Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.     

Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (Apis mellifera)

Axis formation is a key step in development, but studies indicate that genes involved in insect axis formation are relatively fast evolving. Orthodenticle genes have conserved roles, often with hunchback, in maternal anterior patterning in several insect species. We show that two orthodenticle genes, otd1 and otd2, and hunchback act as maternal anterior patterning genes in the honeybee (Apis mellifera) but, unlike other insects, act to pattern the majority of the anteroposterior axis. These genes regulate the expression domains of anterior, central and posterior gap genes and may directly regulate the anterior gap gene giant. We show otd1 and hunchback also influence dorsoventral patterning by regulating zerknült (zen) as they do in Tribolium, but that zen does not regulate the expression of honeybee gap genes. This suggests that interactions between anteroposterior and dorsal-ventral patterning are ancestral in holometabolous insects. Honeybee axis formation, and the function of the conserved anterior patterning gene orthodenticle, displays unique characters that indicate that, even when conserved genes pattern the axis, their regulatory interactions differ within orders of insects, consistent with relatively fast evolution in axis formation pathways.     

Breakdown of abdominal patterning in the Tribolium Kruppel mutant jaws

During Drosophila segmentation, gap genes function as short-range gradients that determine the boundaries of pair-rule stripes. A classical example is Drosophila Krüppel (Dm'Kr) which is expressed in the middle of the syncytial blastoderm embryo. Patterning defects in Dm'Kr mutants are centred symmetrically around its bell-shaped expression profile. We have analysed the role of Krüppel in the short-germ beetle Tribolium castaneum where the pair-rule stripes corresponding to the 10 abdominal segments arise during growth stages subsequent to the blastoderm. We show that the previously described mutation jaws is an amorphic Tc'Kr allele. Pair-rule gene expression in the blastoderm is affected neither in the amorphic mutant nor in Tc'Kr RNAi embryos. Only during subsequent growth of the germ band does pair-rule patterning become disrupted. However, only segments arising posterior to the Tc'Kr expression domain are affected, i.e. the deletion profile is asymmetric relative to the expression domain. Moreover, stripe formation does not recover in posterior abdominal segments, i.e. the Tc'Kr(jaws) phenotype does not constitute a gap in segment formation but results from a breakdown of segmentation past the 5th eve stripe. Alteration of pair-rule gene expression in Tc'Kr(jaws) mutants does not suggest a direct role of Tc'Kr in defining specific stripe boundaries as in Drosophila. Together, these findings show that the segmentation function of Krüppel in this short-germ insect is fundamentally different from its role in the long-germ embryo of Drosophila. The role of Tc'Kr in Hox gene regulation, however, is in better accordance to the Drosophila paradigm.     

Maxillopedia is the Tribolium ortholog of proboscipedia

SUMMARY Null mutations in the Drosophila melanogaster homeotic gene proboscipedia ( pb ) cause transformation of the adult labial palps to legs. The similar phenotype produced by mutations in the Tribolium castaneum homeotic complex (HOMC) gene maxillopedia ( mxp ) has led to suggestions that the two genes may be orthologous. We have cloned the Tribolium ortholog of pb , which predicts a protein with a homeodomain identical to that of Drosophila Pb. The two proteins also share several additional regions of identity, including an N-box, a motif unique to Pb orthologs. We have identified a frameshift mutation within Tribolium pb associated with an mxp null mutation, demonstrating that Tribolium pb corresponds to the mxp genetic locus. Thus, we will refer to the cloned gene as mxp . In addition, we have begun to construct a molecular map of the Tribolium HOMC. Two overlapping BAC clones which span the mxp locus also include the Tribolium labial ortholog ( Tclabial   ) and part of Tczerknüllt , indicating that the order of these genes in the HOMC is conserved between Drosophila and Tribolium.     

The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification

The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.     

Maternal torso signaling controls body axis elongation in a short germ insect

In the long germ insect Drosophila, all body segments are determined almost simultaneously at the blastoderm stage under the control of the anterior, the posterior, and the terminal genetic system . Most other arthropods (and similarly also vertebrates) develop more slowly as short germ embryos, where only the anterior body segments are specified early in embryogenesis. The body axis extends later by the sequential addition of new segments from the growth zone or the tail bud . The mechanisms that initiate or maintain the elongation of the body axis (axial growth) are poorly understood . We functionally analyzed the terminal system in the short germ insect Tribolium. Unexpectedly, Torso signaling is required for setting up or maintaining a functional growth zone and at the anterior for the extraembryonic serosa. Thus, as in Drosophila, fates at both poles of the blastoderm embryo depend on terminal genes, but different tissues are patterned in Tribolium. Short germ development as seen in Tribolium likely represents the ancestral mode of how the primary body axis is set up during embryogenesis. We therefore conclude that the ancient function of the terminal system mainly was to define a growth zone and that in phylogenetically derived insects like Drosophila, Torso signaling became restricted to the determination of terminal body structures.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

A dual role for nanos and pumilio in anterior and posterior blastodermal patterning of the short-germ beetle Tribolium castaneum

Abdominal patterning in Drosophila requires the function of Nanos (nos) and Pumilio (pum) to repress posterior translation of hunchback mRNA. Here we provide the first functional analysis of nanos and pumilio genes during blastodermal patterning of a short-germ insect. We found that nos and pum in the red flour beetle Tribolium castaneum crucially contribute to posterior segmentation by preventing hunchback translation. While this function seems to be conserved among insects, we provide evidence that Nos and Pum may also act on giant expression, another gap gene. After depletion of nos and pum by parental RNAi, Hunchback and giant remain ectopically at the posterior blastoderm and the posterior Krüppel (Kr) domain is not being activated. giant may be a direct target of Nanos and Pumilio in Tribolium and presumably prevents early Kr expression. In the absence of Kr, the majority of secondary gap gene domains fail to be activated, and abdominal segmentation is terminated prematurely. Surprisingly, we found Nos and Pum also to be involved in early head patterning, as the loss of Nos and Pum results in deletions and transformations of gnathal and pre-gnathal anlagen. Since the targets of Nos and Pum in head development remain to be identified, we propose that anterior patterning in Tribolium may involve additional maternal factors.     

The Tribolium ortholog of knirps and knirps-related is crucial for head segmentation but plays a minor role during abdominal patterning

Segment formation in the long germ insect Drosophila is dominated by overlapping gap gene domains in the syncytial blastoderm. In the short germ beetle Tribolium castaneum abdominal segments arise from a cellular growth zone, implying different patterning mechanisms. We describe here the single Tribolium ortholog of the Drosophila genes knirps and knirps-related (called Tc-knirps). Tc-knirps expression is conserved during head patterning and at later stages. However, posterior Tc-knirps expression in the ectoderm is limited to a stripe in A1, instead of a broad abdominal domain covering segment primordia A2-A5 as in Drosophila. Tc-knirps RNAi yields only mild defects in the abdomen, at a position posterior to the abdominal Tc-knirps domain. In addition, Tc-knirps RNAi larvae lack the antennal and mandibular segments. These defects are much more severe than the head defects caused by combined inactivation of Dm-knirps and Dm-knirps-related. Our findings support the notion that the role of gap gene homologs in abdominal segmentation differs fundamentally in long and short germ insects. Moreover, the pivotal role of Tc-knirps in the head suggests an ancestral role for knirps as head patterning gene. Based on this RNAi analysis, Tc-knirps functions neither in the head nor the abdomen as a canonical gap gene.     

Operons Are a Conserved Feature of Nematode Genomes

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.