treatment of these syndromes includes the administration of basic resuscitative measures together with simultaneous cooling aimed at reducing body temperature [1,2]. Cooling may be achieved by several methods, such as immersion in cold water and evaporation of water over the skin [3-7]. Because heat is primarily produced by hyperactivity of muscles in the various syndromes, dantrolene sodium, a muscle relaxant, has also been suggested to accelerate cooling [8-10]. In MH, dantrolene administration resulted in a rapid reduction in mortality rate, and therefore it is now considered as an essential part of treatment in this syndrome [11-13]. Dantrolene has also been recommended for the treatment of NMS [14]. To date, no drug has shown significant efficacy in improving the outcome of patients with HS.
Experience with dantrolene use in HS is in its initial phases (Tables 1,2,3). In some studies a possible benefit from dantrolene treatment was reported, whereas in others it was not found to be effective [15-22]. We reviewed relevant studies concerning dantrolene use in HS in order to establish its role in the treatment of this condition.
Studies included in this review were identified by means of a comprehensive Medline search, limited to the English language literature, using the following keywords: 'dantrolene' and 'hyperthermia' or 'heat'. Each article identified was further examined for relevancy.
Dantrolene
Dantrolene sodium is a hydantoin derivative. It acts directly on muscle contractile elements, attenuating the amount of calcium released from the sarcoplasmic reticulum of skeletal muscle to the cytosol [23-25]. As a result, calcium dependent excitation–contraction coupling and consequent muscle contraction are inhibited.
The mean biological half-life of dantrolene in healthy volunteers or patients was reported to range from 6 to 9 hours, with extremes of 3 and 22 hours [25-30]. It is primarily metabolized in the liver, with 15–25% of the dose administered being excreted by the kidneys [27,29,30]. Dantrolene is marketed as Dantrium (Norwich Eaton Pharmaceuticals, Norwich, NY, USA) and can be administered intravenously (in solution with mannitol and sodium hydroxide) or orally.
When dantrolene is used as an emergency therapy, such as in acute hyperthermic syndromes, it is unlikely to cause major adverse effects [25]. However, prolonged use may be associated with undesirable side effects that include weakness, drowsiness, diarrhea and malaise [25]. Hepatotoxicity, which can be irreversible, is also a major concern. The risk for hepatic injury appears to be about twofold greater in females, in patients receiving doses over 300 mg/day and in those treated for over 60 days [31]. In addition, dantrolene sodium solution is highly alkaline (pH 9.6), and intravenous injection may cause extravasation and tissue necrosis [25].
Heat stroke and 'thermic stress syndrome'
HS is a life-threatening illness characterized by an elevation in core body temperature to above 40°C with central nervous system (CNS) dysfunction that results in delirium, convulsions, or coma [32,33] in the absence of any other cause of CNS dysfunction, and skin dryness. The affected individual may suffer from a characteristic multiple organ clinical and pathological syndrome caused by temperature-induced tissue damage [1,34]. HS results from exposure to a high environmental temperature, in which case it is called 'classic' heat stroke (CHS; or 'nonexertional' HS), or from strenuous exercise, in which case it is called 'exertional' heat stroke (EHS) [34-36]. CHS is usually seen in the very young or elderly and in poor socioeconomic settings with limited access to air conditioning. EHS is seen more commonly in exercising individuals, for instance soldiers in army training. Further reviews on HS are available [32-36].
Epidemiological incidence data for HS is imprecise because of varying definitions of heat-related death and under-diagnosis [35]. One study conducted in urban USA found a variation from 17.6 to 26.5 cases per 100,000 capita. Data from Saudi Arabia show a seasonal incidence pattern, ranging from 22 to 250 cases per 100,000 capita. The crude mortality rate in Saudi Arabia has been estimated at 50%. Treatment consists of immediate cooling and support of organ system function.
Some authors believe that HS, as well as MH and other hyperthermic syndromes, are different manifestations of a broader underlying condition called 'thermic stress syndrome' [9,10,15,17,37]. This common malady, which is triggered by different mechanisms, is characterized by an extreme elevation in body temperature and CNS dysfunction, which may be complicated by bleeding diathesis, elevated serum enzymes levels and renal failure. Because dantrolene was found to be effective in the treatment of MH and NMS, its usage has also been recommended for the treatment of HS [8-10,15].
Dantrolene use in heat stroke
Two reports from the early 1980s demonstrated a beneficial effect from using dantrolene in HS. Lydiatt and Hill [15] reported on a patient with EHS who responded favourably to dantrolene (4 mg/kg intravenously) after sympthomatic therapy had failed. In a case report, Denborough [8] reported that intravenous dantrolene (20 mg bolus) therapy produced rapid recovery in a young soldier suffering from EHS after a march.
In a controlled study, the adjuvant effects of dantrolene in comparison with passive cooling were investigated in a canine model of CHS [16]. Intravenous administration of dantrolene (5 mg/kg) did not significantly improve cooling rates, haemodynamic parameters, pathological changes, or clinical outcome when compared with passive cooling.
Channa and coworkers [17] conducted a study in which 20 EHS patients were randomly assigned to two groups with only one group receiving dantrolene (2.45 mg/kg intravenously). Patients in both groups were cooled either using the Makkah body cooling unit or by 'conventional methods', namely spraying of tap water over the patients while fanning. Cooling rates were found to be significantly higher in the dantrolene group. All patients survived and there was no difference in the incidence of neurological sequelae between the groups.
The effects of dantrolene were also evaluated in 53 patients suffering from CHS [21]. All patients were treated by using a body cooling unit in which atomized water was sprayed over the victim while they were fanned with hot air. In addition, treatment with either dantrolene (2 mg/kg) or placebo was given intravenously after blind, randomized selection.
Dantrolene administration did not result in any enhancement of the cooling rate, and there was no significant difference in the number of hospital stays between the two groups. It should be noted that in this study both treatment groups achieved satisfactory cooling in under 1 hour. Therefore, a possible positive effect of dantrolene in extreme cases of HS, in which cooling times might be longer, cannot be ruled out. It should further be noted that in this study Bouchama and coworkers [21] treated their patients with dantrolene and mannitol simultaneously. Thus, it could not be concluded whether the reported positive effects were attributed to dantrolene or to mannitol.
Mannitol may be beneficial in the setting of HS because of its effect on expanding the intravascular volume and reducing tissue injury after ischemia by scavenging oxygen free radicals [38,39]. In order to address this question, Zuckerman and coworkers [19] designed a randomized controlled study in which heat stroked piglets were treated by one of the following four methods: passive cooling, 'conventional cooling' (intravenous fluid resuscitation, sponging with tap water, mechanical fanning and ice gastric lavage), conventional cooling along with dantrolene, and conventional cooling along with mannitol. Although dantrolene significantly shortened the cooling time when compared with conventional cooling, it did not shorten the cooling time when compared with mannitol. Moreover, it was shown that dantrolene did not improve cardiovascular parameters when administered in addition to conventional methods.
Tayeb and Marzouki [20] investigated whether dantrolene might be advantageous in prophylaxis against HS in exercising sheep. They found that pretreatment with dantrolene (3 mg/kg and 1.5 mg/kg intravenously) caused a significant reduction in body temperature, and decreased the extent of HS signs as well as some of the induced changes in enzymes and hormones. These effects were more significant in the group receiving dantrolene 3 mg/kg than in the group receiving 1.5 mg/kg. The dantrolene group also exhibited a trend toward reduced mortality from HS (0/12) in comparison with the group that did not receive dantrolene treatment (2/6).
Using a rat model of EHS and CHS, Moran and coworkers [18] studied the efficacy of dantrolene both as a prophylactic agent and as a therapeutic drug. Administration of high doses of dantrolene (140 mg/kg intravenously) before heat stress delayed the development of HS in the sedentary animals. Estimated temperatures were lower in the exercising rats that received Dantrolene pretreatment, but this was most reasonably attributed to the limited physical capacity induced by dantrolene. When dantrolene, at the same dose, was administered after the development of HS, it significantly improved the cooling rate in the exercising rats – an effect that was not seen in the sedentary group.
Discussion
The rationale for the use of dantrolene in HS emerged from the overlap in the systemic manifestations of HS and other hyperthermic syndromes, particularly MH. However, some researchers claimed that, although hyperthermia was characteristic of both MH and HS, these syndromes represent two clinical entities with different heat generating cellular mechanisms. Although MH is characterized by muscular rigidity, which causes active heat production, HS usually manifests in the form of flaccidity, and heat production does not continue uncontrolled [17,40]. Therefore, dantrolene, which uncouples the excitation–contraction sequence heat generating mechanism, may not be beneficial in the treatment of HS. Moreover, MH is a genetically transmitted disease, which is triggered by several depolarizing muscle relaxants or volatile anaesthetic agents [41,42]. In comparison, to the best of our knowledge, no genetic predisposition has been definitely incriminated in the typical HS patient.
However, several reports suggest a possible link between HS and MH. Tobin and coworkers [43] reported a case of fatal HS in a patient who experienced MH 8 years earlier and speculated whether other HS victims are MH susceptible. An abnormal caffeine–halothene contracture test, which is considered indicative for MH susceptibility, was found in more than 40% of individuals who survived EHS, whereas less than 1% of the general population exhibited this response [44]. It is important to note that adequate recovery time must be given before performing such tests after a bout of rhabdomyolysis. Muscle function takes considerable time to recover and, inferentially at least, so does muscle metabolism. Therefore, care must be taken before relying on post-HS muscle contracture tests. A possible underlying myopathy in EHS patients was demonstrated by Bendahan and coworkers [45], who found abnormal muscle energetics in such patients by using a 31P magnetic resonance spectroscopy. Therefore, there is some evidence to suggest that a subset of individuals with HS may also be predisposed to MH. Theoretically, this subset of individuals might continue to have a relatively high level of heat production even after collapse. They may also be at increased risk for EHS because an attenuated muscular efficiency can increase muscular caloric production at the same absolute work intensity. The fact that some individuals with HS might have a contribution of MH-type pathophysiology might also explain why there has been anecdotal evidence suggesting a possible benefit from dantrolene in selected cases.
The above studies indicate that the cooling rate was the only parameter among those evaluated that improved when dantrolene was administered after the development of HS [17-19]. However, this effect was not demonstrated in all studies. The diversity of results may be accounted for by the models investigated. When dantrolene was administered in an exercising model, it was found to be more effective than in a CHS model. This is not surprising because dantrolene inhibits muscle contraction, which does not play a role in the pathophysiology of CHS.
Collectively, most of the studies suggest that dantrolene is not beneficial in reducing mortality rate when it is given for the treatment of HS. However, survival rates in EHS currently approach 95%, and the studies were not large enough to exhibit a reduced mortality [1,46]. Moreover, some reports suggest that dantrolene may even prevent death in cases of severe HS [15,17]. For example, one patient in the study conducted by Channa and coworkers [17] who received dantrolene made a complete recovery in spite of an initial core temperature of 44°C. An EHS patient described by Lydiatt and Hill [15] had a rectal temperature of 42°C for more than 30 min and responded only to dantrolene. Noteworthy, the authors did not state the upper recording limit of their thermometer. If a clinical thermometer has an upper limit of 42°C, then it will record 42°C when the actual temperature is 46°C. Despite successful cooling for 30 min, the thermometer still shows 42°C even though the temperature has been reduced by 4°C. Taking dantrolene at this instant will lead to an impressive, albeit misinterpreted, result.
Some researchers indicated that the relative ineffectiveness of dantrolene in some of the studies may be related to an inadequate dose [21,47]. Dantrolene was administered in doses of 2–5 mg/kg, which were selected according to recommended protocols with regard to the use of this agent in MH. However, the optimal dose of dantrolene in MH is still questionable. Although Britt [48] reported a complete recovery from MH when dantrolene was used at a dose of 6 mg/kg, others [31] postulated that an increased dose of dantrolene may be associated with hepatotoxicity. The risk for hepatotoxicity is even more concrete in HS patients because the liver is commonly injured in HS and increasing the dose of dantrolene may worsen this hepatic damage. Nevertheless, hepatotoxicity has never been reported in HS victims who received dantrolene.
Despite the absence of outcome-based data, it might nevertheless be pathophysiologically rational to try dantrolene in selected cases. Dantrolene may be beneficial where there is evidence of ongoing excessive heat production, such as in cases of HS that are accompanied by muscular rigidity. In these cases, the apparent HS may be misdiagnosed, and the patient may actually be suffering from MH or NMS. Finally, dantrolene might also have a role to play in limiting muscle injury and its consequences in rhabdomyolysis by impairing calcium release from the sarcoplasmic reticulum [49,50]. Loss of calcium homeostasis in muscle cells may be associated with muscle injury resulting from impairment in mitochondrial respiration and ATP production, activation of phospholipase A2 with production of leukotrienes and prostaglandins, increased production of free radicals, and activation of calcium-activated proteases [49]. Although there is no evidence that dantrolene reduces the extent of muscle injury in EHS, it was found to protect against muscle injury associated with exercise in several studies [51-54].
Dantrolene pretreatment reduced the severity of HS signs in an exercising animal model. This effect may be attributed to decreased muscular production of metabolic heat or to an unknown modulation of neurotransmitter release [20]. However, the effect of dantrolene on muscle contracture may result in limited physical capacity. Furthermore, the administration of dantrolene before HS is more theoretical than real because the incidence of EHS is negligible in comparison with the number of individuals who are engaged in physical activity and/or are exposed to heat strain.
Conclusion
Therapy with the accepted MH prophylactic dose of 2–5 mg/kg resulted in a possible benefit from dantrolene treatment in HS in some studies, whereas in others it was not found to be effective. The limited literature does not support the routine use of dantrolene as an adjuvant cooling technique in the setting of HS. Nevertheless, because dantrolene does appear to increase cooling rate, furthur studies are advised for dantrolene use in severe cases or cases in which no improvement is observed with other cooling methods. Wider trials are still needed to evaluate the efficacy of dantrolene in reducing mortality, thereby justifying its use in HS. Noteworthy, dantrolene should not be used as a single cooling method, and its administration should only be in addition to the well established 'conventional methods'.
Competing interests
None declared.
Abbreviations
CHS = classic heat stroke; CNS = central nervous system; EHS = exertional heat stroke; HS = heat stroke; MH = malignant hyperthermia; NMS = neuroleptic malignant syndrome.

heat stroke
dantrolene
cooling
hyperthermia
temperature
exertional heat stroke