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).

Caudal homologs in arthropods

To infer similarities and differences in terminal pattern formation in insects, several of the key genes of this process were analyzed in the beetle Tribolium castaneum. Two genes of the terminal pattern cascade, namely tailless (tll) and forkhead (fkh), from Tribolium were cloned and their expression patterns were studied. In addition, the pattern of MAP kinase activation was analyzed at blastoderm stage as a possible signature for torso-dependent signaling. Further, the late expression of the previously cloned Tribolium caudal (Tc-cad) gene was examined. Finally, the upstream region of Tc-tll was used to drive a reporter gene construct in Drosophila. This construct is activated at the terminal regions in Drosophila, suggesting that the torso-dependent pathway is conserved between the species. Most of the expression patterns of the genes studied here are similar in Drosophila and Tribolium, suggesting conserved functions (Schroder, 2000).

A caudal homolog of Bombyx mori encodes a protein of 244 amino acids. The homology between Drosophila and Bombyx homeodomains is 80%. There is a single maternal transcript of 2.3 kb. Bombyx CAD transcripts accumulate first in the nurse cells and are later transferred into the oocyte at a defined time during oogenesis. The maternal transcripts of Bombyx CAD form a concentration gradient spanning the anteroposterior axis during the gastrulation stage and are restricted to the anal pad, the most posterior domain (Xu, 1994).

A homolog to the Drosophila caudal (cad) gene has been isolated from the flour beetle Tribolium castaneum and its expression pattern has been studied. The Tribolium caudal (Tc-cad) gene arrangement is unusual in that there is a partial duplication of the gene resulting in alternative transcripts with identical 5'-exons, but different 3'-exons encoding two different homeoboxes. Expression analysis was carried out using whole-mount in situ hybridization and staining with an antibody raised against the N-terminal part of the protein that is common to both transcripts. At early stages a homogeneously distributed maternal mRNA is observed which is initially also translated throughout the embryo. A little later, a posterior to anterior CAD protein gradient is formed, very similar to that in Drosophila. However, because of the differences in the fate map between Drosophila and Tribolium (see Tribolium early embryonic development), the CAD protein expression at blastoderm stage covers the prospective head and thoracic region and not the abdomen as in Drosophila. Expression of Tc-cad in the prospective abdomen is only seen during further germband growth where it becomes restricted to the growth zone in which the segments are formed. This expression is very similar to the growth zone expression in the somitogenic region seen for cad homologs in vertebrates. After the completion of the segmentation process Tc-cad expression becomes confined to a terminal stripe that resembles a similar stripe at late blastoderm stages in Drosophila (Schulz, 1998).

Insect axis formation is best understood in Drosophila, where rapid anteroposterior patterning of zygotic determinants is directed by maternal gene products. The earliest zygotic control is by gap genes, which determine regions of several contiguous segments and are largely conserved in insects. Isolation of mutations has been used to approach a genetic question: do early zygotic patterning genes control similar anteroposterior domains in the parasitoid wasp Nasonia vitripennis as in Drosophila? Nasonia is advantageous for identifying and studying recessive zygotic lethal mutations because unfertilized eggs develop as males while fertilized eggs develop as females. On first consideration, the Hymenopteran Nasonia and the Dipteran Drosophila appear very similar in their embryonic development, though the Hymenoptera diverged from the Diptera >200 million years ago. Embryos of both species produce larvae in about 1 day at 25°C. In Nasonia, the fertilized egg gives rise to an embryo that undergoes syncytial and cellular blastoderm stages morphologically similar to those of Drosophila. Both Nasonia and Drosophila undergo the long germband mode of embryonic development. Despite these similarities, two observations suggest that the relative importance of maternal versus zygotic patterning functions may differ in the two insects. (1) Although postgastrulation events proceed with very similar timing, the time for early development differs substantially - at 25°C: the events preceding gastrulation take only about 3 hours in Drosophila but almost 10 hours in Nasonia. This difference in timing may allow for greater zygotic control of patterning in Nasonia than in Drosophila. (2) Among the relatives of Nasonia, a polyembryonic mode of development has evolved in which a single fertilized egg gives rise to hundreds or thousands of progeny. Polyembryonic development is likely to rely heavily on zygotic control of patterning. Polyembryony has arisen several times in the Hymenoptera, and the polyembryonic Copidosoma floridanum is in the same superfamily as Nasonia. These considerations pose the following question -- is early development substantially controlled by the zygotic genome in Hymenopterans? This question may be approached genetically, by isolating zygotic mutations that disrupt early anteroposterior patterning in Nasonia. Recessive zygotic mutations have identified three Nasonia genes: head only mutant embryos have posterior defects, resembling loss of both maternal and zygotic Drosophila caudal function; headless mutant embryos have anterior and posterior gap defects, resembling loss of both maternal and zygotic Drosophila hunchback function, and squiggy mutant embryos develop only four full trunk segments, a phenotype more severe than those caused by lack of Drosophila maternal or zygotic terminal gene functions. head only mutant embryos lack all segmentation posterior to the head, in the strongest manifestation of the phenotype, and have only a narrow domain of Ubx-Abd-A expression. head only differs from Drosophila gap genes with respect to the extent of pattern deleted and effects on Ubx-Abd-A. In Drosophila, neither Krüppel nor knirps affects a domain as large as that of head only. Moreover, the wild-type functions of Krüppel and knirps are not required for the positive regulation of Ubx or abd-A in Drosophila (Pultz, 1999).

The characterization is reported of a caudal gene from the rhizocephalan cirripede Sacculina carcini and its embryonic and larval expression patterns. Cirripedes are maxillopodan crustaceans that are devoid of any complete abdominal segment at the adult stage. The genetic basis of this peculiar body plan is being explored. They probably lack the abdominalA gene, while possessing the other Hox genes shared by arthropods. However, at least a part of the genetic program might be conserved, since the engrailed.a and engrailed.b genes are expressed in a posterior region that is interpreted as a relic of an ancestral abdomen. The Sacculina caudal gene is expressed early in embryogenesis, which makes it the earliest genetic marker evidenced in the development of Sacculina and of any other crustacean species. It is expressed later in the embryo in the caudal papilla, a posterior proliferating zone of cells. During the larval stages, the caudal gene is first expressed in the whole thoracic region; then its expression regresses to the posterior end of the larva. Surprisingly, it is never expressed in the vestigial abdomen. This lack of expression of the Sacculina caudal gene in a posterior region, at odds with what is known in all other studied metazoan species, might be correlated with the defective development of the abdomen (Rabet, 2001).

All arthropods share the same basic set of Hox genes, although the expression of these genes differs among divergent groups. In the brine shrimp Artemia franciscana, their expression is limited to the head, thoracic/trunk and genital segments, but is excluded from more posterior parts of the body, consisting of six post-genital segments and the telson (bearing the anus). Nothing is currently known about the genes that specify the identity of these posterior structures. The expression patterns was studied of four candidate genes, Abdominal-B, caudal/Cdx, even-skipped/Evx and spalt, the homologs of which are known to play an important role in the specification of posterior structures in other animals. Abdominal-B is expressed in the genital segments of Artemia, but not in the post-genital segments at any developmental stage. The expression of caudal, even-skipped and spalt in the larval growth-zone suggests they may play a role in the generation of body segments (perhaps comparable with the role of gap and segmentation genes in insects), but not a direct role in defining the identity of post-genital segments. The expression of caudal at later stages suggests a role in the specification of anal structures. A PCR screen designed to isolate Hox genes expressed specifically in the posterior part of the body failed to identify any new Hox genes. It is concluded that the post-genital segments of Artemia are not defined by any of the genes known to play a role in the specification of posterior segments in other arthropods. It is argued that these segments constitute a unique body region that bears no obvious homology to previously characterized domains of Hox gene activity (Copf, 2003).

The number of leg-bearing segments in centipedes varies extensively, between 15 and 191, and yet it is always odd. This suggests that segment generation in centipedes involves a stage with double segment periodicity and that evolutionary variation in segment number reflects the generation of these double segmental units. However, previous studies have revealed no trace of this. The expression of two genes, an odd-skipped related gene (odr1) and a caudal homolog, is described that serve as markers for early steps of segment formation in the geophilomorph centipede, Strigamia maritima. Dynamic expression of odr1 around the proctodaeum resolves into a series of concentric rings, revealing a pattern of double segment periodicity in overtly unsegmented tissue. Initially, the expression of the caudal homolog mirrors this double segment periodicity, but shortly before engrailed expression and overt segmentation, the intercalation of additional stripes generates a repeat with single segment periodicity. These results provide the first clues about the causality of the unique and fascinating "all-odd" pattern of variation in centipede segment numbers and have implications for the evolution of the mechanisms of arthropod segmentation (Chipman, 2004).

These observations invite comparison with the process of segment generation in Drosophila. There, a pattern of double segment periodicity is first generated and then subdivided to yield the final single segment repeat. However, the generation of the 'pair-rule' pattern in Drosophila shows few if any similarities with the early stages of segmentation in Strigamia . Drosophila subdivides the entire body axis into unique domains by activating 'gap genes' under the influence of maternal gradients and then uses the complex promoters of the pair-rule genes to compute a repetitive pattern of gene activity from this underlying aperiodic pattern. The generation of this pattern is almost static with respect to the forming cells of the blastoderm. In Strigamia, the initial patterns of odr1 expression are not static with respect to the underlying cells. It is suggested that the patterns of odr1 gene expression are oscillations of cell state, coordinated as waves that move across the population of cells in the blastodisc, sharpening to encompass fewer cells and stabilizing to double segment periodicity. Thus, despite the fact that odd-skipped is one of the genes expressed in a pair-rule pattern during Drosophila segmentation, it is thought likely that the processes that generate this pair-rule pattern are different in the two species (Chipman, 2004).

Interestingly, odd-skipped family members are downstream targets of the Notch signaling pathway during Drosophila limb segmentation. Recently, it has been shown that the Notch ligand Delta and its target hairy are expressed in a striped pattern during early development and segmentation in the embryo of the spider Cupiennius salei. It has been suggested that Notch signaling in the spider is generating a reiterated pattern through a mechanism analogous to that shown for vertebrate segmentation. These two observations, taken together, suggest the possibility that the odd-skipped family in Strigamia, and possibly in other arthropods, is modulated through a Notch-Delta-mediated oscillator to generate the first serially repeated pattern that begins the segmentation process (Chipman, 2004).

A separate and unresolved issue is whether there are similarities between the process that resolves the pair-rule repeat of Drosophila into a single segment pattern and the process whereby secondary caudal stripes intercalate between primary stripes to generate the single segment repeat in Strigamia. The possibility that such 'frequency doubling' processes may be widespread among the arthropods is supported by the observation of analogous phenomena in chelicerates and short germ insects: in the mite Tetranychus urticae, expression of the paired gene in the prosoma is initially at double segment intervals, with secondary stripes intercalating between them to generate the single segment repeat. In the growing abdomen of the grasshopper Schistocerca americana, paired gene expression also shows a transition from double to single segment periodicity, though in this case the process is one of stripe splitting rather than intercalation. However, in other cases, either no such pattern has been described or the periodicity of gene expression is not yet clear. Of particular relevance in this context is a recent study of segmentation gene expression in the lithobiomorph centipede, Lithobius atkinsoni (only distantly related to Strigamia). The expression of even-skipped in the posterior of the Lithobius germ band shows broad rings around the proctodeum that could reflect dynamic expression, resolving into stripes. However, there is as yet no evidence of subsequent frequency doubling (Chipman, 2004).

These results provide a possible explanation for the observation that, in nature, centipede segment number varies in two-segment increments. It is proposed that variation in segment number among centipedes is caused by variation in the number of cycles of a primary segmentation oscillator, each cycle of which generates two segments. The anteroposterior range of this process may well extend beyond the trunk to include the poison claw and parts of the head and genital regions. Therefore, the occurrence of odd rather than even numbers of leg-bearing segments is not incompatible with this explanation (Chipman, 2004).

Although the molecular mechanisms directing anteroposterior patterning of the Drosophila embryo (long-germband mode) are well understood, how these mechanisms were evolved from an ancestral mode of insect embryogenesis remains largely unknown. In order to gain insight into mechanisms of evolution in insect embryogenesis, the expression and function of the orthologue of Drosophila caudal (cad) was examined in the intermediate-germband cricket Gryllus bimaculatus. A posterior (high) to anterior (low) gradient in the levels of Gryllus bimaculatus cad (Gbcad) transcript is formed in the early-stage embryo, and then Gb' cad is expressed in the posterior growth zone until the posterior segmentation is completed. Reduction of Gb' cad expression level by RNA interference results in deletion of the gnathum, thorax, and abdomen in embryos, remaining only anterior head. The gnathal and thoracic segments are formed by Gb' cad probably through the transcriptional regulation of gap genes including Gb' hunchback and Gb' Krüppel. Furthermore, Gb'cad is found to be involved in the posterior elongation, acting as a downstream gene in the Wingless/Armadillo signalling pathways. These findings indicate that Gb'cad does not function as it does in Drosophila, suggesting that regulatory and functional changes of cad occurred during insect evolution. The Wg/Cad pathway in the posterior pattern formation may be common in short- and intermediate-germband embryogenesis. During the evolutionary transition from short- or intermediate- to long-germband embryogenesis, an ancestral cell-cell signalling system including Wg/Arm signalling may have been replaced by a diffusion system of transcription factors as found in Drosophila. Since Wnt/Cdx pathways are involved in the posterior patterning of vertebrates, such mechanisms may be conserved in animals that undergo sequential segmentation from the posterior growth zone (Shinmyo, 2005).

One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One exception is Bicoid, the master organizer of anterior development in Drosophila and higher dipterans, which is not conserved. Thus, the study of anteroposterior patterning in insects that lack Bicoid can provide insight into the evolution of the diversity of body plan patterning networks. To this end, the long germ parasitic wasp Nasonia vitripennis has been establised as a model for comparative studies with Drosophila. In Nasonia, a gradient of localized caudal mRNA directs posterior patterning, whereas, in Drosophila, the gradient of maternal Caudal protein is established through translational repression by Bicoid of homogeneous caudal mRNA. Loss of caudal function in Nasonia results in severe segmentation defects. Nasonia caudal is an activator of gap gene expression that acts far towards the anterior of the embryo, placing it atop a cascade of early patterning. By contrast, activation of gap genes in flies relies on redundant functions of Bicoid and Caudal, leading to a lack of dramatic action on gap gene expression: caudal instead plays a limited role as an activator of pair-rule gene expression. These studies, together with studies in short germ insects, suggest that caudal is an ancestral master organizer of patterning, and that its role has been reduced in higher dipterans such as Drosophila (Olesnicky, 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 development of most arthropods, the caudal region of the elongating germ band (the growth zone) sequentially produces new segments. Previous work with the spider Cupiennius salei suggested involvement of Delta-Notch signaling in segmentation. This study reports that, in the spider Achaearanea tepidariorum, the same signaling pathway exerts a different function in the presumptive caudal region before initiation of segmentation. In the developing spider embryo, the growth zone becomes morphologically apparent as a caudal lobe around the closed blastopore. Preceding caudal lobe formation, transcripts of a Delta homolog, At-Delta, are expressed in evenly spaced cells in a small area covering the closing blastopore and then in a progressively wider area of the germ disc epithelium. Cells with high At-Delta expression are likely to be prospective mesoderm cells, which later express a twist homolog, At-twist, and individually internalize. Cells remaining at the surface begin to express a caudal homolog, At-caudal, to differentiate as caudal ectoderm. Knockdown of At-Delta by parental RNA interference results in overproduction of At-twist-expressing mesoderm cells at the expense of At-caudal-expressing ectoderm cells. This condition gives rise to a disorganized caudal region that fails to pattern the opisthosoma. In addition, knockdown of Notch and Suppressor of Hairless homologs produces similar phenotypes. It is suggested that, in the spider, progressive activation of Delta-Notch signaling from around the blastopore leads to stochastic cell fate decisions between mesoderm and caudal ectoderm through a process of lateral inhibition to set up a functional caudal lobe (Oda, 2007).

Canonical Wnt signaling has been implicated in an AP axis polarizing mechanism in most animals, despite limited evidence from arthropods. In the long-germ insect, Drosophila, Wnt signaling is not required for global AP patterning, but in short-germ insects including Tribolium castaneum, loss of Wnt signaling affects development of segments in the growth zone but not those defined in the blastoderm. To determine the effects of ectopic Wnt signaling, the expression and function of axin, which encodes a highly conserved negative regulator of the pathway, was analyzed. Tc-axin transcripts maternally localized to the anterior pole in freshly laid eggs. Expression spread toward the posterior pole during the early cleavage stages, becoming ubiquitous by the time the germ rudiment formed. Tc-axin RNAi produced progeny phenotypes that ranged from mildly affected embryos with cuticles displaying a graded loss of anterior structures, to defective embryos that condensed at the posterior pole in the absence of serosa. Altered expression domains of several blastodermal markers indicated anterior expansion of posterior fates. Analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the effects of Tc-axin depletion are mediated through this pathway and that Wnt signaling must be inhibited for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt signaling must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt activity in defining AP polarity and patterning in metazoan development (Fu, 2012).

Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone

Wnt/β-catenin and Hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. This study used the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. Wnt/β-catenin signaling was found to act upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. The target gene sets of the Wnt/β-catenin and Hh pathways were determined; the growth zone signaling center activates a much greater number of genes and the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist (see Drosophila twist). Furthermore, the Wnt pathway is required for hindgut development and Tc-senseless (see Drosophila senseless) as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function (Oberhofer, 2014).

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).

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).

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider: Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen)

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration. In bilaterians, there are at least 12 subfamilies of Wnt genes. Wnt3 and Wnt8 are required for somitogenesis in vertebrates and are thought to be involved in posterior specification in deuterostomes in general. Although TCF and β-catenin have been implicated in the posterior patterning of some short-germ insects, the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. This study investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. These results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, it is proposed that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes (McGregor, 2008).

The posterior truncation phenotypes resulting from pRNAi against Wnt8 in the spider are at least superficially similar to those observed when Wnt8 and/or Wnt3 are perturbed in vertebrate embryos. Removal or blocking Wnt8 and/or Wnt3 in Xenopus, zebrafish, and mouse results in truncated embryos with only a few anterior somites and no tail bud. Although analysis of TCF and β-catenin in Oncopeltus and Gryllus, respectively, indicated that Wnt signaling might be involved in the development of the growth zone and posterior segments in arthropods, the current data show that in fact the same ligand, Wnt8, is employed in posterior development in both vertebrates and arthropods (McGregor, 2008).

In class II and III At-Wnt8pRNAi embryos exhibiting fused L4 limb buds, it also appeared that the most ventral part of this segment is missing. This phenotype shows similarities to the phenotype when short-gastrulation is knocked down in this spider. It suggests that, in addition to A-P patterning, At-Wnt8 is involved in D-V patterning in the spider, a role Wnt8 genes also perform in vertebrates (McGregor, 2008).

There is evidence that Wnt signaling acts upstream of Delta/Notch in vertebrate somitogenesis. Although the expression of Wnt3a and Wnt8 is not cyclical during somitogenesis in vertebrates, some downstream components of Wnt signaling, such as Axin2, are cyclically expressed in mice and possibly are integral to the Delta/Notch-dependent segmentation clock. However, recent experiments have shown that Axin2 and components of the Delta/Notch pathway continue to oscillate in the presence of stabilized β-catenin, which suggests that in mice, Wnt signaling may be permissive for the segmentation clock rather than instructive. Similarly, in zebrafish it is thought that Wnt8 may act to maintain a precursor population of stem cells in the PSM and tailbud rather than directly regulate the segmentation clock. It is proposed that the same ligand, Wnt8, could play a similar permissive role for segmentation in the growth zone of the spider by establishing and possibly maintaining a pool of cells that develop into the opisthosomal segments. When At-Wnt8 activity is reduced, cells are ectopically used in L3/L4 or internalized, depleting the putative growth-zone pool. This depletion manifests as a smaller opisthosoma, separated clusters of cells that give rise to separate irregular germbands, or even no opisthosoma (McGregor, 2008).

It was previously shown that Delta/Notch signaling is also involved in posterior development in the spiders Cupiennius. These new results reveal that in the spider, Wnt8 is required for the clearing of Dl and h expression in the posterior and that this is necessary for repression of twi, activation of cad, and establishment of the growth zone (McGregor, 2008).

The involvement of Wnt8, Delta/Notch signaling, and cad in the posterior development of other arthropods has also been directly demonstrated by functional analysis or inferred from expression patterns, and in vertebrates, Wnt3a and Wnt8 probably act upstream of Delta/Notch and cad during somitogenesis. Taken together, this suggests that a regulatory genetic network for posterior specification including Wnt8, Delta/Notch signaling, and cad could have been present in the last common ancestor of protostomes and deuterostomes, but has subsequently been modified in some lineages. For example, in Drosophila, Delta/Notch signaling is not involved in segmentation, and although the Drosophila Wnt8 ortholog, WntD, is required for D-V patterning, it is not involved in posterior development. Segments arise almost simultaneously in Drosophila, rather than sequentially from a growth zone, so this may suggest that the role of Wnt8 in posterior development was not required for this mode of development and therefore was lost during the evolution of these insects (McGregor, 2008).

These results suggest that Wnt8 regulates formation of the posterior growth zone and then maintains a pool of undifferentiated cells in this tissue required for development of the opisthosoma. Wnt signaling thus regulates the establishment and maintenance of an undifferentiated pool of posterior cells in both vertebrates and spiders and in fact the same Wnt ligand, Wnt8, is used in both phyla. Therefore, Wnt8 could be part of an ancient genetic regulatory network, also including Dl, Notch, h, and cad, that was used for posterior specification in the last common ancestor of deuterostomes and protostomes (McGregor, 2008).

Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors

Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. While the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. This study now shows that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete, and Odd-paired expression (see Comparison of long-germ and short-germ segmentation). In Drosophila these transcription factors are expressed like simple timers within the blastoderm, while in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. It is proposed that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis it was demonstrated that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports (Clark, 2018).

This study has found that segment patterning in both Drosophila and Tribolium occurs within a conserved framework of sequential Caudal, Dichaete and Odd-paired expression. In the case of Opa, there is also evidence for conserved function. However, although the sequence itself is conserved between the two insects, its spatiotemporal deployment across the embryo is divergent. In Drosophila, the factors are expressed ubiquitously within the main trunk, and each turns on or off almost simultaneously, correlating with the temporal progression of a near simultaneous segmentation process. In Tribolium, their expression domains are staggered in space, with developmentally more advanced anterior regions always subjected to a 'later' regulatory signature than more-posterior tissue. These expression domains retract over the course of germband extension, correlating with the temporal progression of a sequential segmentation process built around a segmentation clock (Clark, 2018).

Pair-rule patterning involves several distinct phases of gene expression, each requiring specific regulatory logic. It is proposed that, in both long-germ and short-germ species, the whole process is orchestrated by a series of key regulators, expressed sequentially over time, three of which are the focus of this paper. By rewiring the regulatory connections between other genes, factors such as Dichaete and Opa allow a small set of pair-rule factors to carry out multiple different roles, each specific to a particular spatiotemporal regulatory context. This kind of control logic makes for a flexible, modular regulatory network, and may therefore turn out to be a hallmark of other complex patterning systems (Clark, 2018).

Having highlighted the significance of these 'timing factors' in this paper, the next steps will be to investigate their precise regulatory roles and modes of action. It will be interesting to dissect how genetic interactions with pair-rule factors are implemented at the molecular level. Dichaete is known to act both as a repressive co-factor and as a transcriptional activator; therefore, a number of different mechanisms are plausible. The Odd-paired protein is also likely to possess both these kinds of regulatory activities (Clark, 2018).

Given the phylogenetic distance between beetles and flies (separated by at least 300 million years), it is expected that the similarities seen between Drosophila and Tribolium segmentation are likely to hold true for other insects, and perhaps for many non-insect arthropods as well. It is proposed that these similarities, which argue for the homology of long-germ and short-germ segmentation processes, result from conserved roles of Cad, Dichaete and Opa in the temporal regulation of pair-rule and segment-polarity gene expression during segment patterning. This hypothesis can be tested by detailed comparative studies in various arthropod model organisms (Clark, 2018).

This study provides evidence that a segmentation role for Opa is conserved between Drosophila and Tribolium; clear segmentation phenotypes have also been found for Cad in Nasonia, and for Dichaete in Bombyx. However, as the Tc-opa experiments reveal, functional manipulations in short-germ insects will need to be designed carefully in order to bypass the early roles of these pleiotropic genes. For example, cad knockdowns cause severe axis truncations in many arthropods, whereas Dichaete knockdown in Tribolium yields mainly empty eggs (Clark, 2018).

It was previously thought that Tc-opa was not required for segmentation, and that the segmentation role of Opa may have been recently acquired, in the lineage leading to Drosophila. However, the current analysis reveals that Tc-opa is indeed a segmentation gene, and also has other important roles, including head patterning and blastoderm formation. Given that a similar developmental profile of opa expression is seen in the millipede Glomeris, and even in the onychophoran Euperipatoides, the segmentation role of Opa may actually be ancient (Clark, 2018).

Head phenotypes following Tc-opa RNAi were unexpected, but both the blastoderm expression pattern and cuticle phenotypes that were observed are strikingly similar to those reported for Tc-otd and Tc-ems (Tribolium orthologues of the Drosophila head 'gap' genes orthodenticle and empty spiracles), suggesting that the three genes function together in a gene network that controls early head patterning. This function of Tc-opa might be homologous to the head patterning role for Opa discovered in the spider Parasteatoda, where it interacts with both Otd and Hedgehog (Hh) expression. Opa/Zic is known to modulate Hh signalling, and a role for Hh in head patterning appears to be conserved across arthropods, including Tribolium (Clark, 2018).

Finally, Opa/Zic is also known to modulate Wnt signalling. In chordates, Zic expression tends to overlap with sites of Hh and/or Wnt signalling, suggesting that one of its key roles in development is to ensure cells respond appropriately to these signals. The expression domains of Tc-opa that were observed in Tribolium (e.g. in the head, in the SAZ and between parasegment boundaries) accord well with this idea (Clark, 2018).

Similar embryonic expression patterns of Cad, Dichaete and Opa orthologues are observed in other bilaterian clades, including vertebrates. Cdx genes are expressed in the posterior of vertebrate embryos, where they play crucial roles in axial extension and Hox gene regulation. Sox2 (a Dichaete orthologue) has conserved expression in the nervous system, but is also expressed in a posterior domain, where it is a key determinant of neuromesodermal progenitor (posterior stem cell) fate. Finally, Zic2 and Zic3 (Opa orthologues) are expressed in presomitic mesoderm and nascent somites, and have been functionally implicated in somitogenesis and convergent extension. All three factors thus have important functions in posterior elongation, roles that may well be conserved across Bilateria (Clark, 2018).

In Tribolium, all three factors may be integrated into an ancient gene regulatory network downstream of posterior Wnt signalling, which generates their sequential expression and helps regulate posterior proliferation and/or differentiation. The mutually exclusive patterns of Tc-wg and Tc-Dichaete in the posterior germband are particularly suggestive: Wnt signalling and Sox gene expression are known to interact in many developmental contexts and these interactions may form parts of temporal cascades) (Clark, 2018).

The following outline is suggested as a plausible scenario for the evolution of arthropod segmentation; In non-segmented bilaterian ancestors of the arthropods, Cad, Dichaete and Opa were expressed broadly similarly to how they are expressed in Tribolium today, mediating conserved roles in posterior elongation, while gap and pair-rule genes may have had functions in the nervous system. At some point, segmentation genes came under the regulatory control of these factors, which provided a pre-existing source of spatiotemporal information in the developing embryo. Pair-rule genes began oscillating in the posterior, perhaps under the control of Cad and/or Dichaete, while the posteriorly retracting expression boundaries of the timing factors provided smooth wavefronts that effectively translated these oscillations into periodic patterning of the AP axis, analogous to the roles of the opposing retinoic acid and FGF gradients in vertebrate somitogenesis. Much later, in certain lineages of holometabolous insects, the transition to long-germ segmentation occurred. This would have involved two main modifications of the short-germ segmentation process: (1) changes to the expression of the timing factors, away from the situation seen in Tribolium, and towards the situation seen in Drosophila, causing a heterochronic shift in the deployment of the segmentation machinery from SAZ to blastoderm; and (2) recruitment of gap genes to pattern pair-rule stripes, via the ad hoc evolution of stripe-specific elements (Clark, 2018).

The appeal of this model is that the co-option of existing developmental features at each stage reduces the number of regulatory changes required to evolve de novo, facilitating the evolutionary process. In this scenario, arthropod segmentation would not be homologous to segmentation in other phyla, but would probably have been fashioned from common parts (Clark, 2018).

Caudal homologs in C. elegans

The early asymmetric cleavages of C. elegans embryos produce blastomeres with distinct developmental potentials. The caudal-like homeodomain protein PAL-1 is required to specify the somatic identity of one posterior blastomere in the 4 cell embryo. pal-1 activity is sequentially restricted to this blastomere. Initially, at the 4 cell stage, it is translated only in the two posterior blastomeres. Its function is then restricted to one of these blastomeres. This second targeting step is dependent on the activities of the posteriorly localized SKN-1 and asymmetrically segregated PIE-1 proteins. It is proposed that the segregation of PIE-1, combined with the temporal decay of SKN-1, targets pal-1 activity to this posterior lineage, thus coupling the regulation of this conserved posterior patterning gene to asymmetric cell cleavages (Hunter, 1997).

In Caenorhabditis elegans males, a row of epidermal precursor cells called seam cells generates a pattern of cuticular alae in anterior body regions and neural sensilla (called rays) in the posterior. The Abdominal-B homolog mab-5 is required for two posterior seam cells, V5 and V6, to generate rays. The V5 lineage generates one ray and the V6 lineage generates five rays. In mab-5 mutant males, V5 and V6 do not generate sensory ray lineages but instead generate lineages that lead to alae, cuticular ridges that extend along the two sides of the animal. Alae are normally generated by the V1-V4 cells only. Two independent regulatory pathways can activate mab-5 expression in the V cells. (1) The caudal homolog pal-1 turns on mab5 in V6 during embryogenesis. (2) A Wnt signaling pathway is capable of activating mab-5 in the V cells during postembryonic development, however, during normal development Wnt signaling is inhibited by signals from neighboring V cells. The inhibition of this Wnt signaling pathway by lateral signals between the V cells limits the number of rays in the animal and also determines the position of the boundary between alae and rays (Hunter, 1999).

The Caenorhabditis elegans gene pal-1, a homolog of Drosophila caudal, is required maternally for blastomere specification in the early embryo and postembryonically for tail development in males. Both pal-1 in C. elegans and caudal in Drosophila are transcribed maternally as well as in the embryo. However, whereas either transcription mode alone is sufficient for normal embryonic development in Drosophila, both modes of expression appear to be required in C. elegans. Zygotic transcription of pal-1 is required for posterior patterning during later embryogenesis. Embryos homozygous for strong loss-of-function mutations arrest as nonviable L1 larvae with gross posterior defects. PAL-1 protein produced from zygotic transcripts is expressed dynamically during gastrulation and morphogenesis in specific cells of all major lineages except the germ line. Most expressing cells are undergoing cell movements or forming midline structures or both. Mutant embryos exhibit defects involving most of the expressing cells. Aberrant early cell positions are observed in posterior hypodermis, both in the C-lineage cells that express pal-1 and in the neighboring hypodermal seam cell precursors, which do not, as well as in posterior muscle derived from the C and D lineages. Defects in late gastrulation, ventral hypodermal enclosure, and formation of the rectum result from failures of cell movements of ABp and MS descendants. Limited mosaic analysis supports the view that most of the required pal-1 functions are cell autonomous (Edgar, 2001).

Although PAL-1 seems likely to have several regulatory targets, there is evidence at present for only two. One is the Hox gene mab-5, which is activated in the ABa-derived V6 cells during midembryogenesis. This activation requires pal-1 function, and recent evidence indicates that pal-1 may directly activate mab-5 transcription in the V6 cells. mab-5 could also require pal-1 function for activation in the sex myoblasts descended from the M cell. Regulation by pal-1 of another Hox gene, the posterior-group gene egl-5, would be consistent with the expression patterns of pal-1 and egl-5 in the late embryo, but at present there is no direct evidence for such regulation. Another target for which there is evidence is vab-7, a homolog of the Drosophila pair-rule gene even-skipped. Lack of zygotic vab-7 function causes embryonic defects in embryonic C-lineage hypodermal and muscle patterning, but the embryos are viable. vab-7 is expressed in descendants of the four Cpxx cells, which generate the posterior set of right and left C-derived hypodermal and muscle cells. This expression has been shown to require maternal but not zygotic pal-1 expression. However, the capacity to respond to ectopically expressed pal-1 extends until morphogenesis, and in cosmid-rescued lines, in which maternal pal-1 expression is not detected, zygotically expressed pal-1 may activate vab-7. Moreover, pal-1 mutations and vab-7 mutations cause very similar C-lineage phenotypes, suggesting that pal-1 may function to maintain vab-7 expression in the late embryo (Edgar, 2001 and references therein).

Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. The translational control of the C. elegans caudal homolog, pal-1, has been investigated and it has been found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through a minimal pal-1 3' UTR element to repress pal-1 translation in the distal germline. Data is provided suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, GLD-1 is shown to repress the distal germline expression of the KH domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline (Mootz, 2004).

GLD-1 is homologous to a sub-family of KH domain proteins known as the GSG or STAR domain family, whose members include the evolutionarily conserved Quaking protein, mammalian Sam68 and SF1 and Drosophila How. The ~200 amino acid STAR domain consists of an enlarged KH RNA-binding domain (maxi-KH domain) flanked by conserved residues on both sides. While the functions of these family members are not well understood, they have been implicated in various aspects of RNA metabolism, including mRNA splicing, nuclear export and translation. Other than GLD-1, only one family member, the mouse Quaking I isoform 6, has thus far been implicated as a translational regulator, and this is based on its ability to repress tra-2 expression when expressed in C. elegans (Mootz, 2004 and references therein).

Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multipotent C blastomere lineage in the C. elegans embryo. To identify PAL-1 regulatory target genes, microarrays were used to compare transcript abundance in wild-type embryos with mutant embryos lacking a C blastomere and to mutant embryos with extra C blastomeres. pal-1-dependent C-lineage expression was verified for select candidate target genes by reporter gene analysis, though many of the target genes are expressed in additional lineages as well. The set of validated target genes includes 12 transcription factors, an uncharacterized wingless ligand and five uncharacterized genes. Phenotypic analysis demonstrates that the identified PAL-1 target genes affect specification, differentiation and morphogenesis of C-lineage cells. In particular, cell fate-specific genes (or tissue identity genes) and a posterior HOX gene are activated in lineage-specific fashion. Transcription of targets is initiated in four temporal phases, which together with their spatial expression patterns lead to a model of the regulatory network specified by PAL-1 (Baugh, 2005).

A model is proposed for the structure of the network specified by PAL-1 based only on temporal and spatial expression patterns in wild type. The rationale behind the model is simple: genes activated in one cell cycle affect the expression of genes expressed in the next cell cycle. This premise is supported by both functional analysis of the endodermal network and global analysis of expression dynamics. Furthermore, whereas zygotic pal-1 transcripts are first detected at the 2C-cell stage in Ca and Cp (phase I), loss of zygotic pal-1 function results in a detectable mutant phenotype in their daughters at the 4C-cell stag. In addition, protein for the phase II gene elt-1 is first detected at the end of the 4C-cell stage, and transcription of its confirmed phase III target elt-3 begins in the 8C-cell stage (Baugh, 2005).

That ectopic PAL-1 activity in early blastomeres is sufficient to cause complete transformation of one lineage into another indicates that the regulatory network specified by PAL-1 is modular or self-contained. After maternal PAL-1 specifies the C lineage, embryonically expressed PAL-1 is required for C-lineage development. It is therefore hypothesized that PAL-1 continuously regulates target genes during patterning of the C lineage, as opposed to simply initiating a transcriptional cascade. Although it is not known how far into development PAL-1 function is required, phenotypically mutant pal-1 mosaic animals were recovered corresponding to loss of pal-1 in one Cxx cell at the 4C-cell stage and PAL-1 expression is detectable in the C lineage until the 16C-cell stage, leaving open the possibility that PAL-1 directly activates each of the target genes identified in this study. Combinatorial control of gene expression, where early targets regulate late targets in combination with PAL-1, offers one possible mechanism for the timing of gene expression within this modular network (Baugh, 2005).

There must be additional regulation not predicted by this model. Genes that are not PAL-1 targets are likely to participate in transcriptional regulation and patterning of the C lineage. For example, the Homothorax ortholog unc-62 and the Extradenticle homologs ceh-20 and ceh-40 have superficially similar phenotypes to nob-1 and pal-1, suggesting that these co-factor homeodomain proteins interact with and modify the function of PAL-1 and NOB-1 (Baugh, 2005).

Likewise, the Tcf/Lef factor pop-1 is thought to mediate cell-fate decisions associated with every cell division on the AP axis of the early embryo, and, as has been shown for development of the E lineage, POP-1 is expected to contribute to patterning of PAL-1 target expression, in particular where targets are expressed in only the anterior or posterior daughters following a round of C cell divisions (e.g., hlh-1, elt-1 and vab-7). Repression is completely ignored in the current model, but is probably crucial for patterning, as indicated by the fact that very few targets are expressed in all PAL-1-expressing cells. So there may also be genes repressed by PAL-1. The model does not allow for genes of the same temporal phase to regulate each other, though it is likely that there is mutual repression between genes specifying muscle and epidermis, leading to insulation of the two states. In addition, genes of the same temporal phase expressed in the same cells may activate the expression of one another, and multiple auto-regulatory positive feedbacks are expected in addition to the one demonstrated for pal-1. It will be interesting to compare the structures of different developmental regulatory networks in an effort to understand better how different topological motifs contribute to the functional properties of the regulatory network and ultimately how network structure relates to body plan (Baugh, 2005).

Previous work in C. elegans has shown that posterior embryonic bodywall muscle lineages are regulated through a genetically defined transcriptional cascade that includes PAL-1/Caudal-mediated activation of muscle-specific transcription factors, including HLH-1/MRF and UNC-120/SRF, which together orchestrate specification and differentiation. Using chromatin immunoprecipitation (ChIP) in embryos, direct binding of PAL-1 in vivo to an hlh-1 enhancer element has been detected. Through mutational analysis of the evolutionarily conserved sequences within this enhancer, two cis-acting elements and their associated transacting factors (PAL-1 and HLH-1) were identified that are crucial for the temporal-spatial expression of hlh-1 and proper myogenesis. The data demonstrate that hlh-1 is indeed a direct target of PAL-1 in the posterior embryonic C. elegans muscle lineages, defining a novel in vivo binding site for this crucial developmental regulator. The same enhancer element is also a target of HLH-1 positive auto regulation, underlying (at least in part) the sustained high levels of CeMyoD in bodywall muscle throughout development. Together, these results provide a molecular framework for the gene regulatory network activating the muscle module during embryogenesis (Lei, 2009).

Caudal homologs in other invertebrates

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

Although the tail is one of the major characteristics of animals of the phylum Chordata, evolutionary aspects of the molecular mechanisms involved in its formation are not clear. To obtain insights into these issues, the caudal gene of an ascidian, one of the lower animal groups among chordates, has been isolated and investigated. Ascidian caudal is expressed from the midgastrula stage onward in the lateral walls of the posterior neural tube cell lineage and also in the posterior epidermal cells from the neurula stage. Thus, ascidian caudal expression is restricted to the ectoderm of a tail-forming region throughout embryogenesis. Vertebrate caudal genes are expressed in all three germ layers. Suppression of caudal function by an antisense oligonucleotide or a dominant negative construct causes inhibition of the cell movement required for tail formation. Overexpression of wild-type caudal mRNA in an ascidian animal cap, an animal half explant prepared at the eight-cell stage, causes elongation of the cap. Furthermore, Xenopus embryos injected with dominant negative ascidian caudal exhibit defects in elongation, suggesting a conserved caudal function among chordates. In ascidian embryos, intercalating cell movement along the posterior dorsal midline occurs within neuroectoderm and notochord upon tail elongation. Furthermore, it has been reported that the change of cell shape from columnar to wedge-shaped is first observed in the cells at the lateral borders of the neural plate. The initial expression of ascidian caudal coincides with this cell shape change, which may be responsible for the subsequent dorsal convergence of the ectodermal cell sheet. These results indicate that caudal function is required for chordate tail formation and may play a key role in its evolution (Katsuyama, 1999).

This study reports the characterization of the ortholog of the Xenopus XlHbox8 ParaHox gene from the sea urchin Strongylocentrotus purpuratus, SpLox. It is expressed during embryogenesis, first appearing at late gastrulation in the posterior-most region of the endodermal tube, becoming progressively restricted to the constriction between the mid- and hindgut. The physiological effects of the absence of the activity of this gene have been analyzed through knockdown experiments using gene-specific morpholino antisense oligonucleotides. Blocking the translation of the SpLox mRNA reduces the capacity of the digestive tract to process food, as well as eliminating the morphological constriction normally present between the mid- and hindgut. Genetic interactions of the SpLox gene are revealed by the analysis of the expression of a set of genes involved in endoderm specification. Two such interactions have been analyzed in more detail: one involving the midgut marker gene Endo16, and another involving the other endodermally expressed ParaHox gene, SpCdx. SpLox is able to bind Endo16 cis-regulatory DNA, suggesting direct repression of Endo16 expression in presumptive hindgut territories. More significantly, this study provides the first evidence of interaction between ParaHox genes in establishing hindgut identity, and presents a model of gene regulation involving a negative-feedback loop (Cole, 2009).

Hox genes are known for their coordinated control of patterning of body regions, for example rhombomere patterning under the control of Hoxb genes. Little is known, however, about whether ParaHox genes also function in a coordinated manner to establish boundaries between adjacent territories. The results reveal that the silencing of an anteriorly expressed ParaHox gene (SpLox) leads to the loss of a posterior identity (hindgut), accompanied by the downregulation of a second ParaHox gene (SpCdx) that is normally expressed in the missing territory. These data suggest coordination between these two ParaHox genes at a gene regulatory level (Cole, 2009).

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides leads to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure -- a more severe phenotype than with inhibition of each Wnt alone -- indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediate Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not Bmps or Cyclops, is strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos display complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression is down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling is required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish (Shimizu, 2004).

Cdx-Hox code controls competence for responding to Fgfs and retinoic acid in zebrafish neural tissue

Fibroblast growth factor (Fgf) and retinoic acid (RA) signals control the formation and anteroposterior patterning of posterior hindbrain. They are also involved in development processes in other regions of the embryo. Therefore, responsiveness to Fgf and RA signals must be controlled in a context-dependent manner. Inhibiting the caudal-related genes cdx1a and cdx4 in zebrafish embryos caused ectopic expression of genes that are normally expressed in the posterior hindbrain and anterior spinal cord, and ectopic formation of the hindbrain motor and commissure neurons in the posteriormost neural tissue. Combinational marker analyses suggest mirror-image duplication in the Cdx1a/4-defective embryos, and cell transplantation analysis further revealed that Cdx1a and Cdx4 repress a posterior hindbrain-specific gene expression cell-autonomously in the posterior neural tissue. Expression of fgfs and retinaldehyde dehydrogenase 2 suggested that in the Cdx1a/4-defective embryos, the Fgf and RA signaling activities overlap in the posterior body and display opposing gradients, compared with those in the hindbrain region. Fgf and RA signals were required for ectopic expression. Expression of the posterior hox genes hoxb7a, hoxa9a or hoxb9a, which function downstream of Cdx1a/4, or activator fusion genes of hoxa9a or hoxb9a (VP16-hoxa9a, VP16-hoxb9a) suppressed this loss-of-function phenotype. These data suggest that Cdx suppresses the posterior hindbrain fate through regulation of the posterior hox genes; the posterior Hox proteins function as transcriptional activators and indirectly repress the ectopic expression of the posterior hindbrain genes in the posterior neural tissue. These results indicate that the Cdx-Hox code modifies tissue competence to respond to Fgfs and RA in neural tissue (Shumizu, 2006).

cdx1b regulates expression of downstream factors of Nodal signaling during early endoderm formation

A zebrafish caudal-related homeobox (cdx1b) gene shares syntenic conservation with both human and mouse Cdx1. Zebrafish cdx1b transcripts are maternally deposited. cdx1b is uniformly expressed in both epiblast and hypoblast cells from late gastrulation to the 1-2s stages and can be identified in the retinas, brain and somites during 18-22 hpf stages. After 28 hours of development, cdx1b is exclusively expressed in the developing intestine. Both antisense morpholino oligonucleotide-mediated knockdown and overexpression experiments were conducted to analyze cdx1b function. Hypoplastic development of the liver and pancreas and intestinal abnormalities were observed in 96 hpf cdx1b morphants. In 85% epiboly cdx1b morphants, twofold decreases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors were identified. Furthermore, ectopic cdx1b expression caused substantial increases in the respective numbers of gata5-, cas-, foxa2- and sox17-expressing endodermal precursors and altered their distribution patterns in 85% epiboly injected embryos. Conserved Cdx1-binding motifs were identified in both gata5 and foxa2 genes by interspecific sequence comparisons. Cdx1b can bind to the Cdx1-binding motif located in intron 1 of the foxa2 gene based on an electrophoretic mobility shift assay. Co-injection of either zebrafish or mouse foxa2 mRNA with the cdx1b MO rescued the expression domains of ceruloplasmin in the liver of 53 hpf injected embryos. These results indicate that zebrafish cdx1b regulates foxa2 expression and may also modulate gata5 expression, thus affecting early endoderm formation. This study underscores a novel role of zebrafish cdx1b in the development of different digestive organs compared with its mammalian homologs (Cheng, 2008).

Transcription factors involved in retinogenesis are co-opted by the circadian clock following photoreceptor differentiation

The circadian clock is known to regulate a wide range of physiological and cellular processes, yet remarkably little is known about its role during embryo development. Zebrafish offer a unique opportunity to explore this issue, not only because a great deal is known about key developmental events in this species, but also because the clock starts on the very first day of development. This study identified numerous rhythmic genes in zebrafish larvae, including the key transcriptional regulators neurod and cdx1b, which are involved in neuronal and intestinal differentiation, respectively. Rhythmic expression of neurod and several additional transcription factors was only observed in the developing retina. Surprisingly, these rhythms in expression commenced at a stage of development after these transcription factors are known to have played their essential role in photoreceptor differentiation. Furthermore, this circadian regulation was maintained in adult retina. Thus, once mature photoreceptors are formed, multiple retinal transcription factors fall under circadian clock control, at which point they appear to play a new and important role in regulating rhythmic elements in the phototransduction pathway (Laranjeiro, 2014).

Xenopus Caudal homologs

In vertebrates, the caudal genes begin their expression during gastrulation and they take up a posterior position. By injecting sense and antisense RNA of the Xenopus caudal gene Xcad-2, a number of regulatory interactions were studied among homeobox genes along the anterior-posterior axis. Initially, the Xcad-2 and Otx-2 genes are mutually repressed; by late gastrulation, they mark the posterior- or anterior-most domains of the embryo, respectively. During late gastrulation and neurulation, Xcad-2 plays an additional regulatory function in relation to the Hox genes. Hox genes normally expressed anteriorly are repressed by Xcad-2 overexpression, while those normally expressed posteriorly exhibit more anterior expression. The results show that the caudal genes are part of a posterior determining network that during early gastrulation functions in the subdivision of the embryo into anterior head and trunk domains. Later in gastrulation and neurulation these genes play a role in the patterning of the trunk region (Epstein, 1997).

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

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

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

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

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation, exhibiting a dorsoventral asymmetry in their transcription domains. At mid-gastrulation the ventral preference becomes stronger and the expression of caudal genes takes up a posterior localization, which will be maintained until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes reveals a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis (Pillemer, 1998a).

Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with the exception of a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 efficiently represses during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 is also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor, which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 can still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act either downstream of BMP-4 signaling or independent of it. Xcad-2 can also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 efficiently reverses the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program, which acts during early gastrula stages, and demonstrate that Xcad-2 can execute its ventralizing function in the absence of BMP signaling (Pillemer, 1998b).

A dorsal-ventral difference in the specification of mesoderm in vivo has been discovered by examining the effect of the dominant-negative FGF receptor on a new member of the Xenopus caudal gene family, Xcad-3. Xcad-3 is expressed throughout the marginal zone during the gastrula stages and serves as a useful marker for events occurring within the mesoderm. Disruption of the FGF signaling pathway by the dominant-negative FGF receptor, disrupts the Xcad-3 expression pattern, eliminating expression preferentially from the dorsal regions of the embryo. The expression of the Xenopus brachyury homolog, Xbra, is more readily eliminated from the dorsal than the ventral region of the embryo by the dominant-negative FGF receptor, indicating that the observed dorsal-ventral differences are not unique to Xcad-3. These results demonstrate the importance of regional effects on FGF-mediated induction in vivo and suggest that FGF-dependent expression of mesodermal genes depends upon the localization of other factors which establish dorsal-ventral differences within the embryo (Northrop, 1994).

Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. Fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. A partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. Does the regional localization of bone morphogenetic protein-4 (BMP-4) explain why both MAPKKD and delta FGFR eliminate the dorsal but not the ventral expression of Xcad-3 and Xbra? Ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR, and expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction (Northrop, 1995).

The biological activities of the Xenopus caudal (Cdx) family member Xcad3 have been examined. A series of domain-swapping experiments demonstrate that the N-terminus of Xcad3 is necessary for it to activate Hox gene expression and that this function can be replaced by the activation domain from the viral protein VP16. Injection of 50 pg or more of Xcad3 mRNA leads to activation of HoxC6 and HoxA7, which are normally expressed in both the mesoderm and neuroectoderm, and HoxB7 and HoxB9, which are expressed predominantly in the neuroectoderm. Xcad3 does not upregulate the expression of the general mesodermal marker Xbra, indicating that it does not induce the formation of ectopic mesoderm. Experiments using an Xcad3 repressor mutant (XcadEn-R), which potently blocks the activity of wild-type Xcad3, are reported. Overexpression of XcadEn-R in embryos inhibits the activation of the same subset of Hox genes that are activated by wild-type Xcad3 and leads to a dramatic disruption of posterior development. Xcad3 is shown to be an immediate early target of the FGF signaling pathway: Xcad3 and FGF both posteriorize anterior neural tissue in similar ways. Xcad3 is also required for the activation of Hox genes by FGFs. These data provide strong evidence that Xcad3 is required for normal posterior development and that it regulates the expression of the Hox genes downstream of FGF signaling (Isaacs,1998).

Is there any evidence that the role for caudal-related genes in regulating Hox genes is conserved outside the vertebrates? In Drosophila, homologs of the vertebrate Hox genes (Hom-C complex) are considered to be largely epistatic to caudal, but there is now evidence suggesting that some aspects of expression from the HOM-C complex member Abdominal-B are regulated by caudal. Interestingly, ectopic anterior expression of caudal results in a disruption of head development, part of which appears to be due to the suppression of expression by the deformed gene, which is also a member of the Drosophila Hom-C complex. Certain parallels can be seen with the posterior-promoting/anterior-suppressing activity of the Xenopus Xcad proteins. With regard to the role of caudal-related genes in other invertebrates, it has been suggested that pal-1 is involved in regulation of the C.elegans Abd-B homolog mab-5 (Isaacs, 1998).

The KH domain protein MEX-3 is central to the temporal and spatial control of PAL-1 expression in the C. elegans early embryo. PAL-1 is a Caudal-like homeodomain protein that is required to specify the fate of posterior blastomeres. While pal-1 mRNA is present throughout the oocyte and early embryo, PAL-1 protein is expressed only in posterior blastomeres, starting at the four-cell stage. To better understand how PAL-1 expression is regulated temporally and spatially, MEX-3 interacting proteins (MIPs) have been identified and two that are required for the patterning of PAL-1 expression are described in detail. RNA interference of MEX-6, a CCCH zinc-finger protein, or SPN-4, an RNA recognition motif protein, causes PAL-1 to be expressed in all four blastomeres starting at the four-cell stage. Genetic analysis of the interactions between these mip genes and the par genes, which provide polarity information in the early embryo, defines convergent genetic pathways that regulate MEX-3 stability and activity to control the spatial pattern of PAL-1 expression. These experiments suggest that par-1 and par-4 (see Drosophila Lkb1) affect distinct processes. par-1 is required for many aspects of embryonic polarity, including the restriction of MEX-3 and MEX-6 activity to the anterior blastomeres. PAL-1 is not expressed in par-1 mutants, because MEX-3 and MEX-6 remain active in the posterior blastomeres. The role of par-4 is less well understood. This analysis suggests that par-4 is required to inactivate MEX-3 at the four-cell stage. Thus, PAL-1 is not expressed in par-4 mutants because MEX-3 remains active in all blastomeres. It is proposed that MEX-6 and SPN-4 act with MEX-3 to translate the temporal and spatial information provided by the early acting par genes into the asymmetric expression of the cell fate determinant PAL-1 (Huang, 2002).

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes, among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homolog, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, it has been demonstrated that during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, it has been shown that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. It is therefore suggested that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as midgastrulation (Tour, 2002).

The organizer in vertebrate embryos is responsible for the formation of the primary body axis. In amphibian embryos, the organizer forms in the dorsal marginal zone (prospective dorsal mesoderm) at a location determined by the point of sperm entry. Using inducible versions of axis-inducing proteins, it has been shown that, irrespective of the mode of secondary axis induction, organizer formation in the ventral marginal zone is temporally restricted from the midblastula transition to the onset of gastrulation. The competence of marginal zone cells to respond to organizer-inducing signals is under temporal control, one of the regulators being the homeobox transcription factor Xcad2. Overexpression of Xcad2 restricts the temporal competence for axis induction, whereas partial loss of function expands this competence, supporting the suggestion. It is proposed Xcad2 competes with putative axis-inducing signals within the marginal zone to prevent expression of organizer-specific genes. Elimination of endogenous Xcad2 allows for the activation of organizer genes beyond the normal competence window during early/mid-gastrulation. It is concluded that Xcad2, through its early expression in the ventrolateral marginal zone, terminates the competence of this embryonic region to respond to organizer-inducing signals by preventing the activation of organizer-specific genes (Levy, 2002).

Mammalian Caudal homologs

Continued: Evolutionary Homologs part 2/2

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

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