cis-Regulatory Sequences and Functions (page 1/2)

Even-skipped homologs in other arthropods

Drosophila typifies the so-called long germband mode of insect development, in which the pattern of segments is established by the end of the blastoderm stage. In contrast, the short germband insects, such as the grasshopper, generate all or most of their metameric pattern after the blastoderm stage by the sequential addition of segments during caudal elongation. This difference is discernible at the molecular level in the pattern of initiation of the segment polarity gene engrailed, and the homeotic gene abdominal-A. For example, in both types of insects, engrailed is expressed by the highly conserved germband stage in a pattern of regularly spaced stripes, one stripe per segment. In Drosophila, the complete pattern is visible by the end of the blastoderm stage, although engrailed appears initially in alternate segments in a pair-rule pattern that reflects its known control by pair-rule genes such as even-skipped. In contrast, in the grasshopper, the engrailed stripes appear one at a time after the blastoderm stage as the embryo elongates. Grasshopper even-skipped does not serve a pair-rule function in early development, although it does have a similar function in both insects during neurogenesis later in development (Patel, 1992).

In the honeybee Apis mellifera, the even-skipped ortholog is expressed in both primary and secondary stripes in roughly anterior-to-posterior succession; there are 8 primary (pair-rule) and 16 secondary (parasegmental) stripes. The most posterior primary stripes appear only after the onset of gastrulation. The secondary stripes form by a splitting of primary stripes; they demarcate the parasegmental pattern. While these finding resemble Eve expression in long-germ beetles, the honeybee differs from both beetles and dipterans by two transient pair-rule traits in the parasegmental Eve pattern: the secondary stripes in head and thorax alternate in strength, yet are out of register with the Drosophila pattern: over the whole pattern the odd-numbered stripes vanish earlier than their even-numbered counterparts. As in Drosophila, however, the strong Eve stripes coincide with the weak engrailed stripes. These findings are taken to indicate that (1) honeybee and beetles share a conserved mode of Eve stripe formation while Drosophila has diverged in this respect; (2) honeybee and Drosophila have diverged from the beetles in specific pair-rule traits during the parasegmental expression of both Eve and En, and (3) some of these traits differ in the register of segment pairing and thus may reflect regulatory differences at the pair-rule level between dipterans and the honeybee (Binner, 1997).

Developmental processes have been traditionally viewed to be invariant within higher taxa. However, examples are known whereby closely related species exhibit alterations in early embryogenesis yet appear very similar as adults. Such developmental changes are thought to occur in response to shifts in life history. In insects, the regulation of embryonic development has been intensively studied in model species, such as Drosophila melanogaster. Previous comparative studies suggest that the developmental processes documented in Drosophila adequately describe embryogenesis of advanced, holometabolous, insects generally. There have been few attempts, however, to take into account how life history has influenced early development of insects or to characterize early development of species with life histories fundamentally different from flies. A comparison was carried out of early development in two species from the same family of parasitic wasps that exhibit very different life histories. Bracon hebetor is an ectoparasite that lays large, yolky eggs on the integument of its host that develop much like the free-living honeybee and Drosophila. In contrast, Aphidius ervi is an endoparasite that lays small and apparently yolk-free eggs that develop in the hemocoel of the host. This wasp exhibits a radically different mode of early development at both the cellular and molecular level from B. hebetor. The developmental changes in A. ervi reflect functional adaptations to its derived endoparasitic life history and argue that departures from the fly paradigm may occur commonly among insects whose eggs develop under conditions different from typical terrestrial species (Grbic, 1998).

To compare patterning events at the molecular level, B. hebetor and A. ervi embryos were stained with antibodies that recognize conserved epitopes of Eve, En, and Ubx/Abd-A in different insect species. Eve, a primary pair-rule gene is expressed in the Drosophila syncytium, forms a characteristic seven-stripe pattern with double segment periodicity. En, which is regulated by Eve, is a segment polarity gene that specifies the posterior segmental compartments. Ubx and Abd-A are Drosophila homeotic proteins that specify the posterior thorax and abdomen. In B. hebetor, Eve is expressed in a largely conserved fashion when compared to Drosophila and other long germband insects. Initially, a broad domain of Eve expression splits into broad pair-rule stripes, followed by a split of the individual pair-rule stripes in rapid anteroposterior progression to form segmentally iterated stripes. After germband retraction, Eve localizes in the cells of the dorsolateral mesoderm and neurons; a pattern conserved in all examined insects. In B. hebetor, En is expressed in a rapid anteroposterior progression, forming a mature pattern of segmentally iterated stripes that localize to the posterior segmental compartments. The antibody against Ubx/Abd-A stains the region from the posterior thorax to the penultimate abdominal segment. When A. ervi embryos are stained with anti-Eve, neither a pair-rule nor a segmental pattern is detected. In the extended germband, however, an Eve antigen is detected in dorsolateral mesoderm and neurons. En stripes appear when embryos initiate germband extension. These stripes form sequentially as the germband extends, resulting in a mature pattern of segmentally iterated stripes that localize to the posterior segmental compartments. Ubx/Abd-A is expressed in the posterior thorax and abdomen in the retracted germband stage. Thus, despite the divergence of early patterning, late patterning in B. hebetor and A. ervi includes conserved expression of En and Ubx/Abd-A. This expression pattern in the germband, the phylotypic stage in insects, suggests conservation of this stage irrespective of how development begins (Grbic, 1998).

In short germ insects, the procephalon and presumptive anterior segments comprise most of the embryonic rudiment which lengthens as posterior segments are added during development. The expression pattern of a grasshopper ortholog of the primary pair-rule gene even-skipped suggests that it is not relevant to segmentation in this short germ insect. However in Drosophila, a long germ insect that forms all segments simultaneously, eve plays a vital role in segment formation. The eve ortholog of the beetle Tribolium castaneum (Tc eve) has been cloned. Tribolium has been considered either a short germ insect (because the abdominal segments are added after formation of the germ rudiment) or intermediate germ (because the procephalon, gnathals and thorax are defined in the germ rudiment) but never a long germ insect like Drosophila (see Tribolium early embryonic development). The homeodomain sequence is highly conserved between beetle, fly, and grasshopper eve orthologs. Tc eve is expressed in stripes during segmentation, but in a pattern differing in some details from that of the fly gene. In both insects eve is expressed in the anterior part of each parasegment. However, in Tribolium this expression appears equivalent in both even and odd numbered parasegments. In contrast, Drosophila expression in the even numbered parasegments arises later in development, after expression has taken place in odd numbered parasegments. This pattern is coincident with that detected with a cross-reacting antibody. Tribolium has a fushi tarazu ortholog in its Homeotic complex, but embryos deficient for this region do not have a pair-rule phenotype. Perhaps in the evolution of Drosophila ftz assumed a function in even numbered parasegments that was performed by an ancestral eve ortholog. Thus, an ancestral even-skipped gene appears to have evolved a role in segmentation in a common ancestor of flies and beetles. Central nervous system expression of even-skipped orthologs is of ancient origin. CNS expression preceded the separation of the protostomes and deuterostomes. Unlike vertebrate orthologs but similar to eve, Tc eve is not linked to the homeotic complex. In humans and mice, eve orthologs have been mapped to the 5' end of the homeotic complex consistent with the colinearity between chromosome location and the area of function (the anterior domain). Since Tc eve and Drosophila eve are not linked to the HOM-C complexes of the two species, it appears that eve was lost from the complex before the divergence of beetles and flies, and assumed a new role in segmentation found in Drosophila and Tribolium (Brown, 1997).

There is an ongoing discussion as to whether segmentation in different phyla has a common origin sharing a common genetic program. However, before comparing segmentation between phyla, it is necessary to identify the ancestral condition within each phylum. Even within the arthropods it is not clear which parts of the genetic network leading to segmentation are conserved in all groups. In this paper, the expression of three segmentation genes of the pair-rule class is examined in the spider Cupiennius salei. Spiders are representatives of the Chelicerata, a monophyletic basic arthropod group. During spider embryogenesis, segments are sequentially added at the posterior end of the embryo, which resembles the formation of the abdominal segments in short-germ insect embryos. In spider embryos, the orthologues for the Drosophila primary pair-rule genes hairy, even-skipped, and runt are expressed in stripes in the growth zone, where the segments are forming, suggesting a role for these genes in chelicerate segmentation. These data imply that the involvement of hairy, even-skipped, and runt in arthropod segmentation is an ancestral character for arthropods and is not restricted to a particular group of insects (Damen, 2000).

A remarkable feature of the Cs-Hairy sequence is the change in the conserved carboxyl-terminal tetrapeptide WRPW found in the Hairy family of basic helix-loop-helix transcription factors, which include the Hairy, Deadpan, and Enhancer of split proteins. The WRPW tetrapeptide is changed to WRPF in Cs-H. The WRPW motif is required for interaction with the corepressor Groucho and for transcriptional repression. It is not known whether this 1-aa change in the tetrapeptide affects a putative interaction of Cs-H with Groucho. Runt domain proteins contain a very similar carboxyl-terminal motif, WRPY, which also is required for Groucho-dependent repression in Drosophila (Damen, 2000).

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. Chromophore-assisted laser inactivation (CALI) was employed to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. In this technique, a specific monoclonal antibody against eve that is coupled to a chromophore is injected into the early embryo. The chromophore is then activated by laser light, yielding highly reactive and spatially restricted radicals that destroy the protein to which the antibody is bound. It is possible to generate pair-rule type phenocopies with defects in alternating segments. The defective embryo lacks the mandibular and labial head segments, as well as thoracic segment T2, i.e. alternating segments in the anterior region. Segment T1 is also affected as it shows only one of the two legs. This can be understood in view of the fact that eve is likely to also have a segmental function, although this is apparently less sensitive to a partial loss of the protein. The fact that more posterior segments are not affected can be explained by the successive generation of the eve stripes during germ band elongation. Since the laser treatment occurs only during one particular time of development, one would expect that only those segments are affected that require eve function at that time. That the development of the more posterior segments is independent of the development of the more anterior segments has been shown for short germ embryos. Defects in odd numbered segments are found in Tribolium and not in even numbered ones, as is the case in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve takes place at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. It is concluded that the primary function of eve as a pair rule gene is conserved between the two species (Schroder, 1999).

Morphological studies suggest that insects and crustaceans of the Class Malacostraca (such as crayfish) share a set of homologous neurons. However, expression of molecular markers in these neurons has not been investigated, and the homology of insect and malacostracan neuroblasts, the neural stem cells that produce these neurons, has been questioned. Furthermore, it is not known whether crustaceans of the Class Branchiopoda (such as brine shrimp) or arthropods of the Order Collembola (springtails) possess neurons that are homologous to those of other arthropods. Assaying expression of molecular markers in the developing nervous systems of various arthropods could resolve some of these issues. Expression of Even-skipped and Engrailed, two transcription factors that serve as insect embryonic CNS markers, was examined across a number of arthropod species. This molecular analysis allows verification the homology of previously identified malacostracan neurons and identification of additional homologous neurons in malacostracans, collembolans and branchiopods. Engrailed expression in the neural stem cells of a number of crustaceans was also found to be conserved. It is concluded that despite their distant phylogenetic relationships and divergent mechanisms of neurogenesis, insects, malacostracans, branchiopods and collembolans share many common CNS components (Duman-Scheel, 1999).

To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, the expression of two gap genes hunchback (hb) and Kruppel (Kr) as well as the pair-rule gene even-skipped (eve) were studied in the dipteran Clogmia albipunctata (Nematocera, Psychodidae). Embryogenesis in this species has features of both short- and long-germ modes of development. In Clogmia, hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region. These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis. However, Kruppel expression is rather similar to the Drosophila expression, both at early and late stages. eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension. Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans. Another nematoceran, Coboldia, was therefore studied to address this question and it was found that some segmental stripes form by intercalation as in Drosophila, although belatedly. These results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects (Rohr, 1999).

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. The expression patterns of the genes even-skipped, engrailed, and wingless in a centipede, Lithobius atkinsoni, were examined. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods (Hughes, 2002).

Despite hopes of a 'pair-rule' that could explain the mysterious rules of segment number in centipedes, there is no indication from centipede even-skipped expression that the segments develop in pairs. On the contrary, embryos at various stages represent the presence of new segments, appearing one-by-one in what is apparently a simply sequential manner. Thus, perhaps despite expectations, the Lithobius growth zone is acting as a well-behaved, canonical short-germband posterior proliferation zone. Any mechanism the cells are using to 'count' segments is not apparent from the expression of eve or the appearance of new segments. However, the pair-rule pattern of expression of even-skipped in Tribolium suggests that there may be two separate mechanisms for repressing eve and thus generating segmental primordia. In Tribolium, the primary stripes initially formed span two segments each, and later each stripe splits to form separate secondary stripes for each segment. Thus stripes of even-skipped in alternate segments seem to be demarcated by either an 'initiating' mechanism or a 'splitting' mechanism. If the timing of these two mechanisms was more synchronized, a centipede-like pattern might emerge, which would mask the underlying differences generating subsequent segments. In other words, although each subsequent segment appears to emerge from the growth zone in the same way (as recognized by suppression of even-skipped expression in a band of cells), it is possible that the formation of alternating segments might be controlled by two separate upstream mechanisms which cannot be seen here. With regard to this question of control of even-skipped, a study of the centipede homologs of gap genes could prove to be illuminating (Hughes, 2002).

No evidence is seen for any preformed segments in late centipede embryos that would correspond to the seven leg-bearing segments that arise after hatching. If the tissue of the growth zone were in any way preallocated into segments, one might expect to see very fine stripes of even-skipped and perhaps engrailed and wingless marking these predetermined segments. No such stripes are apparent. Thus, it is suggested that the growth zone must be still active in the juvenile centipede, to produce more segments during the first few weeks after hatching. In essence, the anamorphic centipedes like Lithobius may be hatching precociously, while their growth zones continue producing new segments that are revealed at each molt. This early hatching may make the young centipede less vulnerable to predation, as the eggs of these species are not guarded by the mothers (Hughes, 2002).

The strict regulation of centipede segment number implies that some counting or allocation mechanism exists. Considering the differences between long-germband and short-germband modes of segment formation, it may be instructive to look at mechanisms of vertebrate segmentation for comparison. It is still unclear whether short-germband arthropods may have a clock mechanism similar to the cycles of hairy and Notch expression seen in vertebrates somitogenesis. Even if it were important for short-germband arthropods, such a mechanism would be highly modified in the Drosophila long-germband embryo, and may thus have gone unrecognized (Hughes, 2002).

In the centipede, the concentric circles of even-skipped expression in the extraembryonic membrane seem to suggest traveling waves. These rings of extraembryonic expression are unlikely to have an important function in this membrane, but they may be a revealing side effect of some regulatory mechanism controlling expression. In the bulk of the embryo itself, the expression is clearly periodic but not wave-like, since the expression correlates with the intersegmental furrows and is never seen in the middle of a segment. Within the growth zone, however, it is difficult to precisely describe the temporal dynamic of eve expression by the detection technique used in this study. Thus, further study will be necessary to understand the dynamics of short-germband segmentation (Hughes, 2002).

The pair-rule gene even-skipped is required for the initiation of metameric pattern in Drosophila. But Drosophila segmentation is evolutionarily derived and is not representative of most insects. Therefore, in order to shed light on the evolution of insect segmentation, homologs of the pair-rule gene even-skipped have been studied in several insect taxa. However, most of these studies have reported the expression of eve but not its function. The isolation, expression and function is reported of the homolog of Drosophila even-skipped from the intermediate germband insect Oncopeltus fasciatus. In Oncopeltus, even-skipped striped expression initiates in a segmental and not pair-rule pattern. Weak RNAi suppression of Oncopeltus even-skipped shows no apparent pair-rule like phenotype, while stronger RNAi suppression shows deletion of nearly the entire body. These results suggest that in Oncopeltus, even-skipped is not acting as a pair-rule gene. In almost all insects, prior to its striped expression, even-skipped is expressed in a conserved broad gap-like domain but its function has been largely ignored. This early broad domain is required for activation of the gap genes hunchback and Krüppel. Given the large RNAi deletion phenotype and its regulation of hunchback and Krüppel, even-skipped seems to act as an über-gap gene in Oncopeltus, indicating that it may have both upstream and downstream roles in segmentation (Liu, 2005).

A quantitative atlas of Even-skipped and Hunchback expression in Clogmia albipunctata (Diptera: Psychodidae) blastoderm embryos

Comparative studies of developmental processes are one of the main approaches to evolutionary developmental biology (evo-devo). Over recent years, there has been a shift of focus from the comparative study of particular regulatory genes to the level of whole gene networks. Reverse-engineering methods can be used to computationally reconstitute and analyze the function and dynamics of such networks. These methods require quantitative spatio-temporal expression data for model fitting. Obtaining such data in non-model organisms remains a major technical challenge, impeding the wider application of data-driven mathematical modeling to evo-devo. Antibodies were raised against four segmentation gene products in the moth midge Clogmia albipunctata, a non-drosophilid dipteran species. These antibodies were used to create a quantitative atlas of protein expression patterns for the gap gene hunchback (hb), and the pair-rule gene even-skipped (eve). The data reveal differences in the dynamics of Hb boundary positioning and Eve stripe formation between C. albipunctata and Drosophila melanogaster. Despite these differences, the overall relative spatial arrangement of Hb and Eve domains is remarkably conserved between these two distantly related dipteran species. This study has provided a proof of principle that it is possible to acquire quantitative gene expression data at high accuracy and spatio-temporal resolution in non-model organisms. The quantitative data extend earlier qualitative studies of segmentation gene expression in C. albipunctata, and provide a starting point for comparative reverse-engineering studies of the evolutionary and developmental dynamics of the segmentation gene system (Janssens, 2014).

Maternal torso signaling controls body axis elongation in a short germ insect: The emergence of segments

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. The terminal system in the short germ insect Tribolium was functionally analyzed. 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. It is therefore concluded 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 (Schoppmeier, 2005).

In Drosophila, the anterior- and posterior-most terminal body regions of the embryo depend on the maternal terminal-group genes. One of them, the torso-like (tsl) gene is expressed in somatic follicle cells located at the anterior and posterior pole of the oocyte. In the embryo, tsl contributes to the local activation of the receptor tyrosine kinase Torso at the egg poles. The signal is transduced to the nucleus via a Ras-Raf-MAP-K/Erk phosphorylation cascade, and leads to the expression of the zygotic target genes tailless (tll) and huckebein (hkb) at the posterior terminus of the embryo. Failure to activate Torso signaling results in defects in the head skeleton and loss of all segments posterior to abdominal segment 7, in addition to loss of the hindgut and posterior midgut anlagen (Schoppmeier, 2005 and references therein).

Whether an anteriorly acting terminal system is a general feature of all insects has been challenged because under certain conditions, Torso function at the anterior is dispensable for head development in Drosophila. This hypothesis is supported by the expression of the Tribolium ortholog of tll at the posterior, but not at the anterior pole of blastoderm stage embryos. Thus, in Tribolium, posterior terminal cells appear to be determined before the onset of abdomen formation. It is unknown, however, whether these cells specify posterior fate after axis elongation and abdomen formation is completed or whether they also contribute to earlier steps of segmentation (Schoppmeier, 2005).

The orthologs of the key components of the Torso pathway have been isolated in the short germ beetle Tribolium torso (Tc-tor) and torso-like (Tc-tsl). As in Drosophila, Tc-torso mRNA is maternally inherited by the embryo and expressed ubiquitously in freshly laid eggs, and Tc-tsl is expressed during oogenesis anteriorly and posteriorly in the follicle cells of the oocyte (Schoppmeier, 2005).

Knocking down the function of Tc-torso or Tc-tsl using parental RNA interference leads to identical embryonic phenotypes. Whereas the head and the anterior thorax are unaffected, unexpectedly the most extreme Tc-torsoRNAi and Tc-tslRNAi embryos lack all structures that develop during postblastodermal abdominal growth. Thus, the head and thoracic segments that form in torso or tsl RNAi embryos likely represent the structures, which are determined already during the Tribolium blastoderm stage. Less strongly affected embryos fail to form the full number of abdominal segments (Schoppmeier, 2005).

To determine whether the Tc-torso RNAi phenotype does not reflect a late function of maintaining abdominal fate prior to cuticularization, the expression of Engrailed protein was examined in Tc-torsoRNAi embryos at a stage when abdominal segments should already have developed. Indeed, in strongly affected embryos, Engrailed stripes corresponding to the head and thorax, but not to abdominal segments, are present (Schoppmeier, 2005).

The emergence of segments was visualized in embryos with impaired Torso signaling by analyzing the Tc-even-skipped (Tc-eve) expression pattern. In wild-type embryos, Tc-eve is initially expressed in a double segmental pattern that later resolves into secondary segmental stripes. Tc-tsl RNAi does not interfere with the formation of the first two primary Tc-eve stripes that give rise to the gnathal and the first thoracic (T1) segments. However, although the third primary Tc-eve expression domain (Tc-eve stripe 3) forms normally, this domain does not resolve into segmental stripes, and no additional primary eve-stripes form. In the wild-type, Tc-eve stripe 3 covers the region where the second (T2) and third thoracic (T3) segment will develop. Although Tc-eve stripe 3 does not split in Tc-tsl RNAi embryos, this domain gives rise to the second thoracic segment. Thus, Torso signaling is required for the initiation of axial growth or maintaining the segmentation process (Schoppmeier, 2005).

Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, this study demonstrates regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. The even-skipped locus was sequenced from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. It is concluded that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, a small number of short (20-30 bp) sequences were found to be nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function (Hare, 2008).

Given the extent of non-coding divergence between Drosophila and sepsids across most non-coding DNA, it was surprising to observe small islands of very strong sequence conservation. The finding that there is a significant enrichment of overlapping or adjacent binding sites within conserved blocks lends evolutionary support to long-standing suggestions of the importance of direct competitive and cooperative interactions between bound transcription factors. Numerous studies have demonstrated that appropriate regulation of the eve stripe enhancers (and other enhancers) relies on the close proximity of multiple binding sites for both activators and repressors. Of the 12 footprinted Bicoid, Hunchback, Kruppel, and Giant sites in the minimal stripe 2 element, 8 fall into 2 clusters of about 50 base pairs each containing overlapping activator (Hunchback or Bicoi) and repressor (Kruppel or Giant) sites. In transient transfection experiments using these binding site clusters, Bicoid and Hunchback dependent activation was repressed by DNA binding of Giant or Kruppel, consistent with the short-range repression mechanisms of quenching or competition. Knirps also mediates short-range repression in a range of 50-100 bp through quenching or direct repression of the transcriptional machinery when bound near a promoter (Hare, 2008).

Similarly, Hunchback and Bicoid co-expression in transient transfection experiments results in multiplicative activation of a reporter construct containing a subset of the eve minimal stripe 2 element. Mutation of single activator sites in the minimal stripe 2 element results in a significant reduction in expression, again suggesting that Hunchback and Bicoid bind cooperatively to this enhancer. The local quenching and cooperativity models predict that binding sites in close proximity to each other should be under strong purifying selection to remain close to each other. Under the generally accepted model of binding site turnover, sites are lost in one region of an enhancer when new mutations create a complementary site elsewhere in the same enhancer. The appearance of new sites is the rate-limiting step as there are more mutational steps required to create a new site from random sequence than to destroy an existing site. Since random mutations are far less likely to produce pairs of adjacent sites than single sites, functionally linked pairs of sites are expected to be subject to far lower rates of binding site turnover. In contrast, if binding site turnover is driven by base substitutions, functionally independent sites that are adjacent or even partially overlapping are expected to have essentially the same rates of binding site turnover as isolated sites. The conserved blocks observed between sepsids and Drosophila were generally larger than individual sites, as has been previously reported within Drosophila, consistent with the former model. The observation that proximal sites are preferentially conserved additionally supports their direct functional linkage (Hare, 2008).

However, it is noted that insertions and deletions are a major source of sequence variation in Drosophila, with D. melanogaster having a strong deletion bias and deletion is thought to contribute significantly to binding site turnover. Taking this into account, reduced turnover is expected in even functionally independent binding sites if they are overlapping or adjacent, as some fraction of the deletions that would remove a binding site with a complementary site elsewhere would also affect adjacent, and presumably uncompensated sites. These deletions would be subject to purifying selection, and the rate of turnover for the proximal sites would be reduced. Assessing whether such an effect could explain the observation requires more data on relative rates of nucleotide substitution and insertion and deletions of different sizes in sepsids, which will be accomplished with the sequencing of sepsid genomes (Hare, 2008).

It is possible, however, test the significance of the observation directly. The linked function model predicts that the paired binding sites observed to be conserved between families should be more sensitive to manipulations that alter the spacing between the sites than paired binding sites that are not conserved. Though expression of the sepsid eve enhancers in D. melanogaster embryos is qualitatively very similar to the patterns driven by the D. melanogaster enhancers, there are subtle and interesting differences. Expression of stripe 7 exhibits the most variability across all enhancers in transgenics, including those enhancers from D. melanogaster. It was previously observed that stripe 7 is weakly expressed in D. melanogaster stripe 2 transgenics, and stripe 7 expression is weaker than the endogenous stripe in stripe 3+7 transgenics. Stripe 7 expression is frequently observed in all the non-Drosophila stripe 2 transgenics, and stripe 7 expression did not perfectly recapitulate endogenous expression, suggesting that regulatory information specifying this stripe is distributed across the upstream region, thus challenging the model of enhancer modularity. Information may be more diffusely spread across the locus in sepsids, resulting in missing information in the discrete cloned enhancers, in which case the native D. melanogaster pattern should be more accurately reproduced by cloning a larger regulatory region. Alternately, there could be changes within the non-Drosophila enhancers which result in expression differences in D. melanogaster despite conserved native eve expression, suggesting co-adaptation of each enhancer and its native trans environment (Hare, 2008).

Segmentation gene expression patterns in Bactrocera dorsalis and related insects: regulation and shape of blastoderm and larval cuticle

The oriental fruit fly, Bactrocera dorsalis, is regarded as a severe pest of fruit production in Asia. Despite its economic importance, only limited information regarding the molecular and developmental biology of this insect is known to date. This study provides a detailed analysis of B. dorsalis embryology, as well as the expression patterns of a number of segmentation genes known to act during patterning of Drosophila and compare these to the patterns of other insect families. An anterior shift of the expression of gap genes was detected when compared to Drosophila. This shift was largely restored during the step where the gap genes control expression of the pair-rule genes. The shapes were analyzed of the embryos of insects of different families, B. dorsalis and the blow fly Lucilia sericata and compared with that of the well-characterized Drosophila melanogaster. Distinct shapes were found as well as differences in the ratios of the length of the anterior-posterior axis and the dorsal-ventral axis. These features were integrated into a profile of how the expression patterns of the gap gene Kruppel and the pair-rule gene even-skipped were observed along the A-P axis in three insects families. Since significant differences were observed, how Kruppel controls the even-skipped stripes is discussed. Furthermore, how the position and angles of the segmentation gene stripes differed from other insects is discussed. Finally, the outcome was analyzed of the expression patterns of the late acting segment polarity genes in relation to the anlagen of the naked-cuticle and denticle belt area of the B. dorsalis larva (Suksuwan, 2017).

An Even-skipped homolog in nematodes

The segmental distribution of eve transcription in Drosophila melanogaster appears to be the exception and not the rule. In C. elegans, the eve homolog is required for posterior patterning, a function similar to that obseved for the mammalian homolog. Posterior body muscles and the posterior epidermis are disrupted in vab-7 mutants, supporting the hypothesis that the eve homologs act with genes of the homeobox complex to determine posterior cell fates. egl-5, the most posterior C. elegans homeotic gene requires vab-7 activity for complete expression in muscle cells (Ahringer, 1996).

The transition from maternal to zygotic gene control is a key process in embryogenesis. Although many maternal effect genes have been studied in the C. elegans embryo, how their activities lead to the positional expression of zygotic patterning genes has not yet been established. Evidence is presented showing that expression of the zygotic patterning gene vab-7, a homolog of Drosophila even-skipped, does not depend on cell position or cell contacts, but rather on the production of a C blastomere, which is positioned at the posterior of the embryo. vab-7 expression depends on the production of a C blastomere regardless of postion. pal-1, a caudal homolog with maternal product necessary for the proper development of the C blastomere, is both necessary and sufficient for vab-7 expression. This provides a link between maternal gene activity and zygotic patterning gene expression in C. elegans. The results suggest that zygotic patterning genes might be generally controlled at the level of blastomere fate and not by position (Ahringer, 1997).

In free-living nematodes, developmental processes like the formation of the vulva, can be studied at a cellular level. Cell lineage and ablation studies have been carried out in various nematode species and multiple changes in vulval patterning have been identified. In Pristionchus pacificus, vulva formation differs from Caenorhabditis elegans with respect to several autonomous and conditional aspects of cell fate specification. To understand the molecular basis of these evolutionary changes, a genetic analysis of vulva formation in P. pacificus has been performed. Two mutants in P. pacificus are described in which the vulva is shifted posteriorly, affecting which precursor cells will form vulval tissue. Mutant animals show a concomitant posterior displacement of the gonadal anchor cell, indicating that the gonad and the vulva are affected in a similar way. Mutations in the even-skipped homolog of nematodes, vab-7, cause these posterior displacements. In addition, cell ablation studies in the vab-7 mutant indicate that the altered position of the gonad not only changes the cell fate pattern but also the developmental competence of vulval precursor cells. Investigation of Cel-vab-7 mutant animals shows a similar but weaker vulva defective phenotype to the one described for Ppa-vab-7 (Jungblut, 2001).

Locomotory activity is defined by the specification of motoneuron subtypes. In the nematode C. elegans, DA and DB motoneurons innervate dorsal muscles and function to induce movement in the backwards or forwards direction, respectively. These two neuron classes express separate sets of genes and extend axons with oppositely directed trajectories; anterior (DA) versus posterior (DB). The DA-specific homeoprotein UNC-4 (Drosophila homolog: Unc-4) interacts with UNC-37/Groucho to repress the DB gene, acr-5 (nicotinic acetylcholine receptor subunit). The C. elegans even-skipped-like homoedomain protein, VAB-7, coordinately regulates different aspects of the DB motoneuron fate, in part by repressing unc-4. Wild-type DB motoneurons express VAB-7, have posteriorly directed axons, express ACR-5 and lack expression of the homeodomain protein UNC-4. In a vab-7 mutant, ectopic UNC-4 represses acr-5 and induces an anteriorly directed DB axon trajectory. Thus, vab-7 indirectly promotes DB-specific gene expression and posteriorly directed axon outgrowth by preventing UNC-4 repression of DB differentiation. Ectopic expression of VAB-7 also induces DB traits in an unc-4-independent manner, suggesting that VAB-7 can act through a parallel pathway. This work supports a model in which a complementary pair of homeodomain transcription factors (VAB-7 and UNC-4) specifies differences between DA and DB neurons through inhibition of the alternative fates. Pointing to an ancient origin for homeoprotein-dependent mechanisms of neuronal differentiation in the metazoan nerve cord are the recent findings that Even-skipped transcriptional repressor activity specifies neuron identity and axon guidance in the mouse and Drosophila motoneuron circuit (Esmaeili, 2002).

In vab-7 mutants, both dorsal and ventral nerve cords are defasciculated. This defect is not rescued by restoring proper posterior polarity of DB neurons (by removing unc-4), but is rescued by ectopic VAB-7 expression, suggesting that vab-7, and possibly DB neurons promote process bundling. Interestingly, Even-skipped in Drosophila also has a role in fasciculation. Axonal growth of Eve-expressing neurons (aCC and RP2) in the ISN nerve trunk and their subsequent innervation of dorsal muscles is dependent on Even-skipped. Furthermore, ectopic expression of Even-skipped in the nervous system promotes SN and ISN nerve trunk fasciculation. Indirect evidence has been provided that eve activity is required for expression of an unknown neuronal adhesion molecule. Mutations in a number of genes are known to cause nerve bundle defasciculation in C. elegans. One candidate for a downstream target of vab-7 is the alpha-integrin INA-1, which is expressed in DB (and other) neurons and is required for nerve bundle fasciculation (Esmaeili, 2002).

Genetic studies in C. elegans, Drosophila, and the mouse have shown that Even-skipped homologues function to distinguish alternative fates in the motoneuron circuit. In each case, Eve prevents one class of neuron from adopting traits normally reserved for another. In Drosophila, Eve is expressed in motoneurons that project along the ISN nerve to innervate dorsal muscles. In eve mutants, these motoneurons adopt the axonal trajectory of a different class of ISN motoneurons that synapse onto ventral muscles. Similarly, in vab-7 mutants in C. elegans, DB motoneurons reverse their normal posterior axonal polarity and instead assume the anteriorly directed trajectory of DA motor axons. In the spinal cord of mouse Evx1 mutants, V0 interneurons are apparently transformed into V1 interneurons. At least in C. elegans and in Drosophila, ectopic expression of Even-skipped is also sufficient to impose axonal trajectories normally associated with eve-expressing motoneurons (Esmaeili, 2002).

A common element of Eve function in all three species is the repression of a downstream HD protein, which is normally expressed in the alternative neuron. In C. elegans, vab-7 prevents expression of the DA gene, unc-4, in DB motoneurons. Evx1 functions in mouse V0 neurons as a negative regulator of the Engrailed homologue, En1, a marker for V1 cells. In Drosophila, ectopic expression of Eve is sufficient to inhibit Islet in ISN motoneurons (Esmaeili, 2002).

In addition to these similarities in Eve function, this work shows that VAB-7 functions within a reciprocally inhibitory network: VAB-7 inhibits the DA fate and UNC-4 inhibits the DB fate. Thus, one way that VAB-7 promotes DB differentiation is by blocking expression of a HD transcription factor that antagonizes DB traits. By extension, it is proposed that HD transcription factors in Drosophila and mouse are likely to antagonize fates promoted by Eve. For example, EN1 might exert a negative effect on V0 interneuron differentiation when ectopically expressed in V1 cells in Evx1 mutants just as UNC-4 inhibits DB fates in vab-7 mutant animals. In this case, normal V0 cell migration and axonal trajectory might be restored in Evx1;En1 double mutant mice. Both UNC-4 and EN1 include EH-1 domains that have been shown to recruit the transcriptional co-repressor protein, Groucho. In the case of UNC-4, interactions with the nematode Groucho homolog, UNC-37, repress B-class motoneuron traits. Reciprocal inhibition by EH-1-containing HD proteins that recruit Groucho might be common, as recent work has revealed that such a mechanism in the vertebrate spinal cord defines distinct domains of neural progenitor cells. Thus, this work demonstrates that important elements of both the logic and molecular mechanisms employed by HD proteins in the specification of neuronal fates in the motor circuit have been preserved in evolution from nematodes to mammals (Esmaeili, 2002).

T-box genes form a large family of conserved transcription factors with diverse roles in animal development, but so far functions for only a few have been studied in detail. Four Caenorhabditis elegans T-box genes and the even-skipped-like homeobox gene vab-7 function within a regulatory network to control embryonic patterning and morphogenesis. tbx-8 and tbx-9 have functionally redundant roles in the intercalation of posterior dorsal hypodermal cells, in muscle cell positioning and in intestinal development. Inhibiting tbx-9 alone using RNA interference (RNAi) produces worms that have a thickened, 'bobbed tail' phenotype, similar to that seen in mutants of vab-7, which itself has been shown to pattern posterior muscle and hypodermal cells. In support of the view that these genes function in the same pathway, it has been found that tbx-8 and tbx-9 are both necessary and sufficient for vab-7 expression. In addition, a third T-box gene, tbx-30, acts to repress vab-7 expression in the anterior of embryos. It is further shown that vab-7 itself represses the T-box gene mab-9 in posterior cells. Thus, during posterior patterning in C. elegans, there are multiple interactions between T-box genes and the vab-7 homeobox gene. Evolutionary parallels in other organisms suggest that regulatory interactions between T-box genes and even-skipped homologs are conserved (Pocock, 2004).

The activation of even-skipped in posterior cells by T-box genes has been reported in other systems, suggesting that this may be a conserved mechanism for controlling posterior pattern formation. For example, brachyenteron is required for eve expression in the hindgut and anal pad primordia of Drosophila, and in mouse, evx-1 expression has been shown to be dependent on Brachyury in the posterior tail bud. Likewise, no tail (ntl-zebrafish Brachyury) is required for the maintenance of eve1 expression during tail extension in zebrafish. Whether these examples of T-box gene mediated regulation of eve genes involve direct or indirect effects remains to be seen. It is also noteworthy that ectopic Xbra expression in Xenopus causes marked induction of the eve homolog Xhox3, similar to the induction of vab-7 expression caused by tbx-8 or tbx-9 overexpression reported in this study. Perhaps the regulation of eve has been taken over by tbx-8/tbx-9 in C. elegans in the absence of a bona fide Brachyury homolog (Pocock, 2004).

Ancestral resurrection of the Drosophila S2E enhancer reveals accessible evolutionary paths through compensatory change

Upstream regulatory sequences that control gene expression evolve rapidly, yet the expression patterns and functions of most genes are typically conserved. In order to address this paradox, this study has reconstructed computationally and resurrected in vivo the cis-regulatory regions of the ancestral Drosophila eve stripe 2 element and evaluated its evolution using a mathematical model of promoter function. A feed-forward transcriptional model predicts gene expression patterns directly from enhancer sequence. This functional model was used along with phylogenetics to generate a set of possible ancestral eve stripe 2 sequences for the common ancestors of 1) Drosophila simulans and D. sechellia, 2) D. melanogaster, D. simulans, D. sechellia, and 3) D. erecta and D. yakuba. These ancestral sequences were synthesized and resurrected in vivo. Using a combination of quantitative and computational analysis, clear support was foumd for functional compensation between the binding sites for Bicoid, Giant, and Kruppel over the course of 40-60 million years of Drosophila evolution. This compensation is driven by a coupling interaction between Bicoid activation and repression at the anterior and posterior border necessary for proper placement of the anterior stripe 2 border. A multiplicity of mechanisms for binding site turnover exemplified by Bicoid, Giant, and Kruppel sites, explains how rapid sequence change may occur while maintaining the function of the cis-regulatory element (Martinez, 2014).

Even-skipped in annelids

Homologs of the Drosophila pair-rule gene even-skipped have been identified in the glossiphoniid leeches Helobdella robusta and Theromyzon trizonare. In leech embryos, segments arise sequentially from five pairs of embryonic stem cells (teloblasts) that undergo iterated divisions to generate columns (bandlets) of segmental founder cells (primary blast cells), which in turn generate segmentally iterated sets of definitive progeny. In situ hybridization has revealed that Hro-eve is expressed in the teloblasts and primary blast cells, and that these transcripts appear to be associated with mitotic chromatin. In more advanced embryos, Hro-eve is expressed in segmentally iterated sets of cells in the ventral nerve cord. Lineage analysis revealed that neurons expressing Hro-eve arise from the N teloblast. To assess the function of Hro-eve, embryos were examined in which selected blastomeres had been injected with antisense Hro-eve morpholino oligonucleotide (AS-Hro-eve MO), concentrating on the primary neurogenic (N teloblast) lineage. Injection of AS-Hro-eve MO perturbed the normal patterns of teloblast and blast cell divisions and disrupted gangliogenesis. These results suggest that Hro-eve is important in regulating early cell divisions through early segmentation, and that it also plays a role in neuronal differentiation (Song, 2002).

In Helobdella, each segment arises from the interdigitating clones of seven distinct classes of bilaterally paired segmental founder cells (m, nf, ns, o, p, qf and qs blast cells). Blast cells arise in columns (bandlets) by unequal divisions from five bilateral pairs of large identified stem cells: the M, N, O, P and Q teloblasts. Teloblasts divide with a cell cycle time of about 1 hour. Each lineage contributes a stereotyped set of neurons and other cell types to each segment. In the N (and Q) lineages, two classes of blast cells, nf and ns (qf and qs) arise in exact alternation; one of each is required to generate one segment's worth of progeny. In the M, O and P lineages, each blast cell makes one segment's worth of progeny. In each teloblast lineage, the first-born blast cells contribute to anterior segments and blast cells born later contribute to progressively more posterior segments. Thus, in leech, in contrast to vertebrates and insects, there is a strict correlation between the 'cell cycle clock', by which blast cells arise from the teloblasts, and the 'segmentation clock', by which segmental tissues arise in anteroposterior progression (Song, 2002).

RT-PCR experiments have indicated that Hro-eve expression increases sharply beginning at stage 9, by which time the germinal plate is complete and the differentiation of ventral ganglia and other segmental tissues is under way. In situ hybridization using embryos at stages 9-11 has revealed Hro-eve expression in segmentally iterated sets of neurons in the ventral nerve cord. The Hro-eve transcripts in these cells are cytoplasmic rather than nuclear. Two lines of evidence show that neuronal Hro-eve expression is dynamic over time, and that it undergoes the same progression in each of the midbody segments. (1) Within individual embryos, there are differences in staining pattern along the AP axis that correlate with the sequential segmentation process in leech. (2) The same changes in Hro-eve expression are seen by examining any given region of the germinal plate at progressively later stages of development. Thus, the following description applies to any midbody ganglion and is presented in terms of the clonal age of the n blast cells that contribute to the ganglion, since neurons expressing Hro-eve arise from the nf and ns blast cells (Song, 2002).

Neuronal expression of Hro-eve is first observed when n blast cell clones are ~70 hours old. This corresponds to early stage 9 in the anterior germinal plate. Expression was seen in two spots, one anterior and another more posterior, in each hemiganglion. The anterior spot consists of two or three adjacent cells in the ventrolateral portion of the ganglion; the more posterior spot lies about halfway back in the ganglion and consists of two or three cells in the dorsal portion of the ganglion, roughly midway between the ventral midline and the lateral edge of the ganglion. By clonal age ~90-100 hours, the anterior spot of Hro-eve expression starts to disappear, so that some ganglia have only one anterior spot. By clonal age ~130 hours, the anterior spots have disappeared, but the posterior spot of Hro-eve expression remains in each ganglion through clonal age ~130-150 hours. Therefore, the posterior spot is evident throughout the germinal plate in individual embryos at late stage 10 (Song, 2002).

In glossiphoniid leeches, ganglionic neurons arise from each of the five teloblast lineages, with half or more arising from the N lineage. To identify the lineage(s) of origin of the neurons expressing Hro-eve, in situ hybridization was carried out on embryos in which one or more cells (N, OP or OPQ) had been injected with lineage tracer. The confocal images of sectioned embryos revealed that the in situ label colocalized with lineage tracer when the N lineage was labeled and not when any other lineage was labeled. Thus, it is concluded that the cells expressing Hro-eve arise from the N teloblasts. Moreover, by comparison with more detailed lineage analyses, it is concluded that anterior ventrolateral cells exhibiting transient expression of Hro-eve arise from the ns.a clone, and that the posterior neurons arise from the nf.a clone in each segment (Song, 2002).

Two assays were used to assess the effects of AS-Hro-eve MO on specific neuronal phenotypes. For one, in situ hybridization for Hro-eve was used to mark the subsets of nf- and ns-derived neurons. Cells expressing Hro-eve arise from N teloblasts injected with AS-Hro-eve MO, but the pattern is abnormal; such cells were often out of register with their counterparts in the control half of the germinal plate. For another assay, immunostaining was used to screen for the appearance of three pairs of serotonergic neurons, the anteromedial giant Retzius neurons, which normally arise from the ns.a blast cell clones, and smaller ventrolateral and dorsolateral neurons (cells 21 and 61, respectively), which normally arise from nf.a clones. These three neurons arise in their normal positions in control bandlets, but no serotonergic neurons were detected in the experimental bandlets (Song, 2002).

Further examination of lineage tracer in cells derived from the experimental N teloblasts has revealed that they failed to generate appreciable numbers of neurites. In normal development, three prominent segmental nerves exiting each side of the ganglion, contain neurites derived from contralateral N-derived neurons; these nerves were not detected contralateral to AS-Hro-eve MO-injected N teloblasts. Observations of ganglionic Hro-eve expression were made at stage 10, whereas scoring for serotonergic neurons and segmental nerve formation was carried out at stage 11. Together, these results suggest that AS-Hro-eve MO injections result in a widespread failure of neural development at some point prior to terminal differentiation. Consistent with the fact that Hro-eve is expressed in all five teloblast lineages in early development, the defects induced by AS-Hro-eve MO injections are not restricted to neuronal tissues. For example, the O lineage normally makes a mixture of neurons and epidermal cells. Here, AS-Hro-eve MO injections result in an almost total loss of epidermal cells; neural precursors could be recognized by their ganglionic locations, but as in the N lineage, fail to complete differentiation (Song, 2002).

These results suggest that Hro-eve is important in regulating cell divisions in stem cells and segmental founder cells in early development and that it also regulates the differentiation of a subset of ganglionic neurons. However, no evidence was found that Hro-eve plays a pair rule function similar to that of eve-class genes in arthropod segmentation (Song, 2002).

Even-skipped homologs in Amphioxus

Evx genes are widely used in animal development. In vertebrates they are crucial in gastrulation, neurogenesis, appendage development and tailbud formation, whilst in protostomes they are involved in gastrulation and neurogenesis, as well as segmentation at least in Drosophila. The Evx genes of cephalochordate amphioxus (Branchiostoma floridae) have been cloned and their expressions analyzed to understand how the functions of Evx have evolved between invertebrates and vertebrates, and in particular at the origin of chordates and during their subsequent evolution. Amphioxus has two Evx genes (AmphiEvxA and AmphiEvxB) which are genomically linked. AmphiEvxA is prototypical to the vertebrate Evx1 and Evx2 genes with respect to its sequence and expression, whilst AmphiEvxB is very divergent. Mapping the expression of AmphiEvxA onto a phylogeny shows that a role in gastrulation, dorsal-ventral patterning and neurogenesis is probably retained throughout bilaterian animals. AmphiEvxA expression during tailbud development implies a role for Evx throughout the chordates in this process, while lack of expression at the homologous region to the vertebrate midbrain-hindbrain boundary is consistent with the elaboration of the full organiser properties of this region being a vertebrate innovation (Ferrier, 2001).

Even-skipped homologs in fish and frogs

The structural organization and expression of Abd-B related zebrafish HoxA cluster genes (Hoxa-9, Hoxa-10, Hoxa-11 and Hoxa-13) have been examined, as well as the structure and organization of Evx-2, a gene closely linked to the HoxD complex. The genomic organization of Hoxa genes in fish resembles that of tetrapods albeit intergenic distances are shorter. During development of the fish trunk, Hoxa genes are coordinately expressed, whereas in pectoral fins, they display transcript domains similar to those observed in developing tetrapod limbs. Likewise, the Evx-2 gene seems to respond to both Hox- and Evx-types of regulation. During fin development, this latter gene is expressed as are the neighbouring Hox genes (that is complying with colinearity), in contrast Evx-2's expression in the central nervous system, which does not comply with colinearity and extends up to anterior parts of the brain. These results are discussed in the context of the functional evolution of Hoxa versus Hoxd genes and their different roles in building up paired appendages (Sordino, 1996).

Growth and patterning during fin regeneration, like fin development, is dependent on the integrated expression of homeogenes. The expression and regulation of two vertebrate homologs eve1 and evx2 of the Drosophila pair-rule even-skipped gene family has been studied. Upon amputation of pectoral and caudal fins, both genes are turned on. They are expressed transiently in the mesenchyme during early stages of pectoral and caudal fin development. During the formation of the blastema they are transcribed first in the mesenchyme located underneath the wound epidermis and then, their expression is restricted to regions of regenerating rays. These expression patterns are developmentally regulated since both genes are no longer transcribed when the bony rays are differentiating. Exposure of the regenerates to retinoic acid (RA) modifies the boundaries of eve1 and evx2 expression: the signal is down-regulated in the ray region and up-regulated in the interray region. Moreover, expression is induced in the wound epidermis. These results indicate that eve1 and evx2 products are part of the molecular signals involved in pattern formation of the fin and fin rays in connection with outgrowth. RA might alter growth and morphogenesis of the regenerating fins by a fine regulation of these genes, as well as other genes (Brulfert, 1998).

The even-skipped-related homeobox genes (evx) are widely distributed throughout the animal kingdom and are thought to play a key role in posterior body patterning and neurogenesis. evx1 of zebrafish displays a dynamic and restricted expression pattern during neurogenesis. In spinal cord, rhombencephalon, and epiphysis, evx1 is expressed in several subsets of emerging interneurons prior to their axonal outgrowth, identified as primary interneurons and a subset of Pax2.1(+) commissural interneurons. In the hindbrain, evx1 is expressed in reticulospinal interneurons of rhombomeres 5 and 6 as well as in rhombomere 7 interneurons. The latest emerging evx1+ interneurons in the hindbrain correspond to commissural interneurons. evx1 is also dynamically transcribed during the formation of the posterior gut and the uro-genital system in mesenchymal cells that border the pronephric ducts, the wall of the pronephric duct, and later in the posterior gut and the wall of the urogenital opening. In larvae, the ano-rectal epithelium and the muscular layer that surrounds the analia-genitalia region remain stained up to 27 days. In contrast to expression in other vertebrates, zebrafish evx1 displays no early or caudal expression (Thaeron, 2000).

From 12.5 hours-post-fertilization (hpf) to 50 hpf, evx1 is expressed in subsets of spinal chord interneurons prior to their axonal outgrowth. The earlier evx1-positive interneurons appear in the dorsal part of the spinal cord from the early 7-somite to the 11-somite stage and are likely to be primary interneurons. Then, evx1 was transcribed in more ventral interneurons that are likely to be secondary interneurons since most of them co-express Pax2.1. The evx1+ interneurons form progressively discrete clusters and then (about 36 hpf) they form a continuous row on each side of the spinal cord that persists until 50 hpf (Thaeron, 2000).

In the hindbrain, evx1 is also detected in discrete interneurons prior to axonogenesis. The Pax2.1+ Mid2 and Mid3 commissural interneurons, and CocaA commissural interneurons in r7 express evx1 from the 11-somite to the 18-somite stage. In epiphysis anlage, a dorsal cluster of four to six cells expresses evx1 transiently from 17 to 22 hpf before these neurons develop axonal projections to form the dorsoventral diencephalic tract. At the same stage, evx1 is expressed in cells located in the ventral part of the diencephalon, in the constriction between the midbrain and the diencephalon, in regularly spaced lateral neurons in the midbrain, in the midbrain-hindbrain boundary (MHB) and in the posterior limit of the cerebellum anlage. Then, the signal reinforces in ventral midbrain and extends in the cerebellum. At 24 hpf, evx1 is transcribed in ventral and lateral walls of the cerebellum, and in four to six regularly spaced neurons in the forming tegmentum. From 32 to 50 hpf the discrete signal in the tegmentum turns into a continuous one (Thaeron, 2000).

In the rhombomeric region, evx1 transcripts are detected at 24 hpf in a bilateral antero-posterior row of cells from which extend six U-shaped stripes of evx1-expressing cells. These stripes extend from the ventral part of the alar plate to the anlage of the lateral sensory area in the boundaries between all rhombomeres. By 30 hpf, as the medial anteroposterior row fades in the rostral hindbrain, the first evx1 stripe thickens and the five posterior stripes seem to split in two. This gives rise (from 36 to 72 hpf) to a segmentally reiterated pattern of 12 evx1-positive clusters that mark the anterior and posterior borders of rhombomeres 2-7. In the lateral sensory area, the segmental aspect of the evx1 transcription restricts progressively to the rostral most rhombomeres. At 36 hpf, the evx1+ cells are located at the anterior and posterior borders of r1-r5, whereas they extend along the whole dorsal sensory area of rhombomeres 6, 7 and anterior spinal chord. In the following hours, the transcription of evx1 is maintained in the border regions of the rhombomeres 1-4, but fades in the corresponding regions of r5 and vanishes posteriorly. The evx1 positive cells at both medial and lateral levels are neurons that project their axons ventrally in the commissural tract. Comparisons with whole-mount immunodetections performed with the Zn8 (i.e. Zn5) monoclonal antibody show that the lateral evx1+ neurons correspond to the commissural interneurons identified in the border regions of the dorsal sensory area and that the medial ones are likely to correspond to another set of Zn8 faintly positive neurons (Thaeron, 2000).

Recently, a model to explain the mechanism of Xenopus tail bud formation has been proposed. The NMC model proposes that three regions around the late blastopore lip are required to initiate tail formation. These are the posterior-most neural plate, fated to form tail somites (M), the neural plate (N), immediately anterior to M, and the underlying caudal notochord (C). To initiate tail formation, C must underlie (and presumably signal to) the junction of N and M, which subsequently forms the tip of the tail. During normal development, the NMC interaction leading to specification of the tail bud occurs at the end of gastrulation. Outgrowth of the tail bud commences much later, becoming clearly visible by stage 30 (Beck, 1998 and references).

Several domains of the Xenopus tail bud are defined by two phases of gene expression. The first group of genes are already expressed in the tail bud region before its determination at stage 13 and are subsequently restricted in the extending tail bud by stage 30. This group, the early genes, includes the Notch ligand X-delta-1, the lim domain homeobox factor Xlim1, the T-box factor Xbra, and the homeobox factor Xnot2 and Xcad2, a member of the caudal family. X-delta-1 is expressed specifically in the posterior wall of the neuroenteric canal but is excluded from the chordoneural hinge at stage 30, thus maintaining its earlier expression in the lateral and ventral blastopore lips. Xim1 is expressed in the notochord and dorsal blastopore lip at the end of gastrulation, and is maintained in the chordoneural hinge and posterior tip of the differentiated notochord in later stages. Xnot2 is expressed in the ventral neural tube and chordoneural hinge, but not in the posterior notochord. The posterior notochord therefore represents a novel tail bud region by stage 30, marked by Xlim but not Xnot transcripts, whereas the posterior ventral neural tube is marked by Xnot but not Xbra or Xlim1. Xbra is expressed in the chordoneural hinge and posterior wall. Xcad3 expression in the posterior neural plate is later maintained in the posterior wall and posterior dorsal neural tube. Xpo is expressed in all tissues of the tail bud with the exception of the chordoneural hinge, and is expressed in the fin and epidermis (Beck 1998).

Unlike the early genes, the regional expression of the second group of genes in the extended tail bud can not be traced back to the stage of tail bud initiation. These genes have a late onset of localized expression in the tail bud, corresponding to the beginning of tail outgrowth, although they may be expressed elsewhere in the embryo at stage 13. The dorsal roof domain of the tail bud is marked by expression of Xwnt3a and lunatic fringe. Xwnt5a expression is restricted to the tail bud roof. The distal tip of the tail, which comprises part of the posterior wall, is marked by expression of Xhox3, which marks the distal cells of the tail bud. Xhox3 is a vertebrate homolog of Drosophila evenskipped. Other late genes include BMP-4, X-serrate-1 and BMP-2 (Beck, 1998).

The existence of distinct domains in the positions predicted for C and M is proposed. The restriction of Xcad3 and Xlim1 transcripts to the posterior of the notochord in the early neurula demonstrates that the posterior part of the notochord differs from the crest, corresponding to the C region. Novel domains of the tail bud are proposed to express different combinations of genes. These domains include the dorsal roof of the tail bud, the distal tip of the tail, marked by Xhox3, the chordoneural hinge, the posterior tip of the chordoneural hinge, the posterior wall domain, the tip of the posterior wall, the posterior notochord, the posterior wall of the neuroenteric canal and the ventral neural tube (Beck, 1998).

Even-skipped homologs in mammals

Continued: Evolutionary Homologs part 2/2

Interactive Fly, Drosophila even-skipped: Biological Overview | Transcriptional regulation | Post-transcriptional regulation | Targets of activity | Protein interactions | Developmental Biology | Effects of Mutation | References

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