Interactive Fly, Drosophila

zerknüllt


EVOLUTIONARY HOMOLOGS (part 1/2)

Zerknüllt homologs in invertebrates

Beetle and locust genes have been cloned that are homologous to the class 3 Hox genes of vertebrates (These include three isologous genes, (HoxA3, HoxB3 and HoxD3). Any gene homologous to vertebrate class 3 would be expected to lie between proboscipedia (a class 2 homolog) and Deformed (a class 4 homolog). Outside the homeobox class 3 genes share sequence motifs with the Drosophila zerknullt and z2 genes, and like zen, are expressed only in extraembryonic membranes. It would seem that the zen genes of Drosophila derive from a Hox class 3 sequence that once formed part of the common ancestral Hox cluster; in modern insects this (Hox) gene appears to have lost its role in patterning the anterio-posterior axis of the embryo, and has acquired a new function, specification of the amnioserosa. In the lineage leading to Drosophila, the zen genes have diverged with notable rapidity (Falciani, 1996).

The zerknüllt region of the Drosophila subobscura Antennapedia complex shows an irregular distribution of the conserved and diverged regions in comparison with D. melanogaster, with the homeobox and a putative activating domain completely conserved. Comparisons of the promoter sequence and pattern of expression of the gene during development suggest that the regulation of zen has been conserved during evolution. The conservation of zen expression in a subpopulation of the polar cells indicates the existence of an important role in such cells. There is a transitory segmented pattern of expression of zen in both species, suggesting the existence of interactions with a pair-rule gene (Terol, 1995).

In the long-germ insect Drosophila melanogaster, dorsoventral polarity is induced by localized Toll-receptor activation which leads to the formation of a nuclear gradient of the rel/NF-kappaB protein Dorsal. Peak levels of nuclear Dorsal are found in a ventral stripe spanning the entire length of the blastoderm embryo allowing all segments and their dorsoventral subdivisions to be synchronously specified before gastrulation. A nuclear Dorsal protein gradient of similar anteroposterior extension exists in the short-germ beetle, Tribolium castaneum, which forms most segments from a posterior growth zone after gastrulation. Tc-dl mRNA accumulates in the oocytes of mid and late stage egg chambers suggesting that Tc-dl, like Drosophila DL mRNA is maternally provided. In contrast to Drosophila, (1) nuclear accumulation is first uniform and then becomes progressively restricted to a narrow ventral stripe; (2) gradient refinement is accompanied by changes in the zygotic expression of the Tribolium Toll-receptor suggesting feedback regulation and, (3) the gradient only transiently overlaps with the expression of a potential target, the Tribolium twist homolog, and does not repress Tribolium decapentaplegic. The Tribolium Toll homolog however, differs from Drosophila Toll by being transcribed only zygotically. No nuclear Dorsal is seen in the cells of the growth zone of Tribolium embryos, indicating that here dorsoventral patterning occurs by a different mechanism. However, Dorsal is up-regulated and transiently forms a nuclear gradient in the serosa, a protective extraembryonic cell layer ultimately covering the whole embryo. The connection between Tc-dl and the patterning of the ectoderm is even more indirect. In Drosophila low levels of nuclear Dorsal activate the expression of type II target genes which are required for the formation of neuroectoderm. Simultaneously, Dorsal represses the type III target genes of the Dpp group confining their expression to the dorsal side, which gives rise to the amnioserosa and to the non-neurogenic ectoderm. No fate maps have been constructed for the Tribolium blastoderm embryo so far. However, the serosa cells seem to derive from a broad anterior domain that is slightly tilted to the dorsal side. The serosa is likely to be homologous to the amnioserosa of Drosophila. zen and dpp are expressed in both tissues. While in Drosophila these are dorsal expression domains, in Tribolium the zen and dpp domains initially have the shapes of anterior caps, which are symmetric with regard to the DV axis, even though the Tc-dl gradient has already formed. Only later Tc-zen shifts to the dorsal side, and Tc-dpp shifts to the ventral side. The latter is remarkable since it indicates that a target gene, which is repressed by Dorsal in Drosophila, might be activated by Dorsal in Tribolium. In Drosophila, zen and dpp are also expressed in terminal caps, which are not influenced by Dorsal. However, no function has so far been attributed to these terminal caps since all patterning functions seem to reside in the dorsal expression domains. It is tempting to suggest that the terminal caps are evolutionary remnants of short-germ development, where the extraembryonic tissues are derived from anterior rather than dorsally located regions of the blastoderm (Chen, 2000).

Genes of the Hox cluster are restricted to the animal kingdom and play a central role in axial patterning in divergent animal phyla. Despite its evolutionary and developmental significance, the origin of the Hox gene cluster is obscure. The consensus is that a primordial Hox cluster arose by tandem gene duplication close to animal origins. Several homeobox genes with high sequence identity to Hox genes are found outside the Hox cluster and are known as 'dispersed' Hox-like genes; these genes may have been transposed away from an expanding cluster. Three of these dispersed homeobox genes form a novel gene cluster in the cephalochordate amphioxus. AmphiCdx, AmphiXlox and AmphiGsx are in respectively the Caudal, Zerknullt, and Gsh (related to Labial and Proboscipedia) families of homeodomain proteins. The finding that amphioxus Gsx, Xlox and Cdx class genes form a novel homeobox cluster challenges the idea that these homeobox gene classes are 'dispersed' Hox genes. Instead it is argued that this 'ParaHox' gene cluster is an ancient paralogue (evolutionary sister) of the Hox gene cluster. ParaHox and Hox gene clusters arose by duplication of a ProtoHox gene cluster containing Gsx, Xlox and Cdx genes representing an anterior, 'group three' and posterior subfamily. Amphioxus ParaHox genes have co-linear developmental expression patterns in anterior, middle and posterior tissues. It is proposed that the origin of distinct Hox and ParaHox genes by gene-cluster duplication facilitated an increase in body complexity during the Cambrian explosion (Brooke, 1998).

The class 3 Hox gene ortholog in insects, zerknullt (zen), is not expressed along the anterior-posterior axis, but only in extra-embryonic tissues, suggesting that it has lost its function as a normal Hox gene. To analyze whether this loss of Hox gene function has already occurred in a basal arthropod lineage, a Hox3 ortholog was isolated from the spider Cupiennius salei. In contrast to the insect zen sequences, which have a highly diverged homeobox, the spider Hox3 gene ortholog, Cs-Hox3, shows a high sequence similarity to the class 3 Hox genes of other phyla, including chordates. In situ hybridization in early embryos shows that it is expressed in a continuous region covering the pedipalp segment and the four leg-bearing segments. This expression pattern suggests a Hox-gene-like function for Cs-Hox3. However, the expression pattern does not strictly follow the colinearity rule, since it overlaps fully with the expression domain of the class 1 ortholog of the spider, Cs-lab. Still, these data suggest that the ancestor of the arthropods must have had a class 3 Hox gene with a function in anterior-posterior axis specification and that this function has been lost in the lineage leading to the insects (Damen, 1998).

From an oribatid mite, Archegozetes longisetosus, a gene homologous to the zerknullt (zen) insect genes and the Hox 3 genes of vertebrates has been cloned. Hox genes specify cell fates in specific regions of the body in all metazoans studied and are expressed in restricted regions in the anterior to posterior axis of the embryo. This is true of the vertebrate Hox 3 but not of the zen genes, the insect homologs, and it has been proposed that the zen genes lost their Hox-like function in the ancestor of the insects. Expression of a mite Hox 3/zen homolog was studied. It is expressed in a discrete antero-posterior region of the body with an anterior boundary coinciding with that of the chelicerate homolog of the Drosophila Hox gene, proboscipedia. The anterior boundary is found in the pedipalps, one segment forward of an anterior boundary of AlDef (Archegozetes Hox 4 homolog). It is proposed that loss of the Hox function in insect zen genes is due to functional redundancy due to this overlap with another Hox gene (Telford, 1998).

In Drosophila, the gene bicoid functions as the anterior body pattern organizer. Embryos lacking maternally expressed bicoid fail to develop anterior segments, including the head and thorax. In wild-type eggs, Bicoid mRNA is localized in the anterior pole region and the Bicoid protein forms an anterior-to-posterior concentration gradient. bicoid activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal Caudal mRNA in the anterior region of the embryo. caudal as well as other homeobox genes and members of the Drosophila segmentation gene cascade have been found to be conserved in animal evolution. In contrast, bicoid homologs have been identified only in close relatives of the schizophoran fly Drosophila. This poses the question of how the bicoid gene evolved and adopted its unique function in organizing anterior-posterior polarity. bicoid has been cloned from a basal cyclorrhaphan fly, Megaselia abdita (Phoridae, Aschiza). The cyclorrhaphan flies are divided into two subordinate groups: the Aschiza and the Schizophora. The phorid Megaselia is an aschizan fly. Therefore, it is different from the monophyletic group of schizophoran flies that includes Drosophila and the few other species where, to date, bicoid has been identified. Megaselia-bicoid (Ma-bcd) transcripts accumulate first in the oocyte, where a transient ring-shaped pattern is observed. Later, during oogenesis, transcripts are expressed in the nurse cells; they accumulate in the anterior region of the oocyte and transiently at its posterior pole. In the early embryo, Ma-bcd transcripts spread from the anterior pole forming an enlarging anterior cap until the onset of cellularization. Subsequently, transcripts disappear rapidly. No zygotic expression is observed during embryogenesis. These findings establish identical expression patterns for bicoid and Ma-bcd in Drosophila and Megaselia (Stauber, 1999).

A comparison of the Ma-bcd homeodomain and homeodomain proteins of Drosophila indicates that aside from bicoid, Ma-bcd is most similar to zerknüllt (48.3%), whereas the similarity to homeodomains encoded by the other members of the Drosophila Hox-C is less pronounced (45.0%-36.7%). The homeodomain of Ma-bcd is related only distantly to the homeodomains encoded by orthodenticle and the Drosophila homologs of goosecoid and Ptx1 (38.3%-33.3%), which have been classified in the past as bicoid-related genes. These proteins share common DNA-binding properties that depend on the diagnostic lysine in position 50 of the homeodomain. Also notable is the fact that the zerknüllt homologs of other insects and their orthologs in various animal classes (the Hox3 genes of chordates, ribbonworm, and spider) show a higher degree of similarity to the Ma-bcd homeodomain than do the bicoid-related genes. These observations suggest that, in spite of the considerable sequence divergence exhibited by the Drosophila genes, bicoid and zerknüllt are closely related (Stauber, 1999).

To address the hypothesis that bicoid and zerknüllt are the closest relatives among Hox genes, the Megaselia zerknüllt gene (Ma-zen) was cloned to determine if Ma-zen provides a link between bicoid and the Hox3 genes of the vertebrate Hox clusters. Ma-zen was isolated by a PCR approach and the genomic DNA encompassing the transcription unit was isolated. Whole-mount in situ hybridization of Megaselia embryos reveals that Ma-zen transcripts are expressed only zygotically. They form a restricted pattern at the dorsal side of the blastoderm embryo, covering the area of the amnioserosa precursor cells, and disappear in the extended germ-band stage. Thus, Ma-zen is expressed like zerknüllt in Drosophila. This suggests that Ma-bcd and Ma-zen have separate functions in Megaselia, similar to the functions of their homologs in Drosophila (Stauber, 1999).

Sequence comparison of Ma-bcd and Ma-zen proteins clearly establishes a sister relationship between the two proteins. Evidence for this is based on the following findings. The homeodomain of Ma-bcd shows a higher sequence similarity to Ma-zen (50.0%) than to any other nonorthologous homeodomain. In addition, molecular phylogenetic trees involving the homeodomains of the Hox complex genes of Drospohila resolve with high confidence when the Ma-bcd and Ma-zen sequences, instead of the bicoid and zerknüllt sequences, are used for the analysis. It is important to note that the Drosophila homeodomains of the Hox complex evolve very slowly (except fushi tarazu) and can be assumed to be identical or almost identical in amino acid sequence in Megaselia and Drosophila. The alignment of the Ma-bcd and Ma-zen proteins shows conservation of sequences not only in the homeodomain but also N-terminal to it. The conserved sequences in front of the homeodomain are not evident from the comparison of Drosophila bicoid and zerknüllt. Thus, the sister-gene relationship of bicoid and zerknüllt revealed by the Megaselia genes remains hidden when the obviously more diverged sequences of the Drosophila genes are compared (Stauber, 1999).

The newly established sister-gene relationship implies that bicoid genes, like the zerknüllt genes, are direct homologs of the Hox3 genes in the Hox-C of noninsect animal classes. Thus, bicoid is a Hox gene in the phylogenetic sense, and the location of bicoid in the Hox-C of Drosophila is an ancestral trait. The consistent failure to isolate bicoid from insects other than flies, which has been attempted in various laboratories, suggests a recent origin for the bicoid gene. The fact that Ma-bcd is more similar to the zerknüllt genes of higher insects than to other Hox3 homologs is consistent with the assumption that bicoid originated recently during insect radiation (Stauber, 1999).

bicoid is expressed in the anterior egg region, where it exerts its role in patterning the anterior body of the larval fly. In contrast, zerknüllt and its orthologs function in extraembryonic anlagen. Although the extraembryonic anlage in flies, the amnioserosa, is located at the dorsal side of the blastoderm fate map, extraembryonic anlagen in other insects, such as the beetle Tribolium, are formed in an anterior egg position (see Tribolium early embryonic development). This suggests that initially the sister genes bicoid and zerknüllt may have been coexpressed in the anterior egg region. The subsequent recruitment of bicoid in patterning the embryo, instead of determining the dorsally shifted extraembryonic anlagen, changed the selection conditions for the gene. Ensuing adaptations must have resulted in a new set of target genes, as reflected by the characteristic lysine in Bicoid's homeodomain position 50 that specifies DNA recognition. The newly acquired functions of Bicoid entrained a significant change in the developmental mechanism of axis specification and now furnish an outstanding model of molecular evolution in a patterning process (Stauber, 1999).

The members of the evolutionarily conserved Hox-gene complex are required for specifying segmental identity during embryogenesis in various animal phyla. The Hox3 genes of winged insects have lost this ancestral function and are required for the development of extraembryonic epithelia, which do not contribute to any larval structure. Higher flies (Cyclorrhapha) such as Drosophila melanogaster contain Hox3 genes of two types, the zerknüllt type and the bicoid type. The zerknüllt gene is expressed zygotically on the dorsal side of the embryo and is required for establishing extraembryonic tissue. Its sister gene bicoid is expressed maternally and the transcripts are localized at the anterior pole of the mature egg. Bicoid protein, which emerges from this localized source during early development, is required for embryonic patterning. All known direct bicoid homologs are confined to Cyclorrhaphan flies. This study describes Hox3 genes of the non-Cyclorrhaphan flies Empis livida (Empididae), Haematopota pluvialis (Tabanidae), and Clogmia albipunctata (Psychodidae). The gene sequences are more similar to zerknüllt homologs than to bicoid homologs, but they share expression characteristics of both genes. It is proposed that an ancestral Hox3 gene had been duplicated in the stem lineage of Cyclorrhaphan flies. During evolution, one of the gene copies lost maternal expression and evolved as zerknüllt, whereas the second copy lost zygotic expression and evolved as bicoid. These findings correlate well with a partial reduction of zerknüllt-dependent extraembryonic tissue during Dipteran evolution (Stauber, 2002).

Clogmia zerknüllt (Ca-zen) is strongly expressed in the germ-line cells of ovarian egg chambers. The transcripts are detected in the nurse cells and the oocyte, and seem to be evenly distributed in the early embryo. To corroborate maternal expression of Ca-zen, a Northern blot analysis was performed with mRNA prepared from ovaries. A single band of expected size is obtained after hybridization with a Ca-zen probe. Zygotic expression of Ca-zen starts during cellularization of the blastoderm in an anterior and dorsal domain, which corresponds to the anlage of extraembryonic tissue. Extraembryonic Ca-zen expression is maintained during gastrulation, but no zygotic Ca-zen expression outside the extraembryonic anlage/tissue is observed. The expression pattern of Ca-zen differs in two important ways from conserved zerknüllt expression in Cyclorrhaphan flies: (1) Ca-zen is expressed maternally, whereas in Cyclorrhapha zerknüllt genes are expressed strictly zygotically; (2) zygotic Ca-zen expression extends to the anterior tip of the cellular blastoderm, whereas zerknüllt expression in Cyclorrhapha is restricted to a narrow dorsal strip at the same developmental stage. Thus, Ca-zen in Clogmia combines expression characteristics of bicoid and zerknüllt in Cyclorrhapha (Stauber, 2002).

The maternal expression of all three newly identified Hox3/zerknüllt homologs implies selection for this trait in lower Diptera and release from a corresponding specific constraint in Cyclorrhapha. To understand better the changing developmental constraints during the evolution of Diptera, embryonic development throughout this taxon was compared. Embryonic development in Diptera seems rather uniform and resembles that of Drosophila. However, an important difference within Diptera occurs with respect to extraembryonic tissue organization. The establishment of extraembryonic tissue requires the activity of a Hox3/zerknüllt gene not only in Drosophila, but most likely in all winged insects. In species of several insect orders and, in particular, in all non-Cyclorrhaphan flies analyzed so far, including the three species of this study, extraembryonic tissue consists of an amnion and a serosa. These two epithelia do not contribute to the embryo proper but transiently wrap the embryo. In contrast, Cyclorrhapha including Megaselia and Drosophila develop without such wrapping, and the extraembryonic tissue is reduced to a transient dorsal epithelium, termed amnioserosa and, as recently discovered in Drosophila, some additional cells surrounding the yolk. Thus, the transition in extraembryonic tissue organization in the stem lineage of Cyclorrhaphan flies occurs in a period when maternal expression is lost in the zerknüllt-type Hox3 genes (Stauber, 2002).

On the basis of these findings, it is proposed that bicoid and zerknüllt evolved in the stem lineage of Cyclorrhaphan flies from a Hox3 gene with maternal and zygotic expression, which is still found in non-Cyclorrhaphan Diptera. In the common progenitor, zygotic activity was required for extraembryonic development, a feature conserved by the Cyclorrhaphan zerknüllt genes. Maternal activity of Hox3/zerknüllt homologs is not understood currently; some better understanding will require adopting methods for gene inactivation in non-Cyclorrhaphan Diptera. However, because maternal expression of Hox3/zerknüllt homologs is conserved in all non-Cyclorrhaphan Diptera analyzed so far, maternal activities of these genes are important for development. An understanding is lacking of how maternal Hox3/zerknüllt activity turned into maternal bicoid activity, one of the problems being the different DNA- and RNA-binding properties of Bicoid compared with all other Hox genes. In Drosophila, ectopic expression of zerknüllt induces extraembryonic tissue and ectopic expression of bicoid induces anterior embryonic structures. Thus, both genes have counteracting effects and cannot convert their respective activities in the same spatial domain of the embryo. Therefore, a separation of the functional expression domains of bicoid and zerknüllt in time and space, as well as selective loss of maternal versus zygotic enhancer elements, seems to be an important prerequisite for subsequent divergent evolution of both genes in Cyclorrhapha. It is suggested that anterior localization of Bicoid, which is based on specific sequence elements in the 3' untranslated region of the gene, and respecification of anterior blastoderm toward an embryonic fate were important steps toward this goal. In summary, the key features of this model are as follows: a single Hox3 gene with maternal and zygotic activity is present in the stem lineage of Diptera; it was duplicated in the stem lineage of Cyclorrhapha, giving birth to maternal bicoid and zygotic zerknüllt. The functional evolution of Bicoid-specific DNA- and RNA-binding properties became possible after the reduction of the extraembryonic anlage/tissue. It will be challenging to test this model at the levels of genomics, developmental genetics, and morphology (Stauber, 2002).

The expression patterns of Hox genes have not previously been comprehensively analyzed in a myriapod. The expression patterns are presented of the ten Hox genes in a centipede, Lithobius atkinsoni, and these results are compared to those from studies in other arthropods. Three major findings are reported. (1) It has been found that Hox gene expression is remarkably dynamic across the arthropods. The expression patterns of the Hox genes in the centipede are in many cases intermediate between those of the chelicerates (spiders) and those of the insects and crustaceans, consistent with the proposed intermediate phylogenetic position of the Myriapoda. (2) Two 'extra' Hox genes were found in the centipede compared with those in Drosophila. Based on its pattern of expression, Hox3 appears to have a typical Hox-like role in the centipede, suggesting that the novel functions of the Hox3 homologs zen and bicoid were adopted somewhere in the crustacean-insect clade. In the centipede, the expression of the gene fushi tarazu suggests that it has both a Hox-like role (as in the mite), as well as a role in segmentation (as in insects). This suggests that this dramatic change in function was achieved via a multifunctional intermediate, a condition maintained in the centipede. (3) It was found that Hox expression correlates with tagmatic boundaries, consistent with the theory that changes in Hox genes had a major role in evolution of the arthropod body plan (Hughes, 2002).

The results presented in this study are relevant to the functional change from Hox3 (with a Hox-like role) to zen (with a role in extra-embryonic tissues). In spiders and a mite, the Hox3 gene has a typical Hox-like expression pattern, with a broad domain whose anterior boundary is approximately co-linear with the other Hox genes. The homologous genes of the grasshopper Schistocerca and the beetle Tribolium apparently have zen-like roles, with expression in the extra-embryonic serosa. The centipede Hox3 gene presented here has a Hox-like expression pattern in the segments of the embryonic germband, with no hint of an extra-embryonic domain. Thus, the window of the change in developmental function from Hox3 to zen has been narrowed to somewhere in the insect-crustacean clade. Further studies on crustaceans and lower insects may be able to pinpoint more precisely the phylogenetic timing of the change, and perhaps shed light on the context and the process by which this rogue Hox gene escaped from its role in determining segment identity (Hughes, 2002).

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. 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. It was shown otd1 and hunchback also influence dorsoventral patterning by regulating zerknült (zen) as they do in Tribolium, but 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 (Wilson, 2011).

High plasticity in epithelial morphogenesis during insect dorsal closure
.

Insect embryos complete the outer form of the body via dorsal closure (DC) of the epidermal flanks, replacing the transient extraembryonic (EE) tissue. Cell shape changes and morphogenetic behavior are well characterized for DC in Drosophila, but these data represent a single species with a secondarily reduced EE component (the amnioserosa) that is not representative across the insects. This study examined DC in the red flour beetle, Tribolium castaneum, providing the first detailed, functional analysis of DC in an insect with complete EE tissues (distinct amnion and serosa). Surprisingly, it was found that differences between Drosophila and Tribolium DC are not restricted to the EE tissue, but also encompass the dorsal epidermis, which differs in cellular architecture and method of final closure (zippering). EE tissue complement was experimentally manipulated via RNAi for Tc-zen1, eliminating of the serosa and allow examination of viable DC in a system with a single EE tissue (the amnion). It was found that the EE domain is particularly plastic in morphogenetic behavior and tissue structure. In contrast, embryonic features and overall kinetics are robust to Tc-zen1(RNAi) manipulation in Tribolium and conserved with a more distantly related insect, but remain substantially different from Drosophila. Although correct DC is essential, plasticity and regulative, compensatory capacity have permitted DC to evolve within the insects. Thus, DC does not represent a strong developmental constraint on the nature of EE development, a property that may have contributed to the reduction of the EE component in the fly lineage (Panfilio, 2013; full text of article).

Zerknüllt homologs in vertebrates

Continued: zerknüllt Evolutionary homologs part 2/2


zerknüllt: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.