Table of contents

Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. This study determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. A computational system was developed that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and full 8-mer binding profiles were inferred for the majority of known animal homeodomains. The results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success (Berger, 2008).

It was asked whether the homeodomain monomer binding preferences identified in vitro reflect sequences preferred in vivo. Anecdotally, the highest predicted binding sequences do correspond to known in vivo binding sites. For example, in the predicted 8-mer profile for sea urchin Otx, a previously identified in vivo binding sequence (TAATCC, from the Spec2a RSR enhancer), is contained in the top predicted 8-mer sequence, and, more strikingly, it is embedded in the fifth-highest predicted 8-mer sequence (TTAATCCT). At greater evolutionary distance, three of the four Drosophila Tinman binding sites in the minimal Hand cardiac and hematopoietic (HCH) enhancer are contained within the second (TCAAGTGG), fifth (ACCACTTA), and ninth (GCACTTAA) ranked 8-mers (the fourth overlaps the 428th ranked 8-mer [CAATTGAG], but also overlaps with a GATA binding site and may have constraints on its sequence in addition to binding Tinman) (Berger, 2008).

To ask more generally whether occupied sites in vivo contain sequences preferred in vitro, six ChIP-chip or ChIP-seq data sets in the literature were examined that involved immunoprecipitation of homeodomain proteins that were analyzed, or homologs of proteins analyzed that shared at least 14 of the 15 DNA-contacting amino acids. In all cases, enrichment was observed for monomer binding sites in the neighborhood of the bound fragments, with a peak at the center. Two examples, Drosophila Caudal and human Tcf1/Hnf1 are shown. For Caudal, the size of this ratio peak increased dramatically with E score cutoff, indicating that the most preferred in vitro monomer binding sequences correspond to the most enriched in vivo binding sites (51% of bound fragments have such an 8-mer, versus 17% in randomly selected fragments). For Tcf1/Hnf1, however, the majority of sequences bound in vivo do not contain the best in vitro binding sequences, although most do contain at least one 8-mer with E > 0.45 (53%, versus 27% in random fragments), suggesting utilization of weaker binding sites. Similar results were obtained with PWMs. Thus, the requirement for highest-affinity binding sequences may vary among homeodomain proteins, species, or under different physiological contexts. Nonetheless, a large proportion of the in vivo binding events apparently involve the monomeric homeodomain sequence preferences, which can be derived in vitro (Berger, 2008).

Orthodenticle homologs in C. elegans

Temperature is a critical modulator of animal metabolism and behavior, yet the mechanisms underlying the development and function of thermosensory neurons are poorly understood. C. elegans senses temperature using the AFD thermosensory neurons. Mutations in the gene ttx-1 affect AFD neuron function. This study shows that ttx-1 regulates all differentiated characteristics of the AFD neurons. ttx-1 mutants are defective in a thermotactic behavior and exhibit deregulated thermosensory inputs into a neuroendocrine signaling pathway. ttx-1 encodes a member of the conserved OTD/OTX homeodomain protein family and is expressed in the AFD neurons. Misexpression of ttx-1 converts other sensory neurons to an AFD-like fate. This results extend a previously noted conservation of developmental mechanisms between the thermosensory circuit in C. elegans and the vertebrate photosensory circuit, suggesting an evolutionary link between thermosensation and phototransduction (Satterlee, 2001).

Interestingly, while the related genes ttx-1 and vertebrate Crx have been shown to be required for the differentiation of the AFD thermosensory and the vertebrate photoreceptor cells in the retina respectively, another orthologous gene pair has been shown to be required for the development and differentiation of their primary postsynaptic partners. In the vertebrate retina, the paired-type homeobox gene Chx10/Vsx-1 has been implicated in the development of the bipolar cells, a major class of interneurons in the inner nuclear layer. In C. elegans, ceh-10, the ortholog of Chx10, is required for the differentiation of the AIY interneurons, the major postsynaptic partner of the AFD neurons. Photoresponses have been described for C. elegans, although the cellular bases for these responses are unknown, and these responses have not been genetically characterized. It had been proposed that the conservation of expression patterns and function between ceh-10 and Chx10 may suggest a common evolutionary history between the thermosensory circuit and the photosensory circuit. Results in this paper further extend this hypothesis. Alternatively, this conservation of regulatory mechanisms between the thermosensory and photosensory circuits may represent an example of a developmental genetic pathway recruited independently to perform multiple tasks in different developmental contexts (Satterlee, 2001).

The food-associated thermotaxis behavior of C. elegans is regulated by the thermosensory circuit. This circuit consists of two counterbalancing drives that promote movement either upward or downward in a thermal gradient. Crosstalk between these two opposing pathways results in the correct choice of the preferred temperature. Similarly, in the hypothalamus, the anterior and the posterior areas regulate decreases or increases in body temperature, respectively (Satterlee, 2001 and references therein).

Removal of both branches of the thermosensory circuit by ablation of the AFD and AIZ neurons results in athermotactic behavior. However, a lin-11;ceh-14;ttx-3 triple mutant that has been suggested to also abolish the functions of the AFD-AIY-AIZ circuit shows cryophilic behavior. This apparent contradictory result has led to the proposal of an additional underlying cryophilic circuit, whose function is revealed only in the absence of the AFD-AIY-AIZ circuit. A basic assumption of this model is that mutations in lin-11, ceh-14, and ttx-3 completely abolish all thermoregulatory functions of AIZ, AFD, and AIY neurons, respectively. Mutations in ttx-1 as well as in ttx-3 result in strong cryophilic behavior, similar to that of animals in which either the AFD or AIY neurons have been killed. Thus, it is likely that most, if not all, thermoregulatory functions of the AFD neurons in ttx-1 mutants and AIY neurons in ttx-3 mutants are abolished. However, mutations in lin-11 only partly affect AIZ thermoregulatory function. Consistent with this, lin-11;ttx-1 double mutants continue to exhibit strong cryophilic behavior. Similarly, mutations in ceh-14 may only partly affect the thermosensory functions of the AFD neurons. On the basis of these results, it is suggested that the observed cryophilic behavior of the lin-11;ceh-14;ttx-3 triple mutant results from a strong loss of function of only the thermophilic arm (but not the cryophilic arm) of the circuit and that the AFD-AIY-AIZ circuit is likely the major circuit mediating the examined thermotaxis behavior in C. elegans (Satterlee, 2001 and references therein).

A key feature of thermoregulation in endotherms is the maintenance of internal temperature homeostasis by the hypothalamus. Ectotherms may also maintain an internal body temperature (the preferred or selected temperature), primarily through behavioral modifications. In ttx-1 mutants, the arm of the circuit mediating responses to cold temperature is intact, whereas the arm mediating sensation of warm temperatures is affected. It is possible that when ttx-1 mutants are grown at a given temperature in the presence of food, the absence of signals from the AFD-AIY arm of the circuit results in resetting the animal's food-associated preferred temperature to a lower value. Thus, upon being challenged with a thermal gradient, ttx-1 mutants move to lower temperatures and are therefore cryophilic (Satterlee, 2001).

Because several temperature-regulated processes are normal in ttx-1 mutants, multiple thermosensory circuits must function to sense temperature and to relay them to different behavioral, developmental, and endocrine circuits. Under the conditions examined, entry into the dauer stage is only weakly affected in ttx-1 mutants, whereas exit is severely affected. The sensory neurons and pathways regulating entry and exit from the dauer stage are distinct, and their outputs are likely regulated by distinct sources of sensory inputs. Recovery from (but not entry into) the dauer stage may involve cholinergic signaling via a muscarinic acetylcholine pathway. It has been proposed that temperature inputs via the AFD-AIY-AIZ circuit promote acetylcholine release from unknown neurons, which in turn promotes insulin signaling and consequent recovery from the dauer stage. This is analogous to the hypothalamus-mediated temperature control of metabolic rate. Although daf-7;ttx-1 and daf-7;ttx-3 mutants enter the dauer stage, the resetting of the preferred or selected temperature to lower values may result in the inappropriate production of high levels of acetylcholine and the consequent insulin signaling, resulting in rapid recovery. Consistent with this hypothesis, neither daf-2;ttx-1 nor daf-2;ttx-3 double mutants recover at high temperatures, confirming that insulin signaling, but not TGF-ß signaling, is crucial for dauer recovery. Taken together, the AFD-regulated thermosensory circuit appears to act similarly to the hypothalamus in regulating both motor behavior and endocrine signaling in response to temperature cues. Few molecules involved in thermosensation have been identified. A major goal for the future is the identification of downstream targets of TTX-1 because a subset of these is expected to be comprised of genes directly involved in thermosensation (Satterlee, 2001).

The mechanisms by which the diverse functional identities of neurons are generated are poorly understood. C. elegans responds to thermal and chemical stimuli using 12 types of sensory neurons. The Otx/otd homolog ttx-1 specifies the identities of the AFD thermosensory neurons. ceh-36 and ceh-37, the remaining two Otx-like genes in the C. elegans genome, specify the identities of AWC, ASE, and AWB chemosensory neurons, defining a role for this gene family in sensory neuron specification. All C. elegans Otx genes and rat Otx1 can substitute for ceh-37 and ceh-36, but only ceh-37 functionally substitutes for ttx-1. Functional substitution in the AWB neurons is mediated by activation of the same downstream target lim-4 by different Otx genes. Misexpression experiments indicate that although the specific identity adopted upon expression of an Otx gene may be constrained by the cellular context, individual Otx genes preferentially promote distinct neuronal identities (Lanjuin, 2003).

In mice, both Otx2 and otd can substitute for Otx1 for the majority of Otx1-mediated functions. However, the defects in the lateral semicircular canal of the inner ear present in Otx1 mutant mice are not rescued. This result has led to the hypothesis that, although the distinct phenotypes of the Otx1 and Otx2 mutant mice are largely a consequence of divergence in the regulation of spatiotemporal gene expression as opposed to divergence in gene functions, OTX1 may have acquired additional functions. Similarly, the results of this study indicate that while all three Otx-like genes appear to be able to promote AWB and AWC neuronal characteristics, only ceh-37 is capable of substituting for ttx-1 function in the AFD neurons. Divergence in the 3' UTRs of Otx1 and Otx2 has been shown to result in differential translation of these gene transcripts in the visceral endoderm and epiblast. Although similar divergence in posttranscriptional regulatory mechanisms or differences in protein threshold requirements may play a role in the inability of ceh-36 to fully substitute for the ttx-1 mutant phenotype, the partial rescue observed upon expression of the mutant CEH-36(-VIT) protein suggests that divergence in protein function in the context of the AFD neurons is likely to be critical. Thus, as in mammals, divergence in both OTX protein function as well as gene expression patterns may account for the distinct mutant phenotypes of the Otx genes in C. elegans. These differences may result from different rates of evolutionary change in the regulatory and coding sequences of these genes that presumably arose via gene duplication (Lanjuin, 2003).

Interestingly, expression of the Otx genes in the phasmid neurons uncovers intrinsic preferences of the OTX proteins for promoting distinct cell fates. In the phasmid neurons, the cellular context does not appear to constrain the identity selected upon misexpression of an Otx gene, such that an individual phasmid neuron may adopt either AWC or AFD cellular features. Instead, the identity chosen is partly dictated by the Otx gene expressed. Expression of ceh-36 appears to preferentially allow the expression of AWC markers, whereas expression of ceh-37 or ttx-1 preferentially directs the expression of AFD-specific markers. Although the ability of ceh-36 to preferentially direct the AWC fate may be in part due to its relative inefficiency in directing the AFD fate, ceh-37 and ttx-1 appear to be capable of directing either the AWC or AFD fates when expressed in the AWC or AFD neurons, respectively. These results reveal differences in the intrinsic functions of these OTX proteins and suggest that phenotypic equivalence may arise partly as a consequence of restriction of function by the cellular context of expression (Lanjuin, 2003).

The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. This study shows that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. MLS-2 acts in additional neuron types to regulate their development and differentiation. This analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system (Kim, 2010).

Orthodenticle homologs in other arthropods

Orthopedia (Otp), a novel homeobox-containing Drosophila protein is homologous to both the Orthodenticle and Antennapedia homeodomain proteins, and homologous to the Orthopedia protein identified in the mouse. OTP is highly conserved in evolution. In mouse, otp is expressed in restricted domains of the developing forebrain, hindbrain, and spinal cord (Simeone, 1994).

There are two orthodenticle-related genes in the short-germ beetle Tribolium castaneum. One of the genes (Tc otd-1) is more related to both the amino acid sequence and expression pattern of fruitfly otd. Tc otd-1 is expressed in a broad anterior stripe in the blastoderm embryo, suggesting a role in early head segmentation similar to that of the Drosophila gene. The second gene, Tc otd-2, is more similar in sequence to the otd-related genes isolated from different vertebrate species. Tc otd-2 is not transcribed in the blastoderm, but is expressed later in more limited subsets of cells in the anterior brain. Both the Tribolium genes and the Drosophila gene are expressed in the developing midline of the embryo (Li, 1996).

In Drosophila, the morphogen Bicoid organizes anterior patterning in a concentration-dependent manner by activating the transcription of target genes such as orthodenticle (otd) and hunchback (hb), and by repressing the translation of caudal. Homologs of the bicoid gene have not been isolated in any organism apart from the higher Dipterans. In fact, head and thorax formation in other insects is poorly understood. To elucidate this process in a short-germband insect, the functions of the conserved genes orthodenticle-1 (otd-1) and hb were analyzed in the flour beetle Tribolium castaneum. In contrast to Drosophila, Tribolium otd-1 messenger RNA is maternally inherited by the embryo. Reduction of Tribolium otd-1 levels by RNA interference (RNAi) results in headless embryos. This shows that otd-1 is required for anterior patterning in Tribolium. As in Drosophila, Tribolium hb specifies posterior gnathal and thoracic segments. The head, thorax and the anterior abdomen fail to develop in otd-1/hb double-RNAi embryos. This phenotype is similar to that of strong bicoid mutants in Drosophila. It is suggested that otd-1 and hb are part of an ancestral anterior patterning system (Schröder, 2003).

In Drosophila, bicoid is the central gene in the anterior patterning system. Embryos that do not express Bicoid protein fail to develop a head or thorax, and form a second telson at the anterior pole instead. Binding specificity of Bicoid to its DNA and RNA targets is mediated by a lysine (K) at position 50 of its homeodomain (K50HD). Despite its pivotal role in defining anterior pattern in Drosophila, bicoid orthologs have been isolated only from closely related higher Dipterans. In Drosophila and other higher Dipterans, a bicoid ortholog is located in the Hox gene complex close to the zerknüllt/Hox3 locus -- between proboscipedia/ Hox2 and Deformed/Hox4. A bicoid ortholog is not present in this genomic interval in the beetle Tribolium. This finding corroborates the hypothesis that bicoid evolved recently, probably through the divergence of a Hox3 paralog during evolution of the higher Dipterans, and is not part of an ancient anterior system common to more basal insects (Schröder, 2003).

How is anterior patterning in the embryo organized in the absence of bicoid? In Drosophila bicoid mutants, high levels of bicoid-independent zygotic hb are able to direct the formation of a partial thorax. However, since hb cannot induce formation of head structures on its own, the conserved protein Otd, which like Bicoid contains a K50 homeodomain, has been considered to be an ancestral head determinant. This hypothesis has been tested in an insect that develops as a short-germband embryo, and therefore shows a more general type of embryogenesis. Initially, the function of otd-1 was analyzed in the beetle Tribolium (Schröder, 2003).

In Drosophila, otd acts as a zygotic head gap gene that specifies the ocular and the antennal segments14. Of the two otd paralogs known to exist in Tribolium (otd-1 and otd-2), only otd-1 is expressed during early blastoderm formation. Like its ortholog in Drosophila, Zygotic otd-1 in Tribolium is expressed in an anterior position during early embryogenesis. This indicates that otd-1 might specify similar head segments in both species. In contrast to Drosophila, Tribolium otd-1 mRNA is provided maternally to the developing embryo. Tribolium otd-1 mRNA is initially distributed uniformly in the egg, and is present at the beginning of blastoderm formation. During blastoderm stages, otd-1 mRNA and protein recede first from the posterior and later also from the anterior pole, resulting in a belt-like domain in the prospective head region. Dorsal otd-1 mRNA then begins to disappear, leading to a ventrally located head stripe. In Tribolium, RNAi has been shown to phenocopy gene disruption (Schröder, 2003).

To investigate the function of otd-1 during early pattern formation in Tribolium, double-stranded (ds) otd-1 RNA was injected into eggs (otd-1eRNAi) or female pupae (otd-1pRNAi). The goal of applying pRNAi was to knock down maternally supplied otd-1 mRNA and to avoid injection artefacts. Of the larval cuticles analysed, 91% of otd-1eRNAi and 100% of otd-1 pRNAi developed defects in anterior patterning. In the most extreme cases, the embryos lacked all head structures, indicating that Tribolium otd-1 is required for head formation. Depending on severity, these cuticles can be grouped into classes I-VI: from weakly to strongly affected larvae. The range of cuticle defects is reminiscent of weak and intermediate bicoid mutants in Drosophila. Moreover, head defects are detectable before cuticle formation in embryos, as determined by an analysis of Engrailed expression. This shows that Tribolium otd-1 is required for anterior patterning early during embryogenesis (Schröder, 2003).

A significantly greater number of the more strongly affected class III and IV embryos was observed after pRNAi, compared with eRNAi. The pRNAi depletes most of the maternal and zygotic otd-1 mRNA, whereas eRNAi seems to deplete only zygotic otd-1 mRNA. Therefore, otd-1 seems to be required to define the presumptive head region in Tribolium. Furthermore, maternal otd-1 displays functional characteristics of a coordinate gene, as bicoid does in Drosophila, rather than of a canonical head gap gene (Schröder, 2003).

In Tribolium and Drosophila, zygotic otd seems to act as a head gap gene. otd-1 is also involved in the formation of the anterior-most region of the egg -- the extraembryonic serosa. In otd-1pRNAi embryos, the nuclei of the serosa are irregularly arranged or, in more severe cases, reduced in number. hb, which is strongly expressed in the serosa of the wild-type embryos, is also still detectable in the nuclei of the serosa in otd-1pRNAi embryos. otd-1 is therefore not required to activate hb expression in the serosa. Nevertheless, otd-1 could assist other factors in activating hb expression, such as zerknüllt, which is also expressed in this tissue (Schröder, 2003).

The fact that the otd-1RNAi phenotype only incompletely mimics the Drosophila bicoid mutant phenotype implies that otd-1 is only a partial functional equivalent of bicoid in Tribolium. hb expression was disrupted by pRNAi to determine whether this would result in a phenotype overlapping that of otd-1RNAi. Disruption of hb mRNA results in a lack of the maxillary, the labial and all three thoracic segments in 50% of the analyzed embryos, whereas the anteriormost head segments all developed normally. Drosophila embryos that lack maternally derived and zygotic hb have the same phenotype (Schröder, 2003).

This indicates that, in Drosophila and Tribolium, hb acts as a canonical head gap gene. In the wasp Nasonia, the hb mutant phenotype is more severe: the complete head (except for the anterior-most labrum) and the thorax are missing. It appears that the function of hb as a gap gene has been conserved throughout evolution. The head defects observed in the otd-1RNAi experiments indicate that otd-1 and hb are both involved in regulating the development of gnathal segments. To evaluate the extent of their overlapping functions, embryos were generated in which both otd-1 and hb expression were disrupted by pRNAi. These embryos were called otd-1/hbpRNAi double phenocopies. Forty out of 61 (65.5%) of these embryos developed a headless phenotype, indicating that otd-1 and hb function together to regulate head development (Schröder, 2003).

Analysis of the Engrailed expression pattern in the otd-1/hbpRNAi embryos reveals that only two to six abdominal segments of normal polarity develop in otd-1/hbpRNAi embryos. This shows that otd-1 and hb not only direct the development of head and thorax, but also act synergistically during the segmentation process of the anterior abdomen, possibly by regulating posterior gap and/or homeotic genes. A synergistic relationship between bicoid and hb has been described previously in Drosophila, and might represent an evolutionary principle to confer robustness to the segmentation process. In Tribolium, Otd-1 and Hb might substitute for the function of Bicoid as a transcriptional activator in Drosophila (Schröder, 2003).

In Drosophila, caudal is translationally repressed in the anterior half of the egg by Bicoid. Since no ectopic posterior structures form at the anterior pole of otd-1/hbpRNAi embryos, Otd-1 might not serve as a translational repressor in Tribolium, as Bicoid does in Drosophila. In Drosophila, the RNA-binding activity of Bicoid, and therefore its ability to inhibit caudal translation, requires the presence of an arginine at residue 54 of its homeodomain. This amino acid is replaced by an alanine in the homologous position of Tribolium Otd-1, so its RNA-binding specificity (but not DNA-binding specificity) might be lost. A repressor of Tribolium caudal is therefore likely to exist in the beetle, and serves as a further component of the anterior development system. Such a repressor has yet to be identified, but different mechanisms of caudal regulation have been described in other systems. During Dipteran evolution, the function of Bicoid seems to have replaced the function of maternally inherited otd-1 as well as that of a repressor of caudal (Schröder, 2003).

The expression of otd-1 might be controlled early in embryogenesis by repression, and during later stages by autoactivation. Maternal otd-1 mRNA translation could be repressed at the posterior pole, in a manner similar to that of maternal hb in Drosophila. In Drosophila, and similarly in the grasshopper Schistocerca, hb expression is repressed by the Pumilio/Nanos complex. A predicted Nanos-response element (NRE), identified by the sequence TgGTTGTattatAATTGTAggTA (position 1429-1451, capital letters indicating identity to the NRE of hb in Drosophila and Musca), is present in the 3' untranslated region of Tribolium otd-1. Although no ortholog of pumilio or nanos has been isolated from Tribolium, their function in Tribolium has already been implicated by the downregulation of maternal hb at the posterior pole of the blastoderm embryo19. Maternal otd-1 could serve to activate the expression of zygotic otd-1 only in the embryonic region, at the border of the serosa. The zygotic expression could then be maintained by an autoregulatory loop (Schröder, 2003).

To test this possibility, otd-1-binding sites in its regulatory region will have to be identified, and the effects of disrupting this site will have to be tested in vitro and in vivo. bicoid might have evolved from a duplicated zerknüllt-like gene, which was converted by mutation into a gene that codes for a K50HD-containing protein -- thus adopting the same DNA-binding specificity as Otd-1. During evolution of the higher Diptera, otd expression came under the regulation of the maternal gene bicoid, so that maternal Otd-1 function was not longer required. Thus, otd was restricted to become a gap gene in insects derived from this lineage (Schröder, 2003).

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp),Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm the direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost (Telford, 1998).

The Bicoid (Bcd) gradient in Drosophila has long been a model for the action of a morphogen in establishing embryonic polarity. However, it is now clear that bcd is a unique feature of higher Diptera. An evolutionarily ancient gene, orthodenticle (otd), has a bcd-like role in the beetle Tribolium. Unlike the Bcd gradient, which arises by diffusion of protein from an anteriorly localized messenger RNA, the Tribolium Otd gradient forms by translational repression of otd mRNA by a posteriorly localized factor. These differences in gradient formation are correlated with differences in modes of embryonic patterning. Drosophila uses long germ embryogenesis, where the embryo derives from the entire anterior-posterior axis, and all segments are patterned at the blastoderm stage, before gastrulation. In contrast, Tribolium undergoes short germ embryogenesis: the embryo arises from cells in the posterior of the egg, and only anterior segments are patterned at the blastoderm stage, with the remaining segments arising after gastrulation from a growth zone. This study describes the role of otd in the long germband embryo of the wasp Nasonia vitripennis. Nasonia otd maternal mRNA is localized at both poles of the embryo, and resulting protein gradients pattern both poles. Thus, localized Nasonia otd has two major roles that allow long germ development. It activates anterior targets at the anterior of the egg in a manner reminiscent of the Bcd gradient, and it is required for pre-gastrulation expression of posterior gap genes (Lynch, 2006).

otd had been proposed as an ancestral anterior patterning gene in insects for two major reasons. First, it is highly conserved among animals and has an anterior patterning role in most phyla in which its function has been tested. Second, although it is quite distantly related to bicoid, Otd protein has a lysine at position 50 of its homeodomain that gives it the same DNA binding specificity as Bcd (Lynch, 2006).

Because a Tribolium-like system that forms an Otd gradient for patterning anterior structures based on a posteriorly localized source of translational repression would become increasingly inefficient as the germ rudiment extends more anteriorly, attempts were made to understand whether otd is a conserved anterior patterning factor in long germ insects that lack bcd (Lynch, 2006).

To address this question, the expression and function were examined of otd1 in the wasp Nasonia, which undergoes long germ development that seems to be morphologically similar to that of Drosophila, although early development takes longer. The Nasonia otd1 gene is orthologous to Tribolium otd1, which has an early role in axis formation. There is a second otd gene (otd2) in both species that is expressed only later in development (Lynch, 2006).

Nasonia otd1 is expressed maternally in a surprising pattern. In early ovarian follicles, otd1 mRNA is expressed in the nurse cells, and unexpectedly accumulates at the posterior of the oocyte. In later follicles, otd1 mRNA remains localized at the posterior of the oocyte but also begins accumulating at the anterior pole (Lynch, 2006).

In pre-pole cell embryos, otd1 mRNA is seen tightly localized at the anterior pole, whereas posteriorly localized mRNA becomes associated with the oosome, a structure that is thought to be an equivalent of germ plasm. This oosome-associated mRNA can migrate some distance from the posterior extremity, but returns to the pole just before the pole cells begin to form. After pole cell formation and nuclear migration to the surface of the embryo, otd1 mRNA remains in a bi-polar expression pattern, but appears to become less tightly localized to the cortex. Finally, after cellularization of the blastoderm, otd1 mRNA is seen in cap-like domains at both the anterior and posterior poles (Lynch, 2006).

The ovarian and early embryonic expression patterns of Nasonia otd1 mRNA suggest that gradients of Otd1 protein may form by diffusion from localized sources of mRNA. An anterior Otd1 gradient would indicate possible convergent evolution of maternally localized mRNAs giving rise to gradients of K50 homeoproteins in patterning the anterior of both Drosophila and Nasonia. A posterior Otd1 gradient would show that Nasonia has recruited otd1 to perform a novel posterior patterning role not present in any of its known orthologues. To determine whether Otd1 gradients exist in Nasonia, an antibody was generated to Nasonia Otd1 (Lynch, 2006).

No Otd1 protein is seen before pole cell formation, indicating a mechanism of translational repression at this stage. Nasonia Otd1 protein is first detected when nuclei begin arriving at the surface of the embryo, just after pole cell formation. Notably, Otd1 is initially only seen at the anterior, where it forms an anterior to posterior gradient. A posterior to anterior gradient becomes visible at the posterior pole towards the end of the syncytial blastoderm stage. This pattern indicates a second level of translational repression specific to the posterior aspect of Nasonia otd1 mRNA, and is consistent with the timing of posterior translational repression seen for Nasonia hunchback (hb). Interestingly, sequences that are similar to known Nanos response elements (NREs) are found in the 3' untranslated regions (UTRs) of both Nasonia otd1 (gCGTTtcgccGcATTGTAcgag) and hb, indicating that posteriorly localized nanos may be responsible for preventing the translation of both mRNAs during early syncytial blastoderm stages. Finally, in the cellular blastoderm, Otd1 protein is seen in the anterior and posterior domain, mimicking the expression of mRNA (Lynch, 2006).

The maternal localization of otd1 mRNA along with subsequent Otd1 protein gradients are consistent with this gene acting as a morphogen at both poles of the Nasonia embryo. If this were indeed the case, when levels of Otd1 are reduced, the posterior borders of anterior Otd1 target genes should be shifted towards the anterior, whereas the anterior borders of posterior targets should be shifted posteriorly. To test the function of otd1, the parental RNA interference (pRNAi) technique first developed in Tribolium was adapted to Nasonia. The expression of Otd1 was examined in the offspring of otd1 RNAi-injected mothers and varying amounts of reduction was observed; this correlates with variations in resulting phenotypes and effects on the expression patterns of the potential target genes empty spiracles (ems), giant (gt) and hb (Lynch, 2006).

ems requires high levels of Bcd for expression in Drosophila. Nasonia ems is expressed in a very similar pattern to that of Drosophila. As a very anterior target of Otd1, it should be extremely sensitive to a reduction in Otd1 levels. Indeed, when otd1 is knocked down, most embryos lose ems expression entirely, with only a few exhibiting reduced and anteriorly shifted expression (Lynch, 2006).

Similar to Drosophila, the gap gene gt has two domains of expression in Nasonia. In most cases, when otd1 is knocked down both gt stripes are shifted towards their respective poles; in the most strongly affected embryos, expression is lost at the anterior, whereas some residual expression is still seen at the posterior pole (Lynch, 2006).

The gap gene hb responds to low levels of Bcd in flies and shows a broad anterior expression domain, as well as a posterior stripe. Nasonia hb shows a similar pattern in the late blastoderm stages. In otd1 RNAi embryos, the anterior hb domain shows a clear, although modest, anterior shift of its posterior boundary of expression. The degree to which hb is resilient to this knockdown is surprising, although it has been suggested that anterior zygotic hb can be activated by maternal hb. Notably, although one aspect of zygotic hb expression is Bcd-dependent in Drosophila, this expression can be made dispensable because high levels of maternal hb can activate the zygotic function of hb required for thorax formation (Lynch, 2006).

In contrast to the modest effects on anterior hb expression, knocking down otd1 expression has a marked effect on the posterior domain of hb. In Drosophila, this stripe is activated by tailless, whereas in Tribolium it is not expressed until the latest stages of germband extension. In otd1 knocked down embryos, the anterior border of this stripe is either shifted to the extreme posterior pole of the embryo, or completely lost (Lynch, 2006).

Knockdown of otd1 results-presumably as a consequence of changes in expression of its targets-in the loss of both anterior and posterior segments: defects of varying severity are seen in both the expression of Engrailed protein and larval cuticle structures. At the anterior end, Engrailed stripes and their corresponding cuticular segments are lost in an anterior to posterior progression, with RNAi phenotypes ranging from the loss of only the antennal stripe to the complete lack of head segments. At the posterior, Engrailed stripes and denticle belts appear to be lost in a posterior to anterior progression (Lynch, 2006).

Because hb has been shown to cooperate with bcd in Drosophila and with otd in Tribolium, double knockdown of otd1 and hb was performed to see whether a similar interaction exists in Nasonia. In the most severe cases, the entire anterior is lost, including several anterior abdominal segments. This is more severe than the combination of the most severe phenotypes seen by individual knockdown of either otd1 or hb, or in the zygotic null Nasonia hb allele (hbheadless), and the entire range of phenotypes is more severe. This indicates that Nasonia otd1 and hb cooperate in anterior patterning. Although hb is also expressed and functions at the posterior, as in Drosophila, no increase in the severity or frequency of severe phenotypes is observed at the posterior in the double knockdown. The lack of synergy between Otd1 and Hb at the posterior may allow different functions for the anterior and posterior Otd1 gradients: Otd1 acts with Hb at the anterior, whereas it might act alone or in combination with a different factor (for example, Caudal) at the posterior to activate distinct sets of target genes (Lynch, 2006).

These results show that otd1 has broad roles in patterning both the anterior and posterior of the long germ Nasonia embryo. This provides insight into how a long germ type of patterning might have arisen from a short germ embryo. Major changes in patterning mechanisms at both the anterior and posterior ends of the embryo are required to pattern a long germ embryo. At the anterior, the egg fate map must be shifted so that the anterior (that is, head and thoracic) segments are patterned near the anterior pole. In addition, there must be a source of patterning information that is sufficiently strong to allow relatively tight spacing of these segments, in order to allow enough room at the posterior to pattern the abdomen. A system such as that found in Tribolium does not seem compatible with this because the diffusion gradient arising from a posteriorly localized patterning centre will be shallowest at the anterior, where it would carry the least patterning information. Nasonia solves this by localizing otd1 mRNA at the anterior pole; this results in a protein gradient with the most patterning information at the anterior of the embryo. This allows genes for which the orthologues are expressed more posteriorly in Tribolium to be activated close to the anterior pole in Nasonia. This is similar to the strategy used in Drosophila with localized bcd transcript (Lynch, 2006).

Posterior patterning poses another problem for long germ embryos. In short germ insects, genes patterning posterior segments (such as the posterior domains of gt and hb) are expressed after gastrulation in a 'growth zone', whereas long germ embryos must express them at the blastoderm stage, before gastrulation. Again, Nasonia solves this problem by localizing otd1 mRNA, this time at the posterior pole. The posterior gradient of Otd1 seems to be required for proper expression of posterior gap genes before gastrulation. This is a divergent strategy compared to that used in Drosophila, where neither bcd nor otd mRNA is localized at the posterior, and it is not clear which gene(s) provides morphogenetic information at the posterior. It may be that Bcd function extends posteriorly, because it acts with Caudal to activate posterior knirps and hairy stripe seven. Alternatively, or in addition, the terminal system might act through the induction of tailless, which has been shown to have some characteristics of a morphogen at the posterior (Lynch, 2006).

Although these results give insights into mechanisms of developmental evolutionary change, it will be of interest to understand whether what is seen in Nasonia is part of a larger pattern. For example, both Nasonia and Drosophila use the localization of mRNA encoding K50 homeoprotein (Otd1 or Bcd) to pattern the anterior of the egg. Is this a general strategy for long germband patterning? In contrast, there is no clear parallel between the posterior patterning systems of Nasonia and Drosophila. Is either strategy more common in other long germband embryos? To address this, the mechanisms used in other insects that have evolved long germband embryos must be elucidated. A number of key long germband taxa, including flies that lack bcd, a mosquito, moths and a beetle have been used as laboratory organisms, and the application of further gene expression and functional analyses to these organisms should allow the placement of the Nasonia patterning system in a larger context (Lynch, 2006).

mRNA localization is a powerful mechanism for targeting factors to different regions of the cell and is used in Drosophila to pattern the early embryo. The parasitoid wasp Nasonia (Hymenoptera) undergoes long germ development similar to that of Drosophila, yet is evolutionarily very distant from flies (> 200 MY) and lacks bicoid. During oogenesis of Nasonia, mRNA localization is used extensively to replace the function of the bicoid gene for the initiation of patterning along the antero-posterior axis. Nasonia localizes both caudal and nanos to the posterior pole, whereas giant mRNA is localized to the anterior pole of the oocyte; orthodenticle1 (otd1) is localized to both the anterior and posterior poles. The abundance of differentially localized mRNAs during Nasonia oogenesis provided a unique opportunity to study the different mechanisms involved in mRNA localization. Through pharmacological disruption of the microtubule network, it was found that both anterior otd1 and giant, as well as posterior caudal mRNA localization was microtubule-dependent. Conversely, posterior otd1 and nanos mRNA localized correctly to the posterior upon microtubule disruption. However, actin is important in anchoring these two posteriorly localized mRNAs to the oosome, the structure containing the pole plasm. Moreover, knocking down the functions of the genes tudor and Bicaudal-D mimics disruption of microtubules, suggesting that tudor’s function in Nasonia is different from flies, where it is involved in formation of the pole plasm (Olesnicky, 2007).

Both the Drosophila and Nasonia ovariole are meroistic, meaning that the nurse cells and oocyte are both of germ cell descent and originate from the same primordium, but differentiate during subsequent cell divisions. As each ovarian follicle develops and is positioned more distally along the ovariole, the nurse cells remain attached to one another and to the oocyte through ring canals, which arise from incomplete cleavage during cell division. The 16 sister cells that make up each germline cyst result from four of these incomplete divisions. An egg chamber forms comprising of 15 nurse cells and the oocyte, surrounded by the somatic follicle cells, which form an epithelial layer around the oocyte. Nurse cells produce metabolites and other factors that transit through the ring canals to accumulate in the oocyte (Olesnicky, 2007).

The Drosophila oocyte is specified early during oogenesis as a result of the asymmetric segregation of the fusome, an organelle that connects the 16 cells. Once the oocyte has been specified, the polarity of the oocyte microtubule network becomes extremely dynamic and undergoes a major reorganization resulting from communication between the oocyte and follicle cells. This reorganization is essential to localize maternal mRNAs that will generate the axes of the embryo. At first, microtubule minus ends extend from the nurse cells into the oocyte toward a microtubule organizing center (MTOC) localized at the posterior pole of the oocyte, near its nucleus. Later, however, the posterior MTOC disassembles while multiple MTOCs form toward the anterior of the growing oocyte. At this stage, the microtubules are therefore pointing from the plus end at the posterior of the oocyte to the minus end at the anterior. mRNAs and the oocyte nucleus utilize the polarity of the microtubules to localize to the anterior or posterior pole (Olesnicky, 2007).

Nasonia oogenesis presents striking similarities to that of Drosophila. It is divided into five morphologically distinct stages. In stage 1, the nurse cells and oocyte are indistinguishable until they begin to segregate, with the oocyte lying towards the posterior of the follicle. By stage 2, the nurse cells and a smaller oocyte are clearly distinguishable, as a constriction forms between the oocyte and its supporting nurse cells. At this stage, the oocyte nucleus is positioned in the center of the cell. The oocyte continues to grow throughout stage 3 until it becomes larger than its accompanying nurse cells. Concomitantly, the oocyte nucleus migrates to the dorsal anterior cortex of the developing oocyte, as in Drosophila. Later, during stage 4, the nurse cells begin to degenerate as they empty all their contents into the oocyte. In the final stage (5), a vitelline membrane is constructed around the embryo (Olesnicky, 2007).

This study shows that the localization of four maternal mRNAs is achieved using at least 2 distinct mechanisms. It is shown that, during Nasonia oogenesis, microtubules play a major role in oocyte polarity and in the control of anterior localization of otd1 and gt mRNA and the posterior localization of cad mRNA. In contrast, the actin cytoskeleton is important for anchoring the oosome and is therefore essential for the localization of nanos and otd1 mRNA to the posterior pole of the oocyte (Olesnicky, 2007).

It is proposed that Nasonia utilizes two basic mechanisms for the localization of mRNA, a microtubule-dependent mechanism and an actin-dependent, microtubule-independent one. Anterior localization of gt and otd1 mRNA, as well as posterior localization of cad mRNA, all rely on a similar microtubule-dependent mechanism while posterior localization of otd1 and nos mRNAs relies on actin. In wild-type follicles, cad and gt mRNAs are initially localized, while later in oogenesis this localization is relaxed to achieve a more graded mRNA distribution. otd1 anterior mRNA, although not graded, is also localized loosely in wild-type follicles. nos mRNA localization and posteriorly localized otd1 mRNA, however, are tightly localized to the posterior in a microtubule-independent manner. Interestingly, in freshly laid embryos both posterior otd1 mRNA and nos mRNA are localized to the oosome. Maintaining localization of these two posteriorly localized mRNAs relies on the actin cytoskeleton. Additionally, actin might be required to anchor the oosome to the posterior pole of the oocyte, as well as to trap mRNA to the oosome. It is therefore likely that both mRNAs are localized to structures within the germ plasm, resulting in a tight localization that is maintained throughout oogenesis and early embryogenesis and does not rely extensively on microtubules (Olesnicky, 2007).

In the short-germ beetle Tribolium castaneum, the head gap gene orthodenticle (Tc-otd) has been proposed to functionally substitute for bicoid, the anterior morphogen unique to higher dipterans. This study reanalyzed the function of Tc-otd. A similar range of cuticle phenotypes was obtained as in previously described RNAi experiments; however, unexpected effects were noticed on blastodermal cell fates. First, it was found that Tc-otd is essential for dorsoventral patterning. RNAi depletion results in lateralized embryos, a fate map change that by itself can explain the observed loss of the anterior head, which is a ventral anlage in Tribolium. It was found that this effect is due to diminished expression of short gastrulation (sog), a gene essential for establishment of the Decapentaplegic (Dpp) gradient in this species. Second, it was found that gnathal segment primordia in Tc-otd RNAi embryos are shifted anteriorly but otherwise appear patterned normally. This anteroposterior (AP) fate map shift might largely be due to diminished zen-1 expression and is not responsible for the severe segmentation defects observed in some Tc-otd RNAi embryos. As neither Tc-sog nor Tc-zen-1 probably requires Otd gradient-mediated positional information, it is posited that the blastoderm function of Tc-Otd depends on its initial homogeneous maternal expression and that this maternal factor does not provide significant positional information for Tribolium blastoderm embryos (Kotkamp, 2010).

Evidence for a composite anterior determinant in the hover fly Episyrphus balteatus (Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

Most insect embryos develop from a monolayer of cells around the yolk, but only part of this blastoderm forms the embryonic rudiment. Another part forms extra-embryonic serosa. Size and position of the serosa anlage vary between species, and previous work raises the issue of whether such differences co-evolve with the mechanisms that establish anteroposterior (AP) polarity of the embryo. AP polarity of the Drosophila embryo depends on bicoid, which is necessary and sufficient to determine the anterior body plan. Orthologs of bicoid have been identified in various cyclorrhaphan flies and their occurrence seems to correlate with a mid-dorsal serosa or amnioserosa anlage. This paper introduces Episyrphus balteatus (Syrphidae), a cyclorrhaphan model for embryonic AP axis specification that features an anterodorsal serosa anlage. Current phylogenies place Episyrphus within the clade that uses bicoid mRNA as anterior determinant, but no bicoid-like sequence could be identified in this species. Using RNA interference (RNAi) and ectopic mRNA injection, evidence was obtained that pattern formation along the entire AP axis of the Episyrphus embryo relies heavily on the precise regulation of caudal, and that anterior pattern formation in particular depends on two localized factors rather than one. Early zygotic activation of orthodenticle is separated from anterior repression of caudal, two distinct functions which in Drosophila are performed jointly by bicoid, whereas hunchback appears to be regulated by both factors. Furthermore, it was found that overexpression of orthodenticle is sufficient to confine the serosa anlage of Episyrphus to dorsal blastoderm. These findings are discussed in a phylogenetic context, and it is proposed that Episyrphus employs a primitive cyclorrhaphan mechanism of AP axis specification (Lemke, 2009).

This study found that AP axis specification in Episyrphus is strongly dependent on Eba-cad. Throughout the embryo, ectopic Eba-cad expression interferes with segmentation and differentiation, whereas loss of Eba-cad activity interferes with the formation of all but the anterior head segments. In Drosophila, ectopic translation of the ubiquitous maternal caudal mRNA causes temperature-dependent head involution defects. Ubiquitous expression of a caudal transgene in the syncytial blastoderm also causes head involution defects and, in addition, leads to variable fusions of adjacent segment pairs along the entire embryo. The much stronger gain-of-function phenotype of caudal in Episyrphus could reflect differences in the experimental designs that were employed. However, loss-of-function experiments also suggest that embryonic development in Episyrphus relies more heavily on Eba-cad than embryonic development in Drosophila does on caudal. In Episyrphus, Eba-cad RNAi suppresses the formation of all but one of the seven Eba-eve stripes and severely affects or deletes most postoral segments, whereas caudal-deficient Drosophila embryos form four out of the seven even-skipped stripes and show segmentation in the head, thorax and even parts of the abdomen. The comparatively weak dependence of AP axis specification in Drosophila on caudal can be explained by compensatory input from the anterior gradients of bicoid and maternal hunchback. In turn, the high caudal-dependence of AP axis-specification in Episyrphus, which is similarly observed in species that lack the bicoid gene such as Nasonia and the cricket Gryllus, might reflect the absence of maternal hunchback and/or bicoid activities in this species (Lemke, 2009).

Although endogenous Eba-nos appeared to be dispensable for AP axis specification, ectopic Eba-nos expression in gain-of-function experiments could be used as a functional tool to reveal differences in anterior pattern formation between Episyrphus and Drosophila. Drosophila embryos that ectopically express nanos at the anterior pole develop a mirror-image duplication of the posterior abdomen. This effect is due to the translational repression of maternal bicoid and hunchback mRNAs, which control all aspects of anterior development. Both genes contain functionally important Nanos regulatory elements (NREs), although in wild-type embryos Nanos appears to be irrelevant for the regulation of bicoid. In Episyrphus, no trace was observed of abdominal development at the anterior pole after ectopic expression of Eba-nos, although the activity was high enough to completely suppress the formation of all but the most posterior segments (A6-A8). This phenotype would be expected if at least two independent factors determine anterior development in Episyrphus, only one of which is targeted by ectopic anterior Eba-nos activity, whereas the second factor prevents the formation of ectopic posterior structures. It is proposed that the first factor (Factor 1) consists of an anteriorly enriched NRE-containing mRNA that encodes a protein for the early zygotic activation of Eba-otd and Eba-hb, and that the second factor (Factor 2), which is not repressed by ectopic Eba-nos activity, mediates the repression of Eba-cad and part of the anterior Eba-hb activation. Factor 2 appears to function independently of the terminal system, as neither Eba-cad nor Eba-hb display altered anterior expression domains following RNAi against the putative torso homolog of Episyrphus. Candidate genes for Factor 1 could possibly be identified by searching for NRE-containing sequences in an early embryonic Episyrphus EST database (Lemke, 2009).

In summary, AP polarity of the Episyrphus embryo appears to be determined by two distinct factors at the anterior pole. It cannot be excluded that one of these factors shares homology with bicoid, but in any case the model differs significantly from AP axis specification in Drosophila, where a single protein, Bicoid, activates orthodenticle and hunchback, and represses caudal. Furthermore, the Episyrphus model differs from the Nasonia model in that the transcripts of Eba-otd and Eba-gt (the putative Episyrphus ortholog of giant) are of zygotic origin and not localized (Lemke, 2009).

Episyrphus shares various traits of early embryonic development with non-cyclorrhaphan rather than other cyclorrhaphan flies. It features an anterodorsal serosa anlage, strong influence of caudal on the AP axis, a (nearly) ubiquitous early zygotic activation of hunchback, as well as hunchback expression in the serosa anlage, which has been reported for non-cyclorrhaphan insects and is absent in Drosophila, Musca and Megaselia. During late embryonic development, Engrailed expression in the hindgut of Episyrphus embryos is narrow and ring-shaped similar to some non-cyclorrhaphan insects, whereas Engrailed expression in the hindgut of other cyclorrhaphans is much broader and restricted to the dorsal half. Based on the primitive features of Episyrphus development, it is speculated that the ancestral cyclorrhaphan mechanism of AP axis specification was retained in the Episyrphus lineage. The restriction of the serosa anlage to dorsal blastoderm in response to increased Eba-otd activity might therefore indicate the evolutionary mechanism that altered the position of the serosa anlage (Lemke, 2009).

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).

Maternal activation of gap genes in the hover fly Episyrphus

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, loss- and gain-of-function experiments were used in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. It was found that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. It is conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha) (Lemke, 2010).

Therefore, Episyrphus and other lower cyclorrhaphan flies establish global AP polarity only through bicoid and lack sizable input of nanos, although endogenous nanos activity in these species might stabilize the AP axis by repressing anterior development. Despite the absence of a redundant maternal system to generate global AP polarity, Eba-bcd appears to be a less potent transcriptional activator than Bicoid. In contrast to Drosophila, gap gene activation at the anterior pole of the Episyrphus embryo requires a strong contribution of the terminal system, whereas the posterior domains of knirps and giant are strictly dependent on caudal and do not appear to receive a significant activating input by Eba-bcd. Thus, rather than a strong activation potential, the exclusive control of the central Eba-Kr domain by Eba-bcd appears to be the crucial difference to Drosophila, which renders AP polarity in the Episyrphus embryo entirely dependent on bicoid (Lemke, 2010).

Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, this study used the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and several genes were identified that had not been implicated in this process before. Furthermore, it was shown that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, it was demonstrated that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly (Posnien, 2011).

Tc-six3 is expressed in an anterior median domain from earliest stages on and that it acts as an upstream component of anterior median patterning. Drosophila optix/six3 is expressed in an anterior blastodermal ring anterior to otd, which persists at the dorsal side. Its ring like expression does not support an involvement in median patterning but relevant genetic interactions remain to be studied. The later expression in the labrum and in bilateral dorsal domains, however, is similar in both species (Posnien, 2011).

Interestingly, aspects of dorsal median head patterning are controlled by dpp in Drosophila. Shortly before gastrulation, the action of dpp and its downstream target zen at the dorsal midline separate the neuroectoderm into paired anlagen by medial repression of genes and by promoting median cell death. This results in the establishment of bilateral expression of marker genes of the respective brain parts [e.g. Dchx (pars intercerebralis); Fas2 and Drx (pars lateralis); sine oculis and eyes-absent (visual system)]. Actually, many other anterior patterning genes initiate their expression as unpaired domains across the dorsal midline that are subsequently medially subdivided in Drosophila (e.g. otd, fezf, Dsix4. In contrast, the Tribolium orthologs of most of these genes are initiated as separate bilateral domains (Tc-rx and Tc-fez, Tc-chx and Tc-Fas2, Tc-tll, Tc-six4, Tc-sine oculis, Tc-eyes-absent. Tc-otd1 starts out with ubiquitous expression related to axis formation but then resolves into paired head lobe domains which are separate as with the aforementioned genes (Posnien, 2011).

Due to differences in topology of the head anlagen, median repression of anterior patterning genes by Tc-dpp is not required in Tribolium. Nevertheless, it is expressed along the rim of the head anlagen at blastoderm stages, some parts of which will become the site of dorsal fusion. However, Tc-Dpp activity (detected by antibodies against pMad) does not occur at the site of expression and is clearly distant from the arising Tc-rx, Tc-chx, Tc-six4, Tc-sine oculis or Tc-fas2 domains. Also the Tc-dpp RNAi phenotypes differ from Drosophila mutants in that the head anlagen are expanded and appear to have lost their dorso-ventral orientation (shown by expansion of Tc-otd1 and the proneural gene Tc-ASH) in an overall ventralized embryo. Hence, the early expression of dpp at the future dorsal midline might be ancestral, but its function with respect to medially repressing gene expression has probably evolved in Drosophila (Posnien, 2011).

The difference in generation of paired dorsal domains in these two insect species reflects the different location of the head anlagen. In the long germ insect Drosophila, extraembryonic tissues are reduced to the dorsally located amnioserosa while the head anlagen are situated in the anterior dorsal blastoderm from earliest stages on. Consequently, the head lobes are never separated along the midline. In contrast, in the short germ insect Tribolium, the anterior blastoderm gives rise to extraembryonic amnion and serosa, which eventually ensheath the embryo. In contrast to Drosophila, the Tribolium head anlagen are located in the ventral median blastoderm from where they move towards anteriorly and bend dorsally. The head lobes are separate from the beginning but fuse at late stages at the dorsal midline forming the dorsal head (bend and zipper model). During these morphogenetic movements, the initially separate expression domains of the head lobes eventually come into close proximity at the dorsal midline like in Drosophila. Both the anterior dorsal location of extraembryonic tissue anlagen and the ventral location of the head anlagen are found in most insects and in the hemimetabolous milkweed bug Oncopeltus fasciatus, gene expression data show a clear separated origin of the head lobes in the blastoderm. Hence, Tribolium is likely to represent the ancestral state in insects (Posnien, 2011).

In striking analogy to Drosophila, the expressions of vertebrate eye field patterning genes start out as one midline spanning domain (e.g., Rx and Pax6. Later, these domains split medially, which is the prerequisite for the formation of bilateral eye anlagen. shh as well as six3 are involved in medial repression of Pax6 and Rx2 with six3 acting upstream of shh. This appears to be more similar to the derived Drosophila situation than to the ancestral split of head lobe anlagen. However, the molecules involved in median split are different (dpp in Drosophila versus six3 and shh in vertebrates) and involvement of Tribolium six3 but not dpp is found in median patterning. Hence, the molecular data actually suggest a higher degree of conservation between Tribolium and vertebrates and convergent evolution of the similarity between Drosophila and vertebrates (Posnien, 2011).

Regarding the likely difference to Drosophila, it is striking that the role of vertebrate six3 in median separation of anterior expression domains is similar to what was found in Tribolium. In vertebrates, six3 represses midbrain derived Wnt signaling, which was also found in Tribolium. In vertebrates, six3 and its paralog six6 are involved in pituitary and hypothalamus development. Based on its expression, six3 has been predicted to contribute to neuroendocrine brain parts in annelids and Drosophila. More generally, the similarity of bilaterian neuroendocrine systems and their common origin from placode like precursors have been noted. This study has added functional data showing that Tc-six3 is indeed required for the expression of neuroendocrine markers for the pars intercerebralis (Tc-chx) and pars lateralis (Tc-fas2) placing it high in the hierarchy of neuroendocrine development in bilaterians (Posnien, 2011).

In mouse embryos with reduced levels of six3 and shh expression, median head and brain structures are affected (e.g., median nasal prominence) or absent (e.g., nasal septum, the septum, corpus callosum). Such holoprosencephaly phenotypes are also seen in some human six3 mutations. Very similarly, loss of median brain structures is seen in Tribolium after RNAi for Tc-six3 Overall, these similarities functionally confirm that the ancestral role of six3 orthologs was in the anterior median patterning of the Urbilateria (Posnien, 2011).

Orthodenticle homologs in other invertebrates and primitive vertebrates

Otx genes have been identified in a variety of organisms and are commonly associated with the patterning of anterior structures. In some vertebrates, Otx genes are also expressed in the prechordal mesoderm, where they may have a role in cell movement. Characterization of CnOtx, an Otx gene in hydra, provides evidence that Otx genes appeared early in metazoan evolution. CnOtx is expressed at high levels in developing buds and aggregates, where it appears to have a role in the cell movements that are involved in the formation of new axes. Further, the gene is expressed at a low level throughout the body column of hydra. This latter pattern may reflect a role for CnOtx in specifying tissue as competent to be anterior, although the gene does not have a direct role in the formation of the head (Smith, 1999).

In many bilaterian animals, members of the Otx gene family are expressed in head or brain structures. Cnidarians, however, have no clearly homologous head and no distinct brain; but an Otx homolog from the jellyfish Podocoryne carnea is highly conserved in sequence and domain structure. Sequence similarities extend well beyond the homeodomain; Podocoryne Otx can be aligned over its entire length to human OTX1, OTX2, and CRX. The overall structure of Otx is better conserved from Podocoryne to deuterostomes while protostomes appear to be more derived. The Otx homeodomain, which extends in the Podocoryne protein from residue 56 to 115 exhibits identities ranging from 73% to 85%, as compared to the homeodomains of other Otx family members. The Podocoryne Otx homeodomain is most similar to zebrafish Otx1 with 51 of the 60 (85%) residues identical and 48 to 50 residues conserved in human OTX1, OTX2, and CRX and Drosophila otd. The homeodomains of Otx homologs from flatworms or Hydra appear to be more derived from the common ancestor and share only 44 of 60 (73%) residues with Podocoryne Otx. Based on the homeodomain alone the Caenorhabditis elegans Otx-like genes ceh-36 and ceh-37 could not be included with confidence in the Otx family, but since then a true Otx homolog has appeared in the unfinished part of the almost completed genome of C. elegans (GenBank Accession No. AL020985). In contrast to Drosophila otd, the sequence of Podocoryne Otx can be aligned over the entire length with vertebrate family members. Other deuterostomes, such as sea urchins, fit less well than vertebrates, and all known protostomes appear to be even more derived. In addition to the homeodomain, another sequence motif, the WSP motif, is highly conserved in deuterostome Otx family members and Podocoryne. Furthermore, a hydrophobic C-terminal tail motif can be recognized in Podocoryne and deuterostomes. Duplication of a C-terminal domain in vertebrates is observed when amphioxus Otx is compared to vertebrate sequences. A 12-amino-acid motif in CRX corresponding to part of these domains has been called Otx tail. When Podocoryne Otx is included in multiple sequence alignments, this tail motif seems not to be so well conserved; rather, the tip of the tail with a tryptophan and the collinear C-terminal ends of the proteins are conserved from Podocoryne to man (WXF/YXXL/M*). Both motifs are missing in Drosophila otd, which also appears to be highly derived in other aspects and is much longer (671 amino acids) than other Otx family members. This is not representative of insects as seen in the flour beetle T. castaneum. However, Tribolium has two Otx genes and only in otd2 can a WSP motif be found, but still no tail motif. The inclusion of a leech Otx homolog would disturb the alignment. The leech sequence is again very long (539 amino acids), appears to have a partially conserved tail motif, but lacks a WSP motif. Hydra Otx appears to lack similarity at the N-terminus, in a highly derived WSP motif, and at the tail motif. The phylogenetic analysis of the entire sequences suggests a tree different from that obtained with the homeodomains alone. Cnidarian and deuterostome sequences appear to be closer related, with high bootstrap values, but protostome sequences cannot be aligned reliably (Muller, 1999).

Functions seem to be conserved from protostomes to vertebrates but not in Podocoryne or echinoderms. Podocoryne Otx is expressed only during medusa bud formation and becomes restricted to the striated muscle of medusae. Cnidaria are the most basal animals with striated muscle. Podocoryne polyps have no striated muscle and no Otx expression; both appear only during the asexual medusa budding process. The common ancestor of all animals that gave rise to cnidarians, protostomes, and deuterostomes already had an Otx gene more similar to today's Podocoryne and human homologs than to Drosophila otd, while the head-specific function appears to have evolved only later (Muller, 1999).

At first sight the Otx family seems to be a good example of conserved, homologous gene function throughout animal evolution. Drosophila and human genes seem to do the same thing and are, at least partially, exchangeable. These truly amazing consistencies fit with other observations about the body axis and Hox genes, eye development and Pax genes, or the evolution of appendages and Dlx genes. However, in all these cases clear homologies can at best be assigned only within bilaterian animals and to answer questions about the origin of animal evolution the more basal phyla such as Porifera and Cnidaria will have to be included. In sea urchins and jellyfish, Otx genes show rather different expression patterns than expected. The sea urchin anomaly can be explained by the animal's highly derived body plan; this argument could eventually be extended to jellyfish. However, the overall conservation of the Otx domain structure from cnidarians to deuterostomes and the derived state of protostomes is in contrast to the lack of functional similarities (Muller, 1999).

Homeobox genes such as orthodenticle in Drosophila and its mouse homologs, Otx1 and Otx2, are known to be essential for rostral brain development. To investigate the molecular basis of brain evolution, a search was carried out for otd/Otx-related homeobox genes in the planarian Dugesia japonica, and two genes were identified: DjotxA and B, whose expression appears to be restricted to the cephalic ganglion (brain). The two Dugesia genes are the product of a recent duplication. DjotxA is expressed more medially, in the region containing the termini of the visual axons, and in the visual cells, suggesting involvement in establishment of the visual system. DjotxB is expressed in a discrete region just lateral to the DjotxA-positive domain, but not in the more lateral branch structures, which in turn are characterized by the expression of Djotp, a planarian homeobox gene related to mouse Orthopedia (Otp). In transverse sections of planarians, DjotxA and B expression are observed only at the anterior ends of the stumps, corresponding to the regional pattern of the regenerating brain. These findings suggest that the planarian brain is composed of structurally distinct and functionally diverse domains that are defined by the discrete expression of the three evolutionarily conserved homeobox genes (Umesono, 1999).

Echinoderms possess one of the most highly derived body architectures of all metazoan phyla, with radial symmetry, a calcitic endoskeleton, and a water vascular system. How these dramatic morphological changes evolved has been the subject of extensive speculation and debate, but remains unresolved. Because echinoderms are closely related to chordates and postdate the protostome/deuterostome divergence, they must have evolved from bilaterally symmetrical ancestors. The expression domains in echinoderms are reported for three important developmental regulatory genes (distal-less, engrailed and orthodenticle), all of which encode transcription factors that contain a homeodomain. The reorganization of body architecture involves extensive changes in the deployment and roles of homeobox genes. These include modifications in the symmetry of expression domains and the evolution of several new developmental roles, as well as the loss of roles conserved between arthropods and chordates. New developmental roles include roles for en, in skeletogenesis, otd and dll in podia (tube-feet that function in locomotion, feeding and sensory perception and are extensions of the water vascular system), dll in larvaL brachiolar arms and subtrochal cells, engrailed in rudiment invagination, and otd in the ciliated band. Some of these modifications seem to have evolved very early in the history of echinoderms, whereas others probably evolved during the subsequent diversification of adult and larval morphology. These results demonstrate the evolutionary lability of regulatory genes that are widely viewed as conservative (Lowe, 1997).

In the sea urchin Strongylocentrotus purpuratus, an Orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specific Spec2a gene, and more generally, the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from other species, cDNA and genomic clones were isolated containing SpOtx sequences. SpOtx exists in two forms (alpha and beta) that are generated by alternative RNA splicing from a single SpOtx gene. SpOtx(alpha) and SpOtx(beta) have identical C-termini and homeoboxes but are entirely different in their N-terminal domains. SpOtx(alpha) mRNAs are transcribed from a single start site and accumulated in all cells during cleavage, but are gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contrast, three distinct SpOtx(beta) mRNAs result from two separate transcriptional initiation events, and these transcripts begin to accumulate at mesenchyme blastula stage, primarily in ectoderm; later this is largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx(beta) appears later in development than SpOtx(alpha). Overexpression of SpOtx(alpha) and SpOtx(beta) induced in sea urchin embryos by mRNA injection demonstrates that SpOtx(alpha) is able to repress the accumulation of SpOtx(beta) transcripts, whereas SpOtx(beta) has no effect on the accumulation of SpOtx(alpha) transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates (Li, 1997).

The homeodomain transcription factor SpOtx is required for endoderm and aboral ectoderm formation during sea urchin embryogenesis. SpOtx target genes are repressed by fusing the SpOtx homeodomain to an active repression domain of Drosophila Engrailed. The Engrailed-SpOtx fusion protein reduces the expression of endoderm- and aboral ectoderm-specific genes, and inhibits the formation of endoderm and aboral ectoderm cell types. SoOtx is a strong candidate for a gene whose action is modulated by vegetal signaling carried out by the Wnt pathway. Coexpressing activated beta-catenin with Engrailed-SpOtx does not overcome the inhibition of endoderm and aboral ectoderm formation, suggesting that SpOtx functions either downstream of or parallel to nuclear beta-catenin. Embryos expressing C-cadherin, which blocks nuclear translocation of beta-catenin, have defects in endoderm and aboral ectoderm formation. Coexpressing SpOtx with C-cadherin restores aboral ectoderm-specific gene expression and aboral ectoderm morphology, but with C-cadherin present, SpOtx is not sufficient for endoderm formation. These results show that SpOtx plays a key role in the activation of aboral ectoderm- and endoderm-specific gene expression and, in addition, suggest that SpOtx mediates some of beta-catenin's functions in endoderm and aboral ectoderm formation (Li, 1999).

Two distinct types of Orthodenticle-related proteins (HpOtxE/L) have been implicated as transcription activators of the aboral ectoderm-specific arylsulfatase (Ars) gene of the sea urchin. The structure of HpOtx gene is described and evidence is presented that mRNAs of HpOtxE/L are transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing. By transactivation experiments, it has been demonstrated that HpOtxL activates the Ars promoter in the gastrula-stage embryo (Kiyama, 1998).

A sea urchin homolog of Drosophila Orthodenticle, HpOtxL has been implicated as a transcription activator of the aboral ectoderm-specific arylsulfatase (Ars) gene during early development of the sea urchin embryo. Using an in vivo transactivation system, evidence is presented that HpOtxL activates the target gene by interacting with co-factors. Otx binding sites alone have little effect on the activity of an Ars promoter, but when both Otx binding sites and CAAT sequences are present in the enhancer region of Ars, the fragment shows a high enhancer activity. A gel mobility shift assay reveals that a nuclear protein binds to the CAAT sequences present near the Otx binding sites in the enhancer region of Ars. The activation domain of HpOtxL resides in the C terminal region between amino acids 218 and 238. The N-terminal region is responsible for the enhancement of transactivation of the Ars promoter, although the region itself does not function as an activation domain. HpOtxE, which possesses an N-terminal region different from HpOtxL, does not activate the Ars promoter even in the presence of CAAT sequences. These results suggest that Otx regulates different genes by interacting with different co-factors in sea urchin development (Kiyama, 2000).

Strongylocentrotus purpuratus Otx (SpOtx) is required simultaneously in sea urchin development for the activation of endo16, which encodes a secreted protein that is activated at the end of the cleavage stage, exclusively in cells of the emerging vegetal plate, the progenitors of endomesoderm, and for the activation of spec2a in the aboral ectoderm. spec2a encodes an intracellular calcium binding protein that is expressed exclusively in aboral ectoderm cells. Because Otx binding sites alone do not appear to be responsible for the spatially restricted expression of spec2a, additional DNA elements were sought. Consensus Otx binding sites fused to basal promoters are sufficient to activate CAT reporter gene expression in all cell types, although expression in endomesoderm progenitors is enhanced. However, three non-Otx elements derived from the spec2a enhancer are needed together with Otx sites for specifically aboral ectoderm expression. A DNA element termed Y/CBF, lying just downstream from an Otx site within the spec2a enhancer, mediates general activation in the ectoderm. A second element lying between the Otx and Y/CBF sites, called OER, functions to prevent expression in the oral ectoderm. A third site, called ENR, overlapping another Otx site, is required to repress endoderm expression. Three distinct DNA binding proteins interact sequence specifically at the Y/CBF, OER, and ENR elements. The spec2a enhancer thus consists of closely linked activator and repressor elements that function collectively to cause expression of the spec2a gene in the aboral ectoderm (Yuh, 2001).

Interspecific sequence conservation can provide a systematic guide to the identification of functional cis-regulatory elements within a large expanse of genomic DNA. The test was carried out on the otx gene of a. This gene plays a major role in the gene regulatory network that underlies endomesoderm specification in the embryo. The cis-regulatory organization of the otx gene is expected to be complex, because the gene has three different start sites, and it is expressed in many different spatial domains of the embryo. BAC recombinants containing the otx gene were isolated from Strongylocentrotus purpuratus and Lytechinus variegatus libraries, and the ordered sequence of these BACs was obtained and annotated. Sixty kilobases of DNA flanking the gene, and included in the BAC sequence from both species, were scanned computationally for short conserved sequence elements. For this purpose, a newly constructed software package ('FamilyRelations') was used. This tool allows detection of sequence similarities above a chosen criterion within sliding windows set at 20-50 bp. Seventeen partially conserved regions, most a few hundred base pairs long, were amplified from the S. purpuratus BAC DNA by PCR, inserted in an expression vector driving a CAT reporter, and tested for cis-regulatory activity by injection into fertilized S. purpuratus eggs. The regulatory activity of these constructs was assessed by whole-mount in situ hybridization (WMISH) using a probe against CAT mRNA. Of the 17 constructs, 11 constructs displayed spatially restricted regulatory activity, and 6 were inactive in this test. The domains within which the cis-regulatory constructs were expressed are approximately consistent with results from a whole mount in situ hybridization study on otx expression in the embryo, in which probes specific for the mRNAs generated from each of the three transcription start sites were used. Four separate cis-regulatory elements that specifically produce endomesodermal expression were identified, as well as ubiquitously active elements, and ectoderm-specific elements. Predictions from other work were confirmed with respect to target sites for specific transcription factors within the elements that express in the endoderm (Yuh, 2002).

Micromeres and their immediate descendants have three known developmental functions in regularly developing sea urchins: immediately after their initial segregation, they are the source of an unidentified signal to the adjacent veg2 cells that is required for normal endomesodermal specification; a few cleavages later, they express Delta, a Notch ligand which triggers the conditional specification of the central mesodermal domain of the vegetal plate; and micromeres exclusively give rise to the skeletogenic mesenchyme of the postgastrular embryo. This paper demonstrates the key components of the zygotic regulatory gene network that accounts for micromere specificity. This network is a subelement of the overall endomesoderm specification network of the Strongylocentrotus purpuratus embryo. A central role is played by a newly discovered gene encoding a paired class homeodomain transcription factor which in micromeres acts as a repressor of a repressor: the gene is named pmar1 (paired-class micromere anti-repressor). pmar1 is expressed only during cleavage and early blastula stages, and exclusively in micromeres. It is initially activated as soon as the micromeres are formed, in response to Otx and ß-Catenin/Tcf inputs. The repressive nature of the interactions mediated by the pmar1 gene product was shown by the identical effect of introducing mRNA encoding the Pmar1 factor, and mRNA encoding an Engrailed-Pmar1 (En-Pmar1) repressor domain fusion. In both cases, the effects are derepression of the delta gene and of skeletogenic genes, including several transcription factors normally expressed only in micromere descendants. Deprepression also occurs in a set of downstream skeletogenic differentiation genes. The spatial phenotype of embryos bearing exogenous mRNA encoding Pmar1 factor or En-Pmar1 is expansion of the domains of expression of the downstream genes over most or all of the embryo. This results in transformation of much of the embryo into skeletogenic mesenchyme cells that express skeletogenic markers. The normal role of pmarl is to prevent, exclusively in the micromeres, the expression of a repressor that is otherwise operative throughout the embryo. This function accounts for the localization of delta transcription in micromeres, and thereby for the conditional specification of the vegetal plate mesoderm. It also explains why skeletogenic differentiation gene batteries normally function only in micromere descendants. More generally, the regulatory network subelement emerging from this work shows how the specificity of micromere function depends on continuing global regulatory interactions, as well as on early localized inputs (Oliveri, 2002).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Ten genes expressed in midlevel neural domains were examined, namely tailless (tll), paired box homeobox 6 (pax6), emptyspiracles-like (emx), barH, orthopedia (otp), developing brain homeobox (dbx), lim domain homeobox 1/5 (lim1/5), iroquois (irx), orthodenticle-like (otx), and engrailed (en). These genes are all expressed in chordates at least in the midbrain of the central nervous system, and thus, as a group, their domains are more posteriorly located than the anterior set. Some have the anterior border of the domain in the forebrain (tll, pax6, emx, lim1/5, and otx), and some have their anterior border in the midbrain (otp, barH, dbx, irx, and en). Most have posterior borders in the midbrain, but two (en and irx) have posterior borders in the anterior hindbrain. Thus, while all are expressed in the midbrain, each differs in its anterior and posterior extent. Several of the chordate genes (pax6, dbx, en, and irx) have separate posterior expression domains running the length of the chordate hindbrain and spinal cord at different dorsoventral levels of the neural tube (Lowe, 2003).

In S. kowalevskii, these ten orthologs are expressed in circumferential bands in the ectoderm at least of the mesosome (collar) or anterior metasome, that is, more posteriorly than the anterior group. Each gene differs in the exact anteroposterior extent of its domain -- some are expressed in part or all of the prosome. The most broadly expressed orthologs of this group are pax6, otp, lim1/5, irx, and otx. All are expressed in the prosome (relatively weakly for otx), mesosome (weakly in the case of otp and lim1/5), and anterior metasome, all ceasing by the level of the first gill slit. pax6 is strongest at the base of the proboscis, and lim1/5 is expressed most strongly in a dorsal patch at the base of the proboscis. The most narrowly expressed orthologs are barH, tll, emx, and en. tll is detected in early stages in the anterior prosome, posterior prosome, and anterior mesosome and in later stages restricted to the anterior mesosome. The emx domain is a single ring in the anterior mesosome plus an additional domain in the ciliated band in the posterior metasome, the only gene of the 25 to be expressed in the band cells. barH and en are both expressed in narrow ectodermal bands; barH in the anterior mesosome and en in the anterior metasome. A dorsal view of both en and barH reveals a dorsal narrow gap in expression in the midline. Ventrally, no such gap is observed. Two additional spots of en expression are detected in the ectoderm on either side of the dorsal midline in the proboscis. In the most posterior ring of otx expression in the metasome, a similar gap in expression is observed. otp is expressed predominantly in a punctate pattern in the apical layer of prosome ectoderm and in a diffuse pattern in the basal layer of prosome ectoderm, similar to dlx. It is also expressed in a circumferential ring of intermittant ectodermal cells in the posterior mesosome and then in two parallel lines of cells bilateral to the dorsal axon tract of the anterior metasome. Early dbx expression is most strongly detected in an ectodermal ring in the developing mesosome overlapping the posterior domain of tll. dbx is also expressed in the prosome at low levels throughout the ectoderm and at high levels in scattered individual cells or groups of cells. Later expression is restricted to two ectodermal bands marking the anterior and posterior limits of the mesosome. An additional endodermal domain of expression is observed predominantly in the ventral anterior pharyngeal endoderm (Lowe, 2003).

otx, en, and irx deserve description in more detail because in chordates, especially vertebrates, the products of these regionally expressed genes are thought to interact in setting up the midbrain-hindbrain boundary and the isthmic organizer. Furthermore, the otx domain at the midbrain level is the site from which neural crest cells migrate ventrally to the first branchial arch. In S. kowalevskii, otx is expressed at low but readily detectable levels in the prosome ectoderm and at high levels in four closely spaced ectodermal rings: one at the base of the prosome, two in the mesosome, and one in the anterior metasome. This fourth stripe of otx expression crosses the site where the first gill slit perforates the ectoderm. As evidence, beyond morphology, that the hemichordate gill slit is homologous to the chordate gill slit/branchial arch, the pax1/9 ortholog, known to be expressed in chordate gill slits, is expressed in the endoderm of the developing S. kowalevskii gill slit. Gill slit expression of pax1/9 is observed in the adult of P. flava. Thus, chordates and hemichordates have in common the association of the posterior limit of the otx domain with the position of the first gill slit or branchial arch (Lowe, 2003).

In hemichordates, the en domain overlaps the posterior part of the otx domain, and the irx domain runs through both of these, as is also the case in chordates. However, otx expression in S. kowalevskii extends slightly more posteriorly than does en, whereas in chordates the en domain extends slightly more posteriorly (Lowe, 2003).

In summary, for this midlevel group of genes, the S. kowalevskii orthologs are expressed in the mesosome and anterior metasome (with some domains extending anteriorly into the prosome), that is, more posteriorly than those genes of the anterior group. In general, expression domains that end posteriorly near the midbrain-hindbrain boundary in chordates, end in the anterior metasome in hemichordates. Although the anterior metasome is not the site of an obvious morphological boundary, it is the site of the first gill slit. The first gill slit/branchial arch in chordates is at the same body level as the midbrain-hindbrain boundary (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

As non-vertebrates, ascidians (phylum Urochordata) establish their body axis through gastrulation and neurulation, in a similar manner to vertebrate embryos. The ascidian larva possesses a hollow neural tube running most of the body length, under which lies the gut primordium (the endoderm). The notochord extends the length of the tail; three rows of muscle cells lie on each side of the notochord. Hroth, an orthodenticle-related homeobox gene of the ascidian Halocynthia roretzi is expressed in both involuting mesoderm and anterior ectoderm during gastrulation, while later expression is restricted to the sensory vesicle and anterior epidermis. Hroth is apparently distinct from vertebrate orthodoenticle-related genes, suggesting that Hroth diverted earlier than the diversification of otx1 and otx2. It is likely that Hroth expression in endoderm precursors plays a role in the initiation and progression of involuted movement in ascidian gastrulation. A similar suggestion has also been made for Xenopus and chicken otx2's. Recent analysis of chicken otx2 has clearly shown that it is required for formation of an organized node and anterior axial mesoderm in the embryo at the early streak stage. Involvement in the gastrulation movement of mesoendodermal cells may be a common denominator among ascidian Hroth and vertebrate otx2. In vertebrates, both otx2 and lim1 (Drosophila homolog: apterous) are expressed in the organizer and anterior mesodermal regions. In the ascidian, Hroth and Hrlim expression domains overlap in endoderm precursors that start invaginating at the initial stages of gastrulation. During the early gastrula stage, expressions of both genes in the endoderm precursors become weak almost synchronously. These observations suggest that Hroth and Hrlim may be coordinately involuted in the determination of the endoderm fate or in the initiation of the involution movement. Thus Hroth is involved in both involuting mesoendoderm and anterior ectoderm. Hroth appears to possess an added complexity over the expression pattern of Drosophila orthodenticle, whose expression is restricted to the ectoderm and neural tissues (Wada, 1996).

Ascidians and vertebrates belong to the Phylum Chordata and both have dorsal tubular central nervous systems. The structure of the ascidian neural tube is extremely simple, containing less than 400 cells, among which less than 100 cells are neurons. Recent studies suggest that despite its simple organization, the mechanisms patterning the ascidian neural tube are similar to those of the more complex vertebrate brain. However, identification of homologous regions between vertebrate and ascidian nervous systems remains to be resolved. This study describes the expression of the HrPax-258 gene (Drosophila homolog: Sparkling), an ascidian homolog of vertebrate Pax-2, Pax-5 and Pax-8 genes. Molecular phylogenetic analyses indicate that HrPax-258 is descendant from a single precursor gene that gave rise to the three vertebrate genes. The expression pattern of HrPax-258 suggests that this subfamily of Pax genes has conserved roles in regional specification of the brain. Comparison with expression of ascidian Otx (Hroth) and a Hox gene (HrHox1) by double-staining in situ hybridizations indicates that the ascidian brain region can be subdivided into three parts: an anterior region marked by Hroth, and probably homologous to the vertebrate forebrain and midbrain; a middle region, marked by HrPax-258 and probably homologous to the vertebrate anterior hindbrain (and maybe also the midbrain), and a posterior region marked by Hox genes, which is homologous to the vertebrate hindbrain and spinal cord. Later expression of HrPax-258 in atrial primordia implies that basal chordates such as ascidians have already acquired a sensory organ that develops from epidermal thickenings (placodes) and expresses HrPax-258; this organ may be homologous to the vertebrate ear. The atrial primordia develop as a pair of ectodermal invaginations that fuse to form one atrial siphon. This mode of formation is strikingly similar to that of vertebrate placodes. The atrium of adult ascidians develops ciliated sensory cells in cupular organs resembling those of the vertebrate acoustico-lateralis system. Therefore, placodes are not likely to be a newly acquired feature in vertebrates, but may have already been possessed by the earliest chordates. It is suggested that the atrial primordia of ascidian larvae are homologous to the vertebrate inner ear (otic system). HrPax-258 is also expressed in the primordial pharynx. This organ develops into the oral siphon of adult ascidians. The oral siphon of adult ascidians can be regarded as a mouth. The similarity of cell lineage strongly supports the homology between the ascidian and vertebrate mouth (Wada, 1998).

The function and regulation of expression of Hroth, the ascidian homolog of orthodenticle/otx, was studied during embryogenesis to obtain insights into the mechanisms of gastrulation and neural tube formation. Microinjection of synthetic Hroth mRNA into fertilized eggs leads to embryos with an expanded trunk and a reduced tail. In these embryos, development of notochord and muscle is effected. Also, Hroth overexpression causes ectopic formation of anterior neuroectoderm, along with suppression of epidermis development, even in the absence of cell-cell interaction. Ectodermal expression of Hroth requires an inductive influence from the vegetal hemisphere cells. These data suggest roles for Hroth in both specification of mesoendodermal cells and anterior neuroectoderm formation (Wada, 1999).

The cephalochordate member of the Otx homeobox gene family (AmphiOtx) is likely to be present in a single-copy state in the genome. Phylogenetic analysis indicates that there is single ancestral Otx gene in the first chordates which was duplicated in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1 descendant genes (Williams, 1998).

Gnathostomes carry two lineages of Otx genes (Otx1 and Otx2) as cognates of a Drosophila head gap gene, orthodenticle. Previous studies with mutant mice have demonstrated that they play essential roles in the development of rostral head. To shed light on the evolution of the rostral head in vertebrates, otx cognates in the Japanese marine lamprey, Lampetra japonica, have been characterized. The lamprey genome appeared to have two Otx cognates, LjOtxA and LjOtxB. Phylogenetic analyses suggest that LjOtxA clusters with gnathostome Otx2 genes, but LjOtxB does not belong to either the Otx1 or Otx2 lineage. LjOtxA was expressed in the forebrain and midbrain with the caudal limit possibly at the midbrain/hindbrain junction, just as are gnathostome Otx cognates, but LjOtxB is not expressed in the brain. No Otx1 or Otx2 cognates are known in gnathostomes that are not expressed in the brain. Both LjOtxA and LjOtxB are expressed in the olfactory placode, epiphysis, optic stalks, and lower and upper lips. LjOtxB is also expressed in the eyes, where no LjOtxA transcripts are detected. Thus, the roles played by Otx1 and Otx2 in the development of forebrain and midbrain in gnathostomes appear to be shouldered by LjOtxA alone in the lamprey. LjOtxB may have diverged from the stem of the Otx1 and Otx2 lineages and evolved independently (Ueki, 1998).

Agnathan or jawless vertebrates, such as lampreys, occupy a critical phylogenetic position between the gnathostome or jawed vertebrates and the cephalochordates, represented by amphioxus. In order to gain insight into the evolution of the vertebrate head, a homolog of the head-specific gene Otx has been cloned and characterized from the lamprey Petromyzon marinus. This lamprey Otx gene is a clear phylogenetic outgroup to both the gnathostome Otx1 and Otx2 genes. Like its gnathostome counterparts, lamprey Otx is expressed throughout the presumptive forebrain and midbrain. Together, these results indicate that the divergence of Otx1 and Otx2 took place after the gnathostome/agnathan divergence and does not correlate with the origin of the vertebrate brain. Intriguingly, Otx is also expressed in the cephalic neural crest cells as well as mesenchymal and endodermal components of the first pharyngeal arch in lampreys, providing molecular evidence of homology with the gnathostome mandibular arch and insights into the evolution of the gnathostome jaw (Tomsa, 1999).

The Pax6 gene plays a developmental role in various metazoans as the master regulatory gene for eye patterning. Pax6 is also spatially regulated in particular regions of the neural tube. Because the amphioxus has no neuromeres, an understanding of Pax6 expression in the agnathans is crucial for an insight into the origin of neuromerism in the vertebrates. A single cognate cDNA of the Pax6 gene, LjPax6, has been isolated from a Lampetra japonica cDNA library and the pattern of its expression has been observed using in situ hybridization. Phylogenetic analysis has revealed that LjPax6 occurs as an sister group of gnathostome Pax6. In lamprey embryos, LjPax6 is expressed in the eye, the nasohypophysial plate, the oral ectoderm and the brain. In the central nervous system, LjPax6 is expressed in clearly delineated domains in the hindbrain, midbrain and forebrain. The pattern of LjPax6 expression was compared with that of other brain-specific regulatory genes, including LjOtxA, LjPax2/5/8, LjDlx1/6, LjEmx and LjTTF1. Most of the gene expression domains show a conserved pattern, which reflects the situation in the gnathostomes, conforming partly to the neuromeric patterns proposed for the gnathostomes. It is concluded that most of the segmented domains of the vertebrate brain were already established in the ancestor common to all vertebrates. Major evolutionary changes in the vertebrate brain may have involved local restriction of cell lineages, leading to the establishment of neuromeres (Murakami, 2001).

In the evolutionary context, the crucial questions are, therefore, how many segments are arranged in which pattern in the lamprey brain, and which of these patterns are shared between the lamprey and the gnathostomes? In gnathostomes, the positions of the nerve tracts are conserved between species. Such anatomical conservation is known to be associated with compartmentalization of the neural tube: P1 is characterized by the posterior commissure and P2 by the habenular commissure. Caudal to the optic chiasm, tuberal and mammillary hypothalamic territories are clearly identifiable. In the present study, the posterior commissure, the habenular commissure and the optic chiasm were found to have homologous topography, implying the presence of P1 and P2 in all vertebrate brains. This is also consistent with the expression of regulatory genes. In the stage 26 lamprey brain, the rostral domain of LjOtxA expression overlaps the caudal part of the LjPax6-expressing domain. Considering the positions of the epiphysis, the posterior commissure and the habenular commissure, and comparing the pattern with that known for the embryonic mouse brain, the LjOtxA-LjPax6 co-expressing domain most probably corresponds to the dorsal thalamus plus the pretectum (P1+P2). Gene expression patterns and anatomical structures are the only clue to the boundaries of the more rostral segments (Murakami, 2001).

The LjOtxA-expressing domain terminates rostrally at the presumptive P2/P3 boundary (zona limitans), which is assumed from the position of the epiphysis. Rostral to this, LjPax6 and LjDlx1/6 are co-expressed in the dorsal diencephalon, and no expression is seen in the ventral diencephalon (hypothalamus). The expression of LjTTF1 appears to be complementary to the latter (in the ventral diencephalon or hypothalamus), and no transverse segmental boundaries in the forebrain can be detected for the telencephalon. Furthermore, the boundary between the LjPax6- LjDlx1/6 co-expressing domain and the LjTTF1-expressing domain may correspond to the alar-basal plate boundary (Murakami, 2001).

The morphology of the telencephalon is also problematic. Based on the expression patterns of the regulatory genes, the gnathostome telencephalon is assumed to be composed of three major components: the pallium (medial, dorsal, and lateral pallium), the intermediate zone (ventral pallium) and the subpallium (striatum). Emx and Pax6 are expressed in the pallium, and Dlx in the subpallium. In the stage 26 lampery forebrain, a transverse (morphologically horizontal) furrow is found, designated as the sulcus intraencephalicus anterior. Characteristic gene expression is observed in the part of the brain that is rostral and dorsal to this sulcus. LjPax6 is expressed in the dorsal part and LjDlx1/6 in the ventral part, possibly corresponding to the pallium and striatum in the lamprey, respectively. Furthermore LjEmx is restricted to a small dorsal domain that expresses this gene plus LjPax6, and resembles the dorsal pallium of the gnathostomes. These patterns of gene expression in this part of the lamprey brain are extremely reminiscent of the gnathostome telencephalon. Although this may also imply the presence of the P3/P4 boundary (pallio-subpallial boundary), it could not be followed into the dorsal diencephalic and hypothalamic regions. Finally, there is a region in the gnathostome telencephalon that includes the pallidum, in which Dlx and TTF1 are both expressed. The loss of TTF1 expression in the ventral telencephalic region of the lamprey forebrain may be related to the apparent absence of a pallidum in this animal (Murakami, 2001).

In conclusion, the present study of the lamprey brain primordium suggests the presence of the P1 and P2 segments, a longitudinally extending sulcus limitans that terminates rostrally, close to the optic chiasm, a hypothalamus and a tripartite telencephalon-like domain. All these features are directly comparable with those in the model established in the mouse. These results have not further clarified the number of segments in the rostralmost part of the brain. The shared morphological patterns described above are assumed to be very old in origin, possibly dating to the divergence of the lampreys and the gnathostomes. Since recent analyses based on several molecules place hagfishes as the sister group of the lamprey, the segments listed above were already present in the common ancestor of all the vertebrates. The recent discovery of the earliest fossil vertebrates in the early Cambrian period (490-545 million years ago) suggests that the segmental plan underlying vertebrate brain development may have an even longer history. The absence of compartments and the presence of similar anteroposterior regulation by various regulatory genes in cephalochordates imply that the vertebrate-specific compartments listed above were acquired by rough regionalization of the neurectoderm already present in the cephalochordates. It may have been proliferation of neurectodermal cells, as well as the restriction of local cell lineages to form boundaries, that facilitated this most curious evolutionary transition (Murakami, 2001).

The origin of molecular mechanisms of cephalic development is an intriguing question in evolutionary and developmental biology. Ascidians, positioned near the origin of the phylum Chordata, share a conserved set of anteroposterior patterning genes with vertebrates. The cross-phylum regulatory potential of the ascidian Otx gene in the development of the Drosophila brain and the head vertex structures is reported in this study. The ascidian Otx gene rescues the embryonic brain defect caused by a null mutation of the Drosophila orthodenticle gene and enhances rostral brain development while it suppresses trunk nerve cord formation. Furthermore, the ascidian Otx gene restores the head vertex defects caused by a viable otd mutation, ocelliless, via specific activation and repression of downstream regulatory genes. These cross-phylum regulatory potentials of the ascidian Otx gene are equivalent to the activities of the Drosophila and human otd/Otx genes in these developmental processes. These results support the notion that basal chordates such as ascidians have the same molecular patterning mechanism for the anterior structures found in higher chordates, and suggest a common genetic program of cephalic development in invertebrate, protochordate and vertebrate (Adachi, 2001).

In chordates, formation of neural tissue from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in vertebrates. Using the early neural marker Otx as an entry point, the neural induction pathway in the simple embryos of Ciona intestinalis was dissected. The regulatory element driving Otx expression in the prospective neural tissue was isolated; this element directly responds to FGF signaling and FGF9/16/20 acts as an endogenous neural inducer. Binding site analysis and gene loss of function established that FGF9/16/20 induces neural tissue in the ectoderm via a synergy between two maternal response factors. Ets1/2 mediates general FGF responsiveness, while the restricted activity of GATAa targets the neural program to the ectoderm. Thus, this study identifies an endogenous FGF neural inducer and its early downstream gene cascade. It also reveals a role for GATA factors in FGF signaling (Bertrand, 2003).

Otx expression starts in the animal a6.5 pair of blastomeres as they become restricted to anterior neural fate, at the onset of the neural induction process. At this stage, Otx is also activated in the animal b6.5 pair of blastomeres (precursors of the posterior dorsal neural tube and of the dorsal midline which constitutes a neurogenic region and in some vegetal B-line blastomeres (precursors of the posterior mesendoderm). Interestingly, Otx activation in b6.5, as in a6.5, requires an induction from vegetal blastomeres (Bertrand, 2003 and references therein).

The region in Otx located between -1541 and -1417 is required for expression in the a6.5 lineage, and is referred to as the a-element. Consistent with the simultaneous induction of Otx in a6.5 and b6.5 by vegetal cells, deletion of the a-element also reduces the activity in the b6.5 lineage. Finally, regions located between positions -1417 to -1133, and -706 to -271 are required for expression in A-line, and B/b-lines respectively (Bertrand, 2003).

Otx activation in the a6.5 neural precursors requires an interaction with the anterior vegetal blastomeres (A-line). Thus, the inducing FGF should be expressed in A-line blastomeres, before the onset of Otx expression at the 32-cell stage. The Ciona intestinalis genome contains 6 members of the FGF family. By in situ hybridization, only detect one FGF, FGF9/16/20, could be detected that was expressed at the right time and place to be the inducer. Its expression starts at the 16 cell-stage in the A-line and some B-line cells. Expression is stronger in the A-line than in the B-line, and this difference is further enhanced at the early 32-cell stage. This expression pattern is similar to that of the Ciona savignyi ortholog and is consistent with a role for FGF9/16/20 as endogenous neural inducer (Bertrand, 2003).

By both gene loss of function and binding sites analysis it has been determined that cooperation between the maternal transcription factors, Ets1/2 and GATAa, mediates the initial transcriptional response to FGF. Ets transcription factors have already been shown to act in the FGF pathway in vertebrates, and the members of the Ets1/2 subfamily can be directly phosphorylated and activated by Erk. A role for GATAa in this process was more unexpected, since GATA factors have so far not been implicated in the FGF pathway. However, the fact that multimerized GATA binding sites mediate FGF responsiveness indicate that, in this system, GATA does not act solely to modify or enhance Ets activity but functions as an FGF-activated transcription factor. Consistent with the proposal of a direct involvement of GATA factors in the FGF pathway in vivo, it has recently been shown, in vitro, that vertebrate GATA4 can be directly phosphorylated and activated by Erk (Bertrand, 2003).

Could members of the Ets1/2 and GATA families also play a role in neural induction in vertebrates? Ets2 messenger is present maternally in Xenopus eggs and has recently been shown to be required for the induction of Brachyury by FGF in mesodermal cells. It will be interesting to test whether it also acts in the neural induction pathway. Vertebrate GATA factors are thought to antagonize rather than promote neural tissue formation; GATA1/2/3 family members are expressed during gastrulation in the nonneural ectoderm in zebrafish, Xenopus, and chick and GATA1 has an antineuralizing activity when overexpressed in Xenopus. However, GATA2 has no antineuralizing activity, showing that this is not a general property of GATA factors. GATA2 and GATA5 are present in Xenopus eggs but the early role of these maternal GATA factors has not been studied, leaving open the possibility of an involvement in neural induction. Finally, it is proposed that, in ascidians, the use of different response factors accounts for the activation of different target genes in neuroectoderm and mesoderm. It will be interesting to test whether the same logic is used in vertebrates or whether the increase in gene number has led to the recruitment of different FGF inducers or receptors in these two lineages (Bertrand, 2003 and references therein).

Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis, which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, the regulation of Otx, a gene with a shared complex expression pattern, was studied in both species. It was found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved, in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. It is proposed that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates (Oda-Ishii, 2005).

In Ciona, it has been shown that Otx is activated in the animal hemisphere (a- and b-line) by the neural inducer Ci-Fgf9/16/20. This signal is mediated by the transcription factors Ci-GATAa and Ci-Ets1/2 via a cluster of GATA- and Ets-binding sites in the Ci-Otx ab-module. In Halocynthia, it has also been shown that Hr-Ets, the ortholog of Ci-Ets1/2, is required for Otx activation in the a- and b-line, and this study shows that the modules driving Hr-Otx expression in this line (#1, #2, #5) also contain clusters of GATA- and Ets-BSs. This suggests that the regulatory logic in this line is conserved between the two species. In addition, #2 and #5 also drive expression in a- and b-line cells when tested in Ciona. However, #1 does not drive expression in Ciona, indicating that the syntax of the module is not entirely degenerate and that it partly differs between the two species. Given the important sequence divergence of at least one of the factors binding to the a-module, Ets1/2 (50% amino acid identity between Halocynthia and Ciona), some co-evolution of the module and its binding factors is not unexpected (Oda-Ishii, 2005).

Inderstanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the ascidian mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal β-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ciona friend of GATA gene (Ci-fog) is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. This study has identified a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program (Rothbacher, 2007).

The tripartite organization of the central nervous system (CNS) may be an ancient character of the bilaterians. However, the elaboration of the more complex vertebrate brain depends on the midbrain-hindbrain boundary (MHB) organizer, which is absent in invertebrates such as Drosophila. The Fgf8 signaling molecule expressed in the MHB organizer plays a key role in delineating separate mesencephalon and metencephalon compartments in the vertebrate CNS. This study presents evidence that an Fgf8 ortholog establishes sequential patterns of regulatory gene expression in the developing posterior sensory vesicle, and the interleaved 'neck' region located between the sensory vesicle and visceral ganglion of the simple chordate Ciona intestinalis. The detailed characterization of gene networks in the developing CNS led to new insights into the mechanisms by which Fgf8/17/18 patterns the chordate brain. The precise positioning of this Fgf signaling activity depends on an unusual AND/OR network motif that regulates Snail, which encodes a threshold repressor of Fgf8 expression. Nodal is sufficient to activate low levels of the Snail repressor within the neural plate, while the combination of Nodal and Neurogenin produces high levels of Snail in neighboring domains of the CNS. The loss of Fgf8 patterning activity results in the transformation of hindbrain structures into an expanded mesencephalon in both ascidians and vertebrates, suggesting that the primitive MHB-like activity predates the vertebrate CNS (Imai, 2009).

This study provides a number of key insights into the compartmentalization of the chordate CNS. First, a localized Fgf8 signaling center was probably used by the last shared ancestor of ascidians and vertebrates to delineate two regions of the chordate brain (mesencephalon and metencephalon). Second, Fgf8 signaling in Ciona leads to restricted expression of Otx and FoxB in the PSV, as well as restricted expression of Pax2/5/8-A in the neck. Otx and FoxB might inhibit Hox1 expression in the forebrain via Cyp26, whereas Pax2/5/8-A might coordinate the expression of the regulatory genes required for the differentiation of metencephalon motoneurons, such as Phox2a/Arix. Finally, although the regulatory genes responsible for the compartmentalization of the vertebrate CNS (e.g. Otx, Pax2, Neurogenin, etc.) exhibit comparable patterns of expression in the Ciona CNS, there are both conserved and distinctive features of the underlying mechanism. Localized Fgf8 signaling is used to deploy these expression patterns in both systems, even though different regulatory mechanisms are used to restrict Fgf8 (Imai, 2009).

Genomic cis-regulatory networks in the early Ciona intestinalis embryo

Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. This determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, it was found that most of the previously determined interactions are direct. Focus was placed on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. Many interactions were found that had not been predicted by simple gene knockdown experiments, and a significant fraction of TF-DNA interactions were found to make major contributions to the regulatory control of target gene expression (Kubo, 2010).

The developmental fates of blastomeres in the Ciona embryo have been determined by the gastrula stage. A comprehensive study has revealed that 53 TF genes are zygotically expressed and regulate one another in complex networks before gastrulation begins. To dissect the architecture of these networks at the level of protein-DNA interactions, focus was placed on 11 TF genes that play core roles in gene regulatory networks for endomesoderm specification: Brachyury, FoxA-a, FoxD, MyoD, Neurogenin, Otx, Snail, SoxC, Tbx6b, Twist-like1 and ZicL. Because the Ciona genome contains multiple copies of FoxD, Tbx6b and ZicL as gene clusters and their precise copy numbers have not yet been determined, these genes are collectively referred to FoxD, Tbx6b and ZicL in this paper. Likewise, there are two copies of Twist-like1, which are highly similar to each other, and these are collectively referred to as Twist-like1 (Kubo, 2010).

Eleven gene-fusion constructs were prepared that encode GFP-tagged TFs expressed under the control of their own promoters (e.g. a fusion gene that encodes GFP-tagged Brachyury driven by the Brachyury promoter). When these constructs were introduced into eggs, the resultant embryos expressed the fusion genes at the same time and in the same blastomeres as the endogenous genes. Exceptions were the Twist-like1 and the Snail constructs. Twist-like1 is normally expressed in three cell lineages (A7.6, B7.7 and B8.5), but the construct drove Twist-like1-GFP expression only in the B7.7 and B8.5 lines. Snail expression in the notochord lineage is normally very weak. The Snail construct did not recapitulate this expression in the notochord lineage but did drive Snail-GFP expression in the remaining lineages (Kubo, 2010).

Expression of these genes did not affect embryonic morphology at the stage when the embryos were fixed. The fixed embryos were subjected to ChIP using anti-GFP antibodies, and subsequently to microarray analysis. To define significant regions, two programs were used employing totally different algorithms. DNA segments regarded as positive by both programs were defined as significant. To confirm that this approach successfully identified TF binding sites, the sequences of ZicL and Tbx6b binding regions defined with three different false discovery rates (FDRs) were analyzed, as the consensus binding motifs of these two TFs are known. The frequencies of matches to the consensus binding sequences for ZicL and Tbx6b around peaks in 0.1% FDR were generally better than in 0.01% and 1% FDRs. As expected, the frequencies of the consensus binding sequences for ZicL and Tbx6b were markedly higher around peaks in the identified regions, suggesting that the method was able to successfully identify the TF binding regions (Kubo, 2010).

Brachyury and Ci-tropomyosin-like are the only known direct targets of ZicL and Brachyury, respectively. As an independent confirmation, the TF binding sites of these genes was expected. The ZicL ChIP profile showed a sharp peak around two known strong ZicL binding sites. The Brachyury ChIP profile also showed a peak around the known Brachyury binding site in the Ci-tropomyosin-like promoter. These peaks were included in significant regions identified with all the FDRs described above. ChIP-qPCRs were performed for these two known interactions. The ChIP-qPCR results showed excellent agreement with the ChIP-chip results (Kubo, 2010).

Next, the promoters were examined of genes that were identified in previous studies as likely direct targets of one of the 11 TFs on the basis of expression assays and gene knockdown assays. Among 29 interactions that had been found in the gene knockdown assays and for which both the source and target genes are expressed in the same cells, 28, 23 and 19 interactions were indicated to be direct under the FDRs of 1%, 0.1% and 0.01%, respectively. The remainder of the interactions were not regarded as direct. Otx expression in the A-line lineage requires a cis-regulatory module that includes Fox binding sites and is suppressed in FoxA-a morphants. The FoxA-a binding to this cis-regulatory element was counted with FDRs of 1% and 0.1%, but not with the most stringent FDR (0.01%). Similarly, several lines of evidence have suggested that MyoD is directly regulated by ZicL. First, MyoD expression is suppressed in ZicL morphants. Second, MyoD and ZicL are both expressed in presumptive muscle cells and the time windows of their expression overlap. Lastly, there is a putative ZicL binding site near to the peaks found in the MyoD upstream region. This putative binding was observed under the FDRs of 1% and 0.1%, but not under the most stringent FDR of 0.01%. On the basis of the above observations, in the following sections the results obtained at an FDR of 0.1% are generally described (Kubo, 2010).

The frequencies of the consensus sequences for ZicL and Tbx6b binding were markedly higher around peaks in the identified regions. Since the consensus binding motifs of the other nine TFs had not been determined previously, similar analyses was performed with motifs of homologs in other animals. The frequencies of the consensus binding motifs for six of the TFs, but not FoxD, SoxC or Twist-like1, were markedly higher around peaks. Because the position weight matrices (PWMs) for FoxD, SoxC and Twist-like1 gave higher background, no significant changes were seen. However, the number of matches to the motifs was markedly higher around peaks than in flanking regions and the background. These observations suggested that the method was able to successfully identify the TF binding regions (Kubo, 2010).

As has been reported in other animals, it was found that the regions bound by Brachyury, MyoD, Neurogenin, Snail, Tbx6b, Twist-like1 and ZicL, especially around the peaks, showed a marked GC bias. This bias is likely to be related to the consensus sequences, because the consensus sequences for these TFs are generally more GC-rich than those of the remaining TFs. The observed enrichment of recognition sequences was unlikely to be an artifact of GC bias because even if background sequences were picked with a base composition comparable to the averaged GC content of the bound regions (the difference between the average GC content of the bound and background regions was less than 0.8%), matches to the PWMs were enriched around peaks versus each of the GC-adjusted backgrounds (Kubo, 2010).

Next, attempts were made to discover overrepresented motifs in the regions (360 bp) around the peaks identified by each ChIP experiment using the Trawler program. It was found that overrepresented motifs were similar to the PWMs that were determined experimentally (Tbx6b and ZicL) or to those of homologs in other animals (the remaining nine TFs). This further supported the conclusion that the results of the ChIP experiments were of high quality (Kubo, 2010).

It is generally believed that TFs tend to bind near promoters, although many examples are known in which TFs bind to enhancers far from promoters. The distributions of peaks in all experiments, except Snail ChIP, were higher around transcription start sites. The reason why Snail binding sites were not enriched around transcription start sites is unclear, but this does not necessarily indicate that the results of the Snail ChIP were of low quality. Altogether, these observations support the conclusion that all of the ChIP experiments revealed in vivo occupancies of the TFs (Kubo, 2010).

TF genes were significantly enriched among the target genes of the 11 TFs. Among 670 potential TF genes in the Ciona genome, at least 607 encode proteins with known TF motifs or proteins with two or more zinc-finger motifs that potentially bind to DNA. A significantly greater number of TF genes were found among the targets than would be expected from random sampling. This enrichment indicates that the TFs examined bind targets selectively and not randomly (Kubo, 2010).

The ChIP data was compared with the results of the comprehensive gene knockdown experiments of a previously study. Among 76 interactions previously found in the early embryo, the ChIP assays indicated that 58 are direct. In addition, 251 novel interconnections were found. Among 121 (11×11) possible interconnections, 84 were observed in the present study. The data indicate that these genes are tightly interconnected with one another (Kubo, 2010).

Because the gene regulatory network model previously constructed from comprehensive expression profiles and comprehensive knockdowns of regulatory genes is of single-cell resolution, the ChIP data was interpred into this network by assuming that the examined TFs bind to the targets wherever their mRNAs are expressed. The reconstructed networks had a complex architecture (Kubo, 2010).

The reconstructed regulatory networks allow tracing of development at the single-cell level. Figs S8 and S9 in the supplementary material show the interconnections among the core 11 TFs in A-line and B-line blastomeres, which give rise to endomesodermal tissues, from the 8-cell to the early gastrula stage. Two of the three mesenchymal lineages (B-line mesenchymal cells) and 28 out of 36 muscle cells (B-line muscle cells) in the tadpole larvae are derived from B4.1 blastomeres at the 8-cell stage. Thirty-two and eight notochord cells are derived from A4.1 and B4.1 blastomeres, respectively. Previous studies demonstrated that Twist-like1, MyoD and Brachyury are essential for specification of the mesenchyme, muscle and notochord, respectively (Kubo, 2010).

Twist-like1 is expressed exclusively in the mesenchymal lineage and is regulated by FoxA-a, Otx and ZicL, as indicated by the fact that knockdown of any of these three genes results in loss or reduction of Twist-like1 expression. No direct binding was detected of FoxA-a to the Twist-like1 promoter, but it was found that FoxA-a binds to the upstream regions of Otx and ZicL, and that ZicL and Otx bind to the promoter of Twist-like1. Therefore, it is highly likely that FoxA-a mainly activates Twist-like1 indirectly through activating Otx and ZicL (Kubo, 2010).

Twist-like1 expression begins in B7.7 (the posterior B-line mesenchyme) at the 64-cell stage and in B8.5 (the anterior B-line mesenchyme) at the early gastrula stage. These two mesenchymal lines contribute to distinct adult tissues after metamorphosis. ZicL might be associated with the differences between these two lineages because the contribution of ZicL to Twist-like1 activation is weaker than that of Otx. To confirm this idea, a mutant Twist-like1 promoter was tested, from which a 150 bp segment containing the identified ZicL binding region was deleted. Because the Otx ChIP result indicated that the Otx binding region is distinct from the ZicL binding region, Otx was expected to bind to this mutant promoter. When introduced into fertilized eggs by electroporation, the wild-type promoter (1550 bp) drove reporter expression in 65% of the embryos, whereas the mutant promoter drove reporter expression in 36% of the embryos. In addition to the significant decrease in the number of embryos expressing the reporter, the overall fluorescence was weaker and the posterior B-line mesenchyme did not appear to express the reporter in the mutant construct. To confirm this observation, the experimental embryos were cleavage-arrested at the 110-cell stage. Cells in the arrested embryos cannot divide further, but the developmental programs proceed as in normal embryos. The mutant construct failed to drive reporter expression in the posterior B-line mesenchyme. These results suggest that ZicL contributes to the difference between these two lineages (Kubo, 2010).

A previous study showed that nine mesenchyme-specific non-regulatory genes are under the control of Twist-like1. None of these genes was identified as a direct target in the present study. Even when applied with an FDR of 1%, only one gene was identified as a direct target. Therefore, it is likely that Twist-like1 regulates the expression of mesenchyme-specific genes through its downstream regulatory gene circuit, although there is a possibility that Twist-like1 binds to the regulatory elements of these genes at later stages (Kubo, 2010).

The B6.2 and B6.4 cell pairs in the 32-cell embryo have the potential to give rise to mesenchyme and muscle. At the 64-cell stage, these cells divide, and one of the daughter cells becomes specified to give rise to the muscle cells. Previous functional assays showed that ZicL, Tbx6b and MyoD are essential for specification of muscle cells. Tbx6b begins to be expressed at the 16-cell stage, and cells expressing Tbx6b give rise not only to muscle cells but also to mesenchyme cells. Tbx6b expression declines to undetectable levels before the tailbud stage. ZicL starts to be expressed at the 32-cell stage in a variety of cells, including those with developmental fates of muscle, mesenchyme, notochord and neurons. ZicL expression in the muscle lineage disappears before the late gastrula stage. MyoD expression begins at the 44-cell stage exclusively in the muscle lineage under the control of Tbx6b and ZicL. The present study showed that ZicL, Tbx6b and MyoD constituted a tightly interconnected gene circuit that is responsible for this specification: (1) ZicL bound to the promoters of MyoD and Tbx6b; (2) Tbx6b bound to the promoters of MyoD and ZicL; and (3) MyoD bound to the promoter of Tbx6b and to its own promoter. All of these interactions, except MyoD binding to the Tbx6b promoter, have been confirmed by functional assays (Kubo, 2010).

To understand how this gene circuit regulates downstream muscle-specific genes, the promoters were examined of 13 muscle structural genes that are well annotated and known to be expressed in the larval tail muscle. Of these, ten were directly bound by MyoD and Tbx6, one by MyoD and ZicL, one by Tbx6b and ZicL, and one by MyoD alone (Kubo, 2010).

Both MyoD and Tbx6 bound to the promoters of more than three-quarters of the muscle genes examined. To test the action of this feed-forward loop comprising MyoD and Tbx6b in the regulation of muscle-specific gene expression, the expression patterns of genes under the control of this circuit were examined. Of the 155 genes under the direct control of MyoD and Tbx6b, 50 (including the above ten) were already known to be expressed in muscle cells. From the remaining 105 genes, 20 were randomly chosen, and 15 were found to be expressed in muscle cells, suggesting that this circuit is widely used for the regulation of genes expressed in muscle cells, and also that this circuit might not necessarily be sufficient for driving expression of the target (Kubo, 2010).

Brachyury is activated at the 64-cell stage exclusively in the notochord lineage, and this expression specifies the notochord fate. ZicL directly binds to the Brachyury promoter and activates its expression. It has also been shown that FoxD and FoxA-a are required for Brachyury expression, probably through regulating ZicL expression, and that FGF signaling is also required for Brachyury expression. The present assays showed that not only ZicL, but also FoxD binds to the Brachyury promoter. Although FoxD mRNA is not present in the notochord lineage at the 32-cell and 64-cell stages, when ZicL and Brachyury are activated, respectively (FoxD is expressed in the ancestors of cells in which ZicL and Brachyury are expressed), the ChIP assay indicated that FoxD binds to the promoters of ZicL and Brachyury. Because knockdown of FoxD eliminates ZicL and Brachyury expression and because the FoxD-GFP fusion protein exists in the notochord lineage at the 32-cell stage, it is likely that FoxD protein exists in these cells and binds to the promoters of ZicL and Brachyury when these two genes begin to be expressed (Kubo, 2010).

FoxA-a binding to the Brachyury promoter was not identified under 0.1% FDR. There was, however, a small peak that was counted as significant under 1% FDR. The possibility could not be ruled out that FoxA-a binds weakly to the Brachyury promoter. It is also possible that FoxA-a could bind weakly to a FoxD binding site because the FoxA-a binding peak coincided with that of FoxD. Even if this weak binding occurs in vivo, the regulation of Brachyury by FoxA-a would largely be achieved indirectly through FoxD and ZicL, since strong binding was found of FoxA-a to the promoters of FoxD and ZicL (Kubo, 2010).

Next, 14 non-regulatory genes were examined that are known to be expressed in the notochord under the control of Brachyury. Among them, 11 were identified here as direct targets of Brachyury. The present results suggest that the remaining three genes are regulated indirectly through a gene circuit under the control of Brachyury, although it cannot be ruled out that Brachyury binds to the regulatory elements of these three genes at later stages (Kubo, 2010).

The present study found many interactions between TFs and genomic DNA that were unexpected from preceding gene knockdown assays. Similar observations were also reported in preceding ChIP studies. To estimate what proportion of the binding makes a major contribution to gene regulation in Ciona embryos, MyoD mRNA or an MO against MyoD was injected into eggs, and their effects were analyzed on the expression of the same targets that were analyzed at the gastrula stage or at the tailbud stage, respectively. The mRNA levels of 14 targets, ten of which were expressed in muscle, were significantly increased (>2-fold) in embryos injected with MyoD mRNA, and MyoD MO injection significantly reduced the mRNA levels of three of these targets. The mRNA level of one target (KH.C12.38), which was weakly expressed in muscle at the tailbud stage, was significantly decreased in embryos injected with MyoD mRNA, whereas the mRNA level of one target (KH.C9.27), which was expressed in muscle at the gastrula stage, was significantly increased in embryos injected with the MyoD MO. In total, the mRNA levels of 16 targets were significantly altered by MyoD mRNA overexpression or gene suppression. The remaining four were not significantly affected, although three of these were expressed in muscle, implying that MyoD binding makes a relatively small contribution to activating these target genes. It was also found that eight of 15 Brachyury targets and seven of 12 Twist-like1 targets were significantly affected in the embryos by overexpression or knockdown of Brachyury or Twist-like1, respectively. Therefore, it is estimated that more than half of TF binding makes a major contribution to the regulatory control of gene expression (Kubo, 2010).

A cis-regulatory signature in ascidians and flies, independent of transcription factor binding sites

Transcription initiation is controlled by cis-regulatory modules. Although these modules are usually made of clusters of short transcription factor binding sites, a small minority of such clusters in the genome have cis-regulatory activity. This paradox is currently unsolved. To identify what discriminates active from inactive clusters, attention was focused on short topologically unconstrained clusters of two ETS and two GATA binding sites, similar to the early neural enhancer of Ciona intestinalis Otx. First, 55 such clusters, conserved between the two Ciona genomes, were computationally identified. In vivo assay of the activity of 19 hits identified three novel early neural enhancers, all located next to genes coexpressed with Otx. Optimization of ETS and GATA binding sites was not always sufficient to confer activity to inactive clusters. Rather, a dinucleotide sequence code associated to nucleosome depletion showed a robust correlation with enhancer potential. Identification of a large collection of Ciona regulatory regions revealed that predicted nucleosome depletion constitutes a general signature of Ciona enhancers, which is conserved between orthologous loci in the two Ciona genomes and which partitions conserved noncoding sequences into a major nucleosome-bound fraction and a minor nucleosome-free fraction with higher cis-regulatory potential. This signature was also found in a large fraction of short Drosophila cis-regulatory modules. This study indicates that a sequence-based dinucleotide signature, previously associated with nucleosome depletion and independent of transcription factor binding sites, contributes to the definition of a local cis-regulatory potential in two metazoa, Ciona intestinalis and Drosophila melanogaster (Khoueiry, 2010).

Table of contents

orthodenticle: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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