From genomes to morphology: a view from amphioxus

Holland, P.W.H. 2010. From genomes to morphology: a view from amphioxus. —Acta Zoologica (Stockholm) 91 : 81–86 As complete genome sequences are determined from an ever‐increasing number of animal species, new opportunities are arising for comparative biology. For zoologists interested in the evolution of shape and form, however, there is a problem. The link between genome sequence and morphology is not direct and is obfuscated by complex and evolving genetic pathways, even when conserved regulatory genes are considered. Nonetheless, a large‐scale comparison of genome sequences between extant chordates reveals an intriguing parallel between genotypic and phenotypic evolution. Tunicates have highly altered genomes, with loss of ancestral genes and shuffled genetic arrangements, while vertebrate genomes are also derived through gene loss and genome duplication. The recently sequenced amphioxus genome, in contrast, reveals much greater stasis on the cephalochordate lineage, in parallel to a less derived body plan. The opportunities and challenges for relating genome evolution to morphological evolution are discussed.     

Expression of the Otx2 homeobox gene in the developing mammalian brain: embryonic and adult expression in the pineal gland

Otx2 is a vertebrate homeobox gene, which has been found to be essential for the development of rostral brain regions and appears to play a role in the development of retinal photoreceptor cells and pinealocytes. In this study, the temporal expression pattern of Otx2 was revealed in the rat brain, with special emphasis on the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone-rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, although there was a distinct lag in time of onset. Otx2 protein was identified in pineal extracts and found to be localized in pinealocytes. Total pineal Otx2 mRNA did not show day-night variation, nor was it influenced by removal of the sympathetic input, indicating that the level of Otx2 mRNA appears to be independent of the photoneural input to the gland. Our results are consistent with the view that pineal expression of Otx2 is required for development and we hypothesize that it plays a role in the adult in controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis.     

AmphiNk2-tin,an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express AmphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization.     

Evolutionary change of the numbers of homeobox genes in bilateral animals

It has been known that the conservation or diversity of homeobox genes is responsible for the similarity and variability of some of the morphological or physiological characters among different organisms. To gain some insights into the evolutionary pattern of homeobox genes in bilateral animals, we studied the change of the numbers of these genes during the evolution of bilateral animals. We analyzed 2,031 homeodomain sequences compiled from 11 species of bilateral animals ranging from Caenorhabditis elegans to humans. Our phylogenetic analysis using a modified reconciled-tree method suggested that there were at least about 88 homeobox genes in the common ancestor of bilateral animals. About 50-60 genes of them have left at least one descendant gene in each of the 11 species studied, suggesting that about 30-40 genes were lost in a lineage-specific manner. Although similar numbers of ancestral genes have survived in each species, vertebrate lineages gained many more genes by duplication than invertebrate lineages, resulting in more than 200 homeobox genes in vertebrates and about 100 in invertebrates. After these gene duplications, a substantial number of old duplicate genes have also been lost in each lineage. Because many old duplicate genes were lost, it is likely that lost genes had already been differentiated from other groups of genes at the time of gene loss. We conclude that both gain and loss of homeobox genes were important for the evolutionary change of phenotypic characters in bilateral animals.     

An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution.

The embryology of amphioxus has much in common with vertebrate embryology, reflecting a close phylogenetic relationship between the two groups. Amphioxus embryology is simpler in several key respects, however, including a lack of pronounced craniofacial morphogenesis. To gain an insight into the molecular changes that accompanied the evolution of vertebrate embryology, and into the relationship between the amphioxus and vertebrate body plans, we have undertaken the first molecular level investigation of amphioxus embryonic development. We report the cloning, complete DNA sequence determination, sequence analysis and expression analysis of an amphioxus homeobox gene, AmphiHox3, evolutionarily homologous to the third-most 3' paralogous group of mammalian Hox genes. Sequence comparison to a mammalian homologue, mouse Hox-2.7 (HoxB3), reveals several stretches of amino acid conservation within the deduced protein sequences. Whole mount in situ hybridization reveals localized expression of AmphiHox3 in the posterior mesoderm (but not in the somites), and region-specific expression in the dorsal nerve cord, of amphioxus neurulae, later embryos and larvae. The anterior limit to expression in the nerve cord is at the level of the four/five somite boundary at the neurula stage, and stabilises to just anterior to the first nerve cord pigment spot to form. Comparison to the anterior expression boundary of mouse Hox-2.7 (HoxB3) and related genes suggests that the vertebrate brain is homologous to an extensive region of the amphioxus nerve cord that contains the cerebral vesicle (a region at the extreme rostral tip) and extends posterior to somite four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhox homeobox gene cluster: recent duplication of three family members

We recently reported the discovery of a homeobox gene cluster on the mouse X chromosome, Rhox, whose 12 members are selectively expressed in specific cell types in reproductive organs. Here we report the existence of 20 additional Rhox homeobox genes in this gene cluster. Most of the newly identified Rhox paralogs retain the same order and relative orientation as three of the originally described Rhox genes, suggesting that they arose from recent duplications of this trimer unit. Many of these new Rhox family members are expressed in the testis and placenta. Analysis of synonymous and nonsynonymous substitutions in their homeodomain region suggests that these new Rhox paralogs duplicated so recently that their encoded proteins have not yet acquired distinct DNA-binding specificities. The existence of these new Rhox genes provides an opportunity to examine the initial stages of gene cluster evolution.     

The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution

Background  

The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire.     

Characterization and developmental expression of AmphiNk2-2, an NK2 class homeobox gene from amphioxus (Phylum Chordata; Subphylum Cephalochordata)

 The genome of amphioxus includes AmphiNk2-2, the first gene of the NK2 homeobox class to be demonstrated in any invertebrate deuterostome. AmphiNk2-2 encodes a protein with a TN domain, homeodomain, and NK2-specific domain; on the basis of amino acid identities in these conserved regions, AmphiNk2-2 is a homolog of Drosophila vnd and vertebrate Nkx2–2. During amphioxus development, expression of Amph- iNk2-2 is first detected ventrally in the endoderm of late gastrulae. In neurulae, endodermal expression divides into three domains (the pharynx, midgut, and hindgut), and neural expression commences in two longitudinal bands of cells in the anterior neural tube. These neural tube cells occupy a ventrolateral position on either side of the cerebral vesicle (the probable homolog of the vertebrate diencephalic forebrain). The dynamic expression patterns of AmphiNkx2-2 suggest successive roles, first in regionalizing the endoderm and nervous system and later during differentiation of specific cell types in the gut (possibly peptide endocrine cells) and brain (possibly including axon outgrowth and guidance). Received: 24 November 1997 / Accepted: 2 February 1998     

人睾丸与脑基因表达谱的相似性

人类的物种形成与进化问题一直是研究的一个焦点。近年来,对于人和灵长类以及果蝇等其他一些动物多种组织基因表达谱的研究表明,在人的进化过程中脑基因表达的改变最为显著,并且脑中许多基因的表达呈显著上调。信息学分析显示,在多种组织当中,人的脑与睾丸可能存在最为相似的基因表达谱。这些结果提示睾丸可能与脑类似,也在人的物种形成和进化历程中起着重要作用。本文对人睾丸和脑基因表达谱的研究进行了回顾,并提出了该研究方向今后的一些研究设想。     

Behavioral and synaptic defects in C. elegans lacking the NK-2 homeobox gene ceh-28

C. elegans pharyngeal behavior consists of two distinct types of muscle contractions, termed pumping and peristalsis. Pumping ingests and concentrates bacteria in the anterior pharyngeal lumen, and it is occasionally followed by a transient peristaltic contraction that carries ingested bacteria through the posterior pharyngeal isthmus. These behaviors are controlled by a small pharyngeal nervous system consisting of 20 neurons that is almost completely independent of the extra-pharyngeal nervous system. The cholinergic motor neuron M4 controls peristalsis via synapses with the posterior isthmus muscles. Here we show that the NK-2 family homeobox gene ceh-28 is expressed in M4, where it regulates synapse assembly and peristalsis. ceh-28 mutants exhibit frequent and prolonged peristalses, and treatment with agonists or antagonists of muscarinic acetylcholine receptors can phenocopy or suppress ceh-28 mutant defects, respectively. Synapses in ceh-28 mutant M4 cells are irregularly spaced and sized, and they are abnormally located along the full length of the isthmus. We suggest that CEH-28 inhibits synaptogenesis, and that ceh-28 mutant behavioral defects result from excessive or ectopic stimulation of muscarinic acetylcholine receptors in the isthmus muscles.     

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.     

The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog

 The hedgehog family of intercellular signalling molecules have essential functions in patterning both Drosophila and vertebrate embryos. Drosophila has a single hedgehog gene, while vertebrates have evolved at least three types of hedgehog genes (the Sonic, Desert and Indian types) by duplication and divergence of a single ancestral gene. Vertebrate Sonic-type genes typically show conserved expression in the notochord and floor plate, while Desert- and Indian-type genes have different patterns of expression in vertebrates from different classes. To determine the ancestral role of hedgehog in vertebrates, I have characterised the hedgehog gene family in amphioxus. Amphioxus is the closest living relative of the vertebrates and develops a similar body plan, including a dorsal neural tube and notochord. A single amphioxus hedgehog gene, AmphiHh, was identified and is probably the only hedgehog family member in amphioxus, showing the duplication of hedgehog genes to be specific to the vertebrate lineage. AmphiHh expression was detected in the notochord and ventral neural tube, tissues that express Sonic-type genes in vertebrates. This shows that amphioxus probably patterns its ventral neural tube using a molecular pathway conserved with vertebrates. AmphiHh was also expressed on the left side of the pharyngeal endoderm, reminiscent of the left-sided expression of Sonic hedgehog in chick embryos which forms part of a pathway controlling left/right asymmetric development. These data show that notochord, floor plate and possibly left/right asymmetric expression are ancestral sites of hedgehog expression in vertebrates and amphioxus. In vertebrates, all these features have been retained by Sonic-type genes. This may have freed Desert-type and Indian-type hedgehog genes from selective constraint, allowing them to diverge and take on new roles in different vertebrate taxa. Received: 20 July 1998 / Accepted: 23 September 1998     

Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx

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

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Cloning and characterization of NM23-Bbt2 gene from amphioxus Branchiostoma belcheri tsingtauense

A full-length amphioxus (Branchiostoma belcheri tsingtauense) NM23-Bbt2, NM23-H2 homologue, cDNA was isolated from the cDNA library and sequenced. The obtained amphioxus NM23-Bbt2 cDNA contains an open reading frame coding for 171 amino acids. Sequence analysis showed that the amphioxus NM23-Bbt2 was highly conserved with that of other species, and all of them contained highly conserved motifs that play important roles in the function of NM23. RT-PCR revealed that NM23-Bbt2 is expressed in the neuronal tissues and is expressed in all stages during the embryogenesis. Nucleoside kinases are thought to have a critical role in regulatory processes such as signal transduction, proliferation, and differentiation. Taken together, these results suggest that nucleoside diphosphate kinases have an important role to play in embryogenic development in amphioxus. Phylogenetic analysis showed that the amphioxus group 1 NDPKs (Bf1-4) may be precursors of the human group 1 NDPKs, NM23-Bf5, NM23-Bf6, NM23-Bf7 and NM23-Bf8 may be precursors of NM23-H5, NM23-H6, NM23-H7 and M23-H8, respectively. Our finding of nine NM23 genes in Branchiostoma floride, the precursor of vertebrates, strongly suggests that the ancestral gene corresponding to each of vertebrates NM23 genes generated before the appearance of vertebrates. Comparison of the gene structures of NM23-H2 homologue from invertebrates to vertebrates suggests that the locations of three of the four introns are conserved in amphioxus and vertebrates.