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Integumental color changes and eye pigment movements in crustaceans are regulated by pigmentary-effector hormones. The identified hormones include: an octapeptide RPCH (red pigment-concentrating hormone) and several forms of octadecapeptide PDH (pigment-dispersing hormone: α-PDH, β-PDH). RPCH-related peptides (AKHs, adipokinetic hormones) and PDH-related peptides (PDFs, pigment-dispersing factors) occur in insects, and are recognized as members of AKH/RPCH and PDH/PDF peptide families. The domain for mature peptide is located between the signal peptide and precursor-related peptide in AKH/RPCH precursors, and at the C-terminal end in the PDH/PDF precursors. The precursor-related (associated) peptides in RPCH and PDH precursors in Crustacea show little or no similarity to corresponding domains of AKH and PDF precursors in insects. Although the functions of precursor-related peptides are unknown, the mature peptides are shown to serve diverse functions. RPCH's actions in crustaceans include: pigment concentration in one or more types of chromatophores, dark-adaptational screening pigment movement in distal eye pigment cells, increase of retinal sensitivity, and neuromodulation. The related AKHs largely influence metabolism in insects, although they serve additional functions. PDHs trigger pigment dispersion in chromatophores and induce light-adaptational screening pigment movements in extraretinular eye pigment cells. The related PDFs appear to serve as a transmitter of circadian signals in the regulation of biological rhythms in insects. Evolutionary relationships among the PDH/PDF peptides and directions for future research are discussed.
I present an overview of recent research on the isolation and characterization of members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. Members of this arthropod-specific family include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). There are two subfamilies of this neuropeptide group, based upon the presence or absence of a C-terminal CHH precursor-related peptide. There are also sequence motif differences between these subfamilies. Most of the peptides comprising this neuropeptide family are synthesized and released by the eyestalk X-organ/sinus gland complex. Recent experiments have demonstrated the presence of extra-eyestalk cells that produce CHH and the assignment of additional functions to this hormone family.
This paper summarizes recent work on various aspects of hormonal control of regeneration in the crustacean, Uca pugilator. Hormonal control in this crab is effected by means of the crustactean steroid hormones, the ecdysteroids. New evidence is presented supporting a role for the retinoid hormones, all-trans retinoic acid and 9-cis retinoic acid, in the control of regeneration in these animals. The possible role of fibroblast growth factors in organization of the limb blastema is explored and the similarities between vertebrate and invertebrate control of regeneration are discussed.
Molting and regeneration of lost appendages are tightly-coupled, hormonally-regulated processes in decapod crustaceans. Precocious molts are induced by eyestalk ablation, which reduces circulating molt-inhibiting hormone (MIH) and results in an immediate rise in hemolymph ecdysteroids. Precocious molts are also induced by autotomy of 5–8 walking legs; adult land crabs (Gecarcinus lateralis) molt 6–8 wk after multiple leg autotomy (MLA). Autotomy of one or more of the 1° limb buds (LBs) that form after MLA before a critical period interrupts proecdysis until 2° LBs re-regenerate and grow to the approximate size of those lost. Based on these observations, Skinner proposed that limb buds produce two factors that control proecdysial events. Limb Autotomy Factor–Anecdysis (LAFan), produced by 1° LBs when at least five legs are autotomized, stimulates anecdysial animals to enter proecdysis. Limb Autotomy Factor–Proecdysis (LAFpro), produced by 2° LBs in premolt animals when at least one 1° LB is autotomized, inhibits proecdysial processes. Initial characterizations suggest that LAFpro is a MIH-like polypeptide that inhibits the synthesis and secretion of ecdysteroid by the Y-organs.
In crustaceans, secretion of ecdysteroid molting hormones by Y-organs is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by the X-organ/sinus gland complex of the eyestalks. The current review considers recent research on MIH, with a primary focus on MIH of brachyurans (crabs). New data on the production of recombinant MIH (rMIH) are also included. Available data indicate the MIH gene of brachyurans encodes a 113 amino acid prohormone composed of a 35 residue signal peptide and a 78 residue mature MIH. The primary structure of MIH is highly conserved among brachyurans. The MIH transcript is detectable in eyestalk neural ganglia throughout the molt cycle of the blue crab, Callinectes sapidus. Stage-specific changes in the abundance of MIH mRNA in C. sapidus eyestalks are generally consistent with the hypothesis that MIH negatively regulates ecdysteroid production during the molt cycle. MIH transcripts have also been detected in the brain of two species. Recombinant MIH was produced using prokaryotic (pET vector/Escherichia coli) and eukaryotic (baculovirus/insect cells) expression systems. Recombinant MIH produced in E. coli was of the predicted size and was MIH immunoreactive; it did not have MIH bioactivity. Polyclonal antisera raised against the prokaryotically expressed rMIH bound specifically to neurosecretory cells in the X-organ, their associated axons, and axon terminals in the sinus gland. Recombinant MIH expressed using the baculovirus system was of the predicted size, was MIH immunoreactive, and inhibited ecdysteroid production by Y-organs in vitro.
The Y-organs of crustaceans secrete ecdysteroids (molting hormones) and are regulated (negatively) by a neurosecretory peptide, molt-inhibiting hormone (MIH). Signaling path(s) in Y-organs were explored that connect MIH receptors ultimately with suppression of receptor number for the uptake of cholesterol (ecdysteroid precursor) and of gene expression of steroidogenic enzymes. Experiments were conducted in vitro with Y-organs of crabs (Cancer antennarius, Menippe mercenaria) and crayfishes (Orconectes sp.). It was confirmed in all species that steroidogenesis occurs in the absence of external calcium (Ca), but increases to a maximum as Ca is increased to 1 to 10 mM and is substantially inhibited at higher Ca concentrations. MIH does not require external Ca for inhibitory action, but inhibition is eliminated by high Caconcentrations. Several experimental approaches failed to find evidence of phospholipase C activation, turnover of inositol triphosphate or diacylglycerol generation connected with steroidogenesis. Unbinding or chelation of intracellular Ca with thapsigargin or TMB-8, respectively both caused dose-dependent inhibition of ecdysteroid output. Blockade of Ca channels with verapamil, nifedipine or nicardipine also inhibited steroidogenesis; highest doses inhibited profoundly to below Ca-free basal levels. Inhibition also was obtained with all doses of the Ca channel agonist/antagonist (−) BAY K 8644 in crabs, but in crayfishes lower doses were stimulatory. However, if the crayfish cells were depolarized, allowing greater Ca influx, the previously stimulatory doses of BAY K 8644 became inhibitory. Y-organ protein kinase C (PKC) is Ca-sensitive. Activation of PKC was uniformly stimulatory in crabs, but inhibitory in crayfishes. Cytochalasin D, which disrupts the actin cytoskeleton, and which causes moderate Ca influx, stimulated hormone formation. These results are interpreted to indicate a regulatory role for Ca in ecdysteroidogenesis, involving a local, submembrane circulation of Ca through ion channels and Ca pumps and interaction with PKC in phosphorylating key proteins. An optimal local Ca environment fostering hormone synthesis is evident since too little or too much Ca is inhibitory.
Methyl farnesoate (MF) had no effect on ecdysone production in crab or crayfish Y-organs in 24-hr incubations with MF at 100 pM to 10 μM.
The crustacean mandibular organ (MO) produces methyl farnesoate (MF), a juvenile hormone-related compound thought to have roles in crustacean reproduction and development. Therefore, the control of MF production by the MO has been of considerable interest. Current evidence indicates that the MO is negatively regulated by peptides present in the eyestalk (MO inhibiting factor, MO-IH). Several eyestalk neuropeptides have been identified that inhibit MF synthesis by MO incubated in vitro. The amino acid sequences of these MO-IH peptides are similar to peptides in the crustacean hyperglycemic hormone (CHH) family of neuropeptides. In addition, there appears to be a compound in the eyestalk that lowers hemolymph levels of MF in vivo but does not directly affect the MO in vitro. The inhibition of MF synthesis by eyestalk peptides involves the inhibition of farnesoic acid O-methyl transferase, the last enzyme in the MF biosynthetic pathway. The activity of this enzyme is affected by cyclic nucleotides, suggesting that these compounds may be involved in the signal transduction pathway mediating the effects of MO-IH.
Since the discovery that methyl farnesoate (MF), the unepoxidated form of the insect juvenile hormone (JHIII), is produced by mandibular organs of numerous crustaceans, extensive evidence has accumulated that this compound appears to perform similar functions in the Crustacea as JH performs in insects. A major function of MF appears to be in enhancing reproductive maturation. This was first shown by indirect experimentation with eyestalk ablation, which augmented MF production. Subsequently, direct treatments of several species of crustacea with MF showed that reproductive maturation was enhanced.
A second function of MF, similar to that of the JH of insects, is in the maintenance of juvenile morphology. This is especially true in the late larval transformations into juveniles, where MF plays an inhibitory role, as well as during the transformation of juveniles into adults. These results were inferred from eyestalk removal experiments. In the case of the larval-juvenile transition, inhibitory results were also obtained with MF by direct hormone treatments. However, the transition from very early larval stages, such as one nauplius stage proceeding to the next, which in many cases also involves morphogenetic changes, may be occurring in the presence of MF. Indeed, MF appears to be stimulatory to early postembryonic larval stages of Crustacea. Again, this function of MF in Crustacea appears to be similar to functions of JH in early postembryonic insects. However, it should be pointed out that there are many more “early” stages in Crustacea than there are in insects, and very few of these cases have been investigated.
When considering the animal kingdom and larval metamorphosis, the question may be raised whether there are other members of the JH family regulating metamorphosis and reproduction. One plausible example appears to be among certain annelids. The trochophores of Capitella respond to various juvenoids, but are most responsive, within one hour, to MF and eicosatrienoic acid. This latter compound is present also in adult annelids, where it has been named “Sperm Maturation Factor,” since it seems to function in the maturation of sperm in Arenicola. Therefore, eicosanoids perform in annelids two functions performed in insects by JHs.
In conclusion, it seems that there are morphogenesis promoting responses to JHs in early larval development in crustaceans, annelids, and possibly other forms, which differ from those MF effects in later larvae of Crustacea where MF retards morphogenesis. Such early responses as noted here have recently also been described for insects. Furthermore, it is clear that the polyunsaturated 8,11,14-eicosatrienoic and aracidonic acids seem to be juvenoids, and appear to function as such in annelids, and may also be functionally active in insects and crustaceans. It seems reasonable to conclude therefore that new and novel juvenoids exist, while others still await discovery.
Eyestalk neuroendocrine factors control specific yolk protein synthesis in the ovaries of the shrimp, Penaeus vannamei. A bioassay was developed to measure specific yolk protein synthesis in vitro. The eyestalk neuroendocrine complex may also produce a peptide capable of stimulation of yolk synthesis.
One of the major changes that occurs during the maturation of oocytes is the accumulation of yolk protein, or vitellin (Vn). To better understand how this process is regulated, we characterized the Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea). This Vn is a 322 kDa molecule composed of three subunits. Using purified Vn, we developed an anti-Vn antiserum and used it to characterize vitellogenin by Western blot analysis. The antiserum was also used in an ELISA to measure hemolymph levels of vitellogenin. Previous studies suggested the presence of vertebrate-type steroids might stimulate reproductive processes in decapod crustaceans. Treatment of sexually quiescent female shrimp with progesterone, hydroxyprogesterone, and estradiol did not increase hemolymph levels of yolk protein precursor. The absence of a response to these steroids may reflect the presence of other hormones (such as the gonad-inhibiting hormone) that prevent oocyte development. To examine the molecular basis for the regulation of vitellogenesis, ovarian and hepatopancreas expression cDNA libraries were screened using the anti-Vn antiserum. A 2.9 kilobase clone was isolated from both cDNA libraries suggesting that both tissues are sites of vitellogenin synthesis. These molecular tools should be useful for in vitro studies of vitellogenin synthesis.
The androgenic gland has been described in a variety of crustacean species—isopods, amphipods and decapods. It has been shown to play a role in the regulation of male differentiation and in the inhibition of female differentiation. Upon its application for endocrine manipulation, it inhibits female characteristics. Recently, the androgenic hormone from the isopod Armadillidium vulgare was purified and characterized on the basis of a morphological bioassay. The hormone is a glycosylated protein composed of two peptide chains connected each to the other by two disulfide bridges. The pro-hormone consists of the same two chains connected by a third peptide in a complex that resembles the insulin super family hormones. The study of the androgenic gland in decapods lags behind that in the isopods, and a decapod androgenic hormone has yet to be identified. In this review, five decapod species are described as models, in which the androgenic gland exerts morphological, anatomical, physiological and behavioral effects. These models could serve as the basis of possible bioassays for the study of the structure and mode of action of the androgenic hormone in decapod crustaceans.
Beyond Reconstruction: Using Phylogenies to Test Hypotheses About Vertebrate Evolution
A recent total evidence analysis of the position of cetaceans (whales, dolphins and porpoises and extinct relatives) among mammals indicated that the phylogeny of these taxa remains poorly resolved. Molecular data show that 1) the order Artiodactyla (even-toed ungulates) is paraphyletic unless whales are included within it and 2) that the traditional relationships of clades within Artiodactyla are not supported. This controversy also affects the position of a wholly extinct clade, Mesonychia, which has been argued to be the group of terrestrial mammals most closely related to whales. Here I update a previous total evidence analysis by adding several hundred new informative molecular characters from the literature. Even with the addition of these characters the phylogeny remains unresolved. All most parsimonious trees, however, indicate a paraphyletic Artiodactyla with conflict existing over the exact sister taxon of Cetacea. Congruence between different equally parsimonious cladograms and the stratigraphic record as measured using the modified Manhattan Stratigraphic Measure shows that all of the competing topologies, including those with a paraphyletic Artiodactyla, are significantly congruent with the stratigraphic record. In fossil taxa cladistic optimization can be used as an alternative to “argument from design” (Lauder, 1996) to reconstruct behavior and soft tissues that do not fossilize or osteological characters that have not preserved. In certain scenarios of cetacean phylogeny, optimization indicates that taxa such as the archaic whales Ambulocetus and Pakicetus, and possibly mesonychians, are more correctly reconstructed without hair.
Identifying when homoplasy is due to convergence requires confidence in trees and precise analysis of potentially convergent characters. Some features of mammals that eat mostly ants and termites are used as examples of convergence; the most speciose assemblages of these mammals are in the orders Xenarthra and Pholidota. My studies on cranial muscles in xenarthrans and pholidotans aim to 1) precisely describe the anatomy in ant-eating and non-ant-eating lineages, 2) assess variation among ant-eating lineages, and 3) compare the most derived conditions (in xenarthran anteaters and pholidotan pangolins). These data clarify the nature of morphological adaptation in ant-eating mammals, and when combined with accumulating phylogenetic studies, allow us to distinguish features that have evolved convergently from those that are variable but not correlated with diet. Interpreting the extreme similarity in anteaters and pangolins remains problematic due to lingering disagreement among phylogenetic hypotheses. Prevailing opinion favors interpretation of these similarities as convergent.
Marine fishes that spawn at the water's edge or even out of water provide their eggs with the advantages of the warmer temperatures and high oxygen availability of the high intertidal zone. However, they increase the risks of desiccation and terrestrial predation. Beach spawners are present in at least 6 families of teleost fishes. Two hypotheses about the origin of beach spawning are tested by mapping of reproductive habitat and spawning site utilization onto phylogenies of two families that contain multiple species that spawn on beaches: Osmeridae, the smelts, and Atherinopsidae, the silversides. Our analysis suggests that beach spawning has evolved repeatedly in certain lineages, and that its antecedents are different for each family. Anadromy appears to have been the precursor for at least 3 different evolutionary origins of beach spawning in osmerids, while nearshore marine spawning in association with plant or gravel substrates was probably the precursor for the atherinopsids. Phylogenetic analysis enables us to reject or support specific evolutionary hypotheses for each clade.
Hypotheses of relationships are critical to describing and understanding patterns of evolution within groups of organisms. But rarely has a comparative, historical approach been employed to study developmental change, particularly among anurans. A recent resurgence of interest in collecting basic ontogenetic information provides us with the opportunity to compare ontogenetic trajectories in a phylogenetic framework. Larval skeletons and osteological development were examined for 22 taxa and compared to two hypotheses of relationships—that of Cannatella, and one proposed herein based on 41 morphological characters from larvae and 62 from adults. Larval characters were mapped on the alternate cladograms using the ACCTRAN optimization criterion. Several larval features are highly conserved among some anurans, suggesting that there is some level of canalization of morphology early in ontogeny. In contrast, a number of morphologies vary among groups, supporting the fact that there have been major evolutionary modifications to anuran larval morphologies early in ontogeny and in the early evolutionary history of anurans.
Phenotypes manifest a balance between the inherited tendency to remain the same (phenotypic stability) and the tendency to change in response to current environmental conditions (adaptation). This paper explores the role of functional integration and functional trade-offs in generating phenotypic stability by limiting the responses of individual characters to environmental selection. Evolutionarily stable configurations (ESCs) are systems of functionally interacting characters within which characters are “judged” by their contribution to system-level functionality. This “internal” component of selection differs from traditional “external” selection in that it travels with the organism wherever it goes and is maintained across a wide range of environments. External selection, in contrast, is by definition environment-dependent. The temporal and geographic constancy of internal selection therefore acts to maintain phenotypic stability even as environments change. Functional trade-offs occur when one character participates in more than one function, but can only be optimized for one. Participation of certain (“keystone”) characters in a trade-off potentially causes stabilization of an entire system owing to a cascade of functional dependencies on that character. Phylogenetic character analysis is an essential part of elucidating these processes, but patterns cannot be used as prima facie evidence of particular processes.
Recent phylogenetic analyses of fossil and living crocodylians allow us to compare the taxonomic, geographic, and temporal distributions of morphological features, such as snout shapes. A few basic snout morphotypes—generalized, blunt, slender, deep, and excessively broad (“duck-faced”)—occur multiple times in distantly-related lineages. Some clades—especially those found in the Northern Hemisphere or with minimum origination dates in the Cretaceous or lower Tertiary—are morphologically uniform, but geographically widespread; crocodylian faunas of the early Tertiary tend to be composite, with sympatric taxa being distantly related, and similar-looking taxa on different continents being close relatives. In contrast, crocodylian faunas of the later Tertiary tend to be more endemic, with local adaptive radiations occurring in Africa and Australia containing members of most basic snout shapes. Endemic radiations in Africa and Australia have largely been replaced by Crocodylus, which can be divided into subclades that may individually represent endemic adaptive radiations.
Past approaches to understanding the evolution of locomotory strategies among Paleozoic amniotes (“primitive reptiles” of previous parlance) have been influenced by preservational bias: early occurrences of some amniote taxa were used to polarize the acquisition or development of locomotory structures among the earliest amniotes. Using a phylogeny representing the current consensus in the literature, we investigate the major locomotory strategies that have been posited for Paleozoic amniotes (basal synapsids on one hand and early reptiles on the other) by optimizing the major locomotory styles identified for these taxa onto the consensus tree, in order to present an overview of the pattern of evolution of locomotory strategies inherited and adopted by various amniote lineages.
Adaptive scenarios in evolutionary biology have always been based on incremental improvements through a series of adaptive stages. But they have often been justified by appeal to assumptions of how natural selection must work or by appeal to optimality arguments or notions of evolutionary process. Cladistic methodology, though it cannot logically falsify hypotheses of process, provides hypotheses of evolutionary pattern independent of other considerations and so provides a useful test of consilience with genealogy. I illustrate the cross-test of hypotheses of the evolution of several functions and adaptations related to the origin of bird flight with independently derived phylogenetic analysis. Consilience does not support ideas that the close ancestors of birds were arboreal or evolved flight from the trees, nor that they were physiologically intermediate between typical reptiles and living birds, nor that feathers evolved for flight. Rather, the ancestors of birds were terrestrial, they were fast-growing, active animals, and the original functions of feathers were in insulation and coloration.
Across major phylogenetic comparisons, the evolution of Hox clusters generally parallels the evolution of axial complexity. Sponges lack a fixed primary body axis and regional axial differentiation. Correspondingly, sponges appear to lack a Hox cluster. Bilaterian animals are characterized, at least primitively, by the presence of an anterior-posterior axis. In many bilaterians, the anterior-posterior axis is finely subdivided into morphologically distinct regions; e.g., consider the many distinct vertebrae of the human vertebral column or the many distinct body segments of the fruitfly. This axial complexity is encoded in part, by the genes of the Hox cluster. Bilaterians possess from seven to upwards of forty Hox genes which sort into four monophyletic classes (anterior, group-3, central, and posterior). Cnidarians (e.g., sea anemones) display an intermediate stage of axial complexity. Unlike sponges, they possess a fixed primary body axis, known as the oral-aboral axis, with a distinct head, body column, and foot. However, the primary axis of cnidarians lacks the degree of axial differentiation found in vertebrates or insects. Cnidarians possess distinct anterior and posterior Hox genes. Cnidarians appear to lack group-3 or central Hox genes. Southern mapping experiments in the sea anemone, Nematostella indicate linkage between an anterior Hox gene, an even-skipped ortholog, and a posterior Hox gene. The linkage of eve to a Hox gene, a condition previously described in a coral, is found in vertebrates but apparently absent in insects. Cnidarians hold the potential to reveal important intermediate stages in the evolution of Hox clusters and axial complexity.
Hox genes in bilaterians specify distinct regions along the anterior-posterior axis. A question of interest is when in metazoan evolution did this class of genes take on this function. Hox genes have been isolated from a number of cnidarian species including hydra. The expression patterns of two of them, Cnox-3 and Cnox-2 have been examined in adult hydra. Cnox-3, a labial homologue, plays a role in oral/anterior patterning, while Cnox-2, a Deformed homologue or a Gsx homologue of the ParaHox cluster appears to repress anterior patterning in the body column. The two genes play a role in axial patterning that is consistent with the tissue dynamics of an adult hydra.
Underlying any analysis on the evolution of development is a phylogenetic framework, whether explicitly stated or implied. As such, differing views on phylogenetic relationships lead to variable interpretations of how developmental mechanisms have changed through time. Over the past decade, many long-standing hypotheses about animal evolution have been questioned causing substantial changes in the assumed phylogenetic framework underlying comparative developmental studies. Current hypotheses about early metazoan history suggest that three, not two, major lineages of bilateral animals originated in the Precambrian: the Deuterostomes (e.g., seastars, acorn worms, and vertebrates), the Ecdysozoans (e.g., nematodes and arthropods), and the Lophotrochozoans (e.g., annelids, mollusks, and lophophorates). Although information in Hox-genes bears directly on our understanding of early metazoan evolution and the formation of body plans, research effort has been focused primarily on two taxa, insects and vertebrates. By sampling a greater diversity of metazoan taxa and taking advantage of biotechnological advances in genomics, we will not only learn more about metazoan phylogeny, but will also gain valuable insight as to the key evolutionary forces that established and maintained metazoan bauplans.
The Hox genes are widely regarded as candidates for involvement in major evolutionary changes in body plan organization. We examine Hox gene expression data for several taxa, in relation to recent work on the polychaete annelid Chaetopterus. The work in Chaetopterus shows the basic conservation of colinearity of anterior expression boundaries seen in other groups. It also reveals novel patterns including early expression in the larval growth zone and later formation of posterior boundaries that correlate with morphological transitions in the polychaete body plan. The polychaete gene expression pattern is compared with those of Hox gene homologs in other taxa to reveal differences that represent evolutionary changes in Hox gene regulation between lineages. Correlations between Hox gene expression differences and morphological differences are examined, focussing on a number of cases in which posterior Hox gene expression boundaries correlate with morphological transitions. Differential regulation of these posterior expression boundaries is proposed as a possible mechanism for changes body plan regionalization.
Flatworms (phylum Platyhelminthes) are favourite organisms in Developmental Biology and Zoology because of their extraordinary powers of regeneration and because they may hold a pivotal place in the origin and evolution of the Bilateria. Hox genes play key roles in both processes: setting up the new anteroposterior pattern in the former, and as qualitative markers of phylogenetic affinities among bilaterian phyla in the latter. We have searched for Hox and ParaHox genes in several flatworm groups spanning from freshwater triclads to marine polyclads and, more recently, in the acoels, the likely earliest extant bilaterian. We have isolated and sequenced eight Hox genes from the freshwater triclad Girardia tigrina and three Hox and two ParaHox genes from the polyclad Discocelis tigrina. Data from the acoels Paratomella rubra and Convoluta roscoffensis is also reported. Flatworm Hox sequences and 18S rDNA sequence data support clear affinities of Platyhelminthes to spiralian lophotrochozoans. The basal position of acoel flatworms supported from recent 18S rDNA data, remains still uncertain. Expression of Hox genes in intact and regenerating adult organisms show nested patterns with graded anterior expression boundaries, or ubiquitous expression. New approaches to study the function of Hox genes in flatworms, such as RNA interference are briefly discussed.
Ascidians, along with other urochordates, are the most evolutionary distant group from vertebrates to display definitive chordate-specific characters, such as a notochord, dorsal hollow nerve cord, pharynx and endostyle. Most solitary ascidians have a biphasic life history that has partitioned the development of these characters between a planktonic microscopic tadpole larva (notochord and dorsal nerve cord) and a larger sessile adult (pharynx and endostyle). Very little is known of the molecular axial patterning processes operating during ascidian postlarval development. Two axial patterning homeobox genes Otx and Cdx are expressed in a spatially restricted manner along the ascidian anteroposterior axis during embryogenesis and postlarval development (i.e., metamorphosis). Comparisons of these patterns with those of homologous cephalochordate and vertebrate genes suggest that the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a heterochronic shift in tissue-specific expression. Studies examining the role of all-trans retinoic acid (RA) in axial patterning in chordates also contribute to our understanding of the role of homeobox genes in the development of larval and adult ascidian body plans. Our studies demonstrate that RA does not regulate axial patterning in the developing ascidian larval neuroaxis in a manner homologous to that found in vertebrates. Although RA may regulate the expression of some ascidian homeobox genes, ectopic application of RA does not appear to alter the morphology of the larval CNS. However, treatment with similar or lower concentrations of RA, have a profound effect on postlarval development and the juvenile body plan. These changes are correlated to a dramatic reduction of Otx expression. Through these RA-induced effects we infer that while RA may regulate the expression of some homeobox genes during embryogenesis it has a far more dramatic impact on postlarval development where regulative processes predominate.
The early origin of four vertebrate Hox gene clusters during the evolution of gnathostomes was likely caused by two consecutive duplications of the entire genome and the subsequent loss of individual genes. The presumed conserved and important roles of these genes in tetrapods during development led to the general assumption that Hox cluster architecture had remained unchanged since the last common ancestor of all jawed vertebrates. But recent data from teleost fishes reveals that this is not the case. Here, we present an analysis of the evolution of vertebrate Hox genes and clusters, with emphasis on the differences between the Hox A clusters of fish (actinopterygian) and tetrapod (sarcopterygian) lineages. In contrast to the general conservation of genomic architecture and gene sequence observed in sarcopterygians, the evolutionary history of actinopterygian Hox clusters likely includes an additional (third) genome duplication that initially increased the number of clusters from four to eight. We document, for the first time, higher rates of gene loss and gene sequence evolution in the Hox genes of fishes compared to those of land vertebrates. These two observations might suggest that two different molecular evolutionary strategies exist in the two major vertebrate lineages. Preliminary data from the African cichlid fish Oreochromis niloticus compared to those of the pufferfish and zebrafish reveal important differences in Hox cluster architecture among fishes and, together with genetic mapping data from Medaka, indicate that the third genome duplication was not zebrafish-specific, but probably occurred early in the history of fishes. Each descending fish lineage that has been characterized so far, distinctively modified its Hox cluster architecture through independent secondary losses. This variation is related to the large body plan differences observed among fishes, such as the loss of entire sets of appendages and ribs in some lineages.
The colinear, anterior to posterior expression domains of the Hox genes in vertebrate embryos is strongly correlated with regional changes in vertebral morphology. The limbs of tetrapods are consistently aligned with specific areas of the vertebral column. However, control of limb development is apparently situated in the lateral plate mesoderm, and has been experimentally shown to be independent of an axial Hox code (Cohn et al., 1997, Nature 387:97–101). We have used experimental manipulation of chick embryos to test the causal role of Hox genes in patterning derivatives of the paraxial mesoderm. Hox expression in heterotopically transplanted segmental plate responds in a manner consistent with a patterning role for these genes in the morphological behavior of the transplants. Expression is maintained in dorsal paraxial regions where patterning is also intrinsic to the donor site of the graft. However, expression is apparently lost in somite cells that migrate into the host lateral plate environment and form appropriate host-level muscles. This arrangement could enable increased plasticity in the evolution of transpositional variation in the vertebrate body plan.