Amphioxus Branchiostoma Lanceolatum Research Articles

Recognition of protein folds via dipolar couplings

Alignment of proteins in dilute liquid crystalline medium gives rise to residual dipolar couplings which provide orientational information of vectors connecting the interacting nuclei. Considering that proteins are mainly composed of regular secondary structures in a finite number of different mutual orientations, main chain dipolar couplings appear sufficient to reveal structural resemblance. Similarity between dipolar couplings measured from a protein and corresponding values computed from a known structure imply homologous structures. For dissimilar structures the agreement between experimental and calculated dipolar couplings remains poor. In this way protein folds can be readily recognized prior to a comprehensive structure determination. This approach has been demonstrated by showing the similarity in fold between the hitherto unknown structure of calerythrin and sarcoplasmic calcium-binding proteins from Nereis diversicolorand Branchiostoma lanceolatumwith known crystal structures. Abbreviations:CSA, chemical shift anisotropy; D6PC, dihexanoyl phosphatidylcholine; D7PC, diheptanoyl phosphatidylcholine; DMPC, dimyristoyl phosphatidylcholine; INEPT, insensitive nuclei enhancement by polarization transfer; NOE, nuclear Overhauser enhancement; PDB, Protein Data Bank entries 1cll: calmodulin, 1rec: recoverin, 1tco: serine-threonine phosphatase B2, 2bbm: calmodulin complexed with myosin light chain kinase, 2sas: sarcoplasmic calcium-binding protein from an amphioxus Branchiostoma lanceolatum , 2scp: sarcoplasmic calcium-binding protein from a sandworm Nereis diversicolor; TROSY, transverse relaxation optimized spectroscopy.

The tissue-specific transcription factor Pit-1 is expressed in the spinal cord of the lancelet, Branchiostoma lanceolatum

The spinal cord of the lancelet Branchiostoma lanceolatum was studied by using a monoclonal antibody to the rat tissue-specific transcription factor, Pit-1. Our previous studies have demonstrated Pit-1 immunoreactivity in different nervous and endocrine structures of the head region of adults and in the rostral central nervous system (CNS) of larval lancelet. Our present results show the presence of Pit-1-like protein in dorso-lateral nerve cells and ependymocytes of the adult spinal cord. Using double immunofluorescence techniques, we have revealed the coexistence of the glial fibrillary acidic protein (GFAP) with Pit-1 in groups of laterally located ependymocytes. The occurrence of GFAP, a specific marker of mammalian astrocytes and radial glia, in some lancelet ependymocytes confirms that glial elements are also present in protochordates. Furthermore, other ependymocytes, located in the roof of the central canal and containing Pit-1-like protein exclusively, could be considered as ependymal tanycytes.

Evidence for the presence of the tissue-specific transcription factor Pit-1 in lancelet larvae.

Recent molecular studies have noted the affinity among cephalochordates and vertebrates. In particular, a cluster of vertebrate-like homeobox genes regulates the development of the lancelet Branchiostoma lanceolatum. A previous study has outlined the expression pattern of the pituitary-specific transcription factor Pit-1 in adult lancelets. Pit-1 belongs to the POU family of transcription factors, which, like homeotic proteins, are members of the helix-turn-helix superfamily of proteins. POU is an acronym for Pit-1, Oct-1 and Oct-2, and Unc-86. In the present work, we investigated the head region of premetamorphic larvae of B. lanceolatum, by means of scanning electron microscopy, wholemount and tissue sections immunocytochemistry, and Western blotting assay, to verify the presence and distribution of Pit-1. Immunoreactive Pit-1 protein was detected in the rostral nerves and in a cluster of photoreceptor cells of the frontal eye. At the same time, an electrophoretic band of 33 kDa was shown from extracts of premetamorphic larvae and recognized by a monoclonal antibody to rat Pit-1. On the basis of the immunocytochemical and electrophoretic results, we can assume that Pit-1 may play a neuromodulatory role in the larval central nervous system. Moreover, the spatial and temporal distribution of Pit-1 protein in larva and adult lancelets agrees only in part with that described in embryonic and adult mice, suggesting different molecular controls of regional identity in the nervous system of cephalochordates and vertebrates.

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates.

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.

Localization and Partial Characterization of Melatonin Receptors in Amphioxus, Hagfish, Lamprey, and Skate

Through its secretion of melatonin, the pineal complex of vertebrates exerts a range of physiological effects including regulation of circadian rhythms, seasonal reproduction, metamorphosis, and body color change. Little is known about phylogenetic differences in the distribution and characteristics of melatonin binding sites in fishes. We usedin vitroautoradiography to examine binding of [2-125I]iodomelatonin (IMEL) in 20-μm frozen sections of amphioxus (Branchiostoma lanceolatum), Atlantic hagfish (Myxine glutinosa), larval and adult lamprey (Petromyzon marinus), little skate (Raja erinacea), and rainbow trout (Oncorhynchus mykiss). Tissue was incubated with IMEL in the presence or absence of unlabeled melatonin (1 μM, in order to assess nonspecific binding). A concentration of 32 pM IMEL was used for single point assays and competition studies. No specific binding was found in hagfish or amphioxus, which lack a pineal complex. In the optic tecta of lamprey, skate, and trout, IMEL binding is highly specific (melatonin⪢N-acetylserotonin>5- methoxytryptophol⪢serotonin). Scatchard analysis revealed that the tectal binding sites are of high affinity (Kd=36, 38, and 50 pM) and low capacity (Bmax=8.1, 19.8, and 21.8 fmol/mg protein) in lamprey, skate, and trout, respectively. In adult lampreys, intense specific IMEL binding is found in the optic tectum (layer I>II>III) and preoptic nucleus (pars parvocellularis>magnocellularis). Binding was less intense and consistent in the same areas of ammocoete brain. In skates and trout, intense specific binding is found in optic tectum, lateral geniculate body, diencephalic preoptic and suprachiasmatic nuclei, basal hypothalamus, and the medial pallium. These results indicate that specific melatonin binding sites are present in all craniate taxa examined except in hagfish. Although we cannot rule out the possibility that melatonin receptors are secondarily lost in hagfish, their absence in amphioxus makes this unlikely. We speculate that melatonin actions in early vertebrates may have included regulation of visual and endocrine responses to light.

Actin isoforms in amphioxus Branchiostoma lanceolatum.

Actin is a highly conserved cytoskeletal protein that is ubiquitous in all eukaryotes. Little is known about actin expression in amphioxus, the closest living relative of the vertebrates. In the present study, involving Western blotting and indirect immunofluorescence, we report the characterization and localization of various actin isoforms in amphioxus (Branchiostoma lanceolatum) tissues. Three antibodies against vertebrate actins were used: a polyclonal antibody recognizing beta-cytoplasmic actin (anti-beta actin), a monoclonal antibody against sarcomeric actins (anti-alphaSR-1), and a monoclonal antibody specific for alpha-smooth actin (anti-alphaSM-1). Western blot analysis of amphioxus extracts immunodecorated with these antibodies showed a 43-kDa-positive band co-migrating with respective controls. The amphioxus isoactin expression patterns recognized by these antibodies were similar to those of vertebrates, i.e., anti-beta actin showed positive staining mainly in non-muscle cells, anti-alphaSR-1 labelled dorsolateral myotomal muscles, and anti-alphaSM-1 stained ventral muscles. These results demonstrate that at least two muscle actins are present in amphioxus, suggesting that muscle actin gene duplication events began before vertebrate divergence from the amphioxus lineage.

Two cholinesterase activities and genes are present in amphioxus

To obtain information about the evolution of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the vertebrates, we investigated the cholinesterase (ChE) activity of the cephalochordate amphioxus (Branchiostoma floridae and Branchiostoma lanceolatum). On the basis of evidence from enzymology, pharmacology, and molecular biology, we conclude that amphioxus possesses two ChE activities and two ChE genes. Two covalent inhibitors of cholinesterases were able to pharmacologically isolate the two activities as drug-sensitive ChE and drug-resistant ChE. Kinetically, in terms of substrate specificity, the drug-sensitive ChE resembles vertebrate AChE, and the drug-resistant ChE resembles the BuChE of cartilaginous and bony fish or the intermediate ChE of protostome invertebrates. We also used the polymerase chain reaction with degenerate oligonucleotide primers and genomic DNA to obtain clones of 1,574 and 1,011 bp corresponding to two cholinesterase genes from amphioxus, which we designated as ChE1 and ChE2. ChE2 codes for an enzyme with an acyl-binding pocket sequence, a portion of the protein that plays an important role in determining substrate specificity, typical of invertebrate ChE. ChE1, which contains a 503-bp intron, encodes a protein with a novel acyl binding site. Phylogenetic analysis of the sequences suggests that the two genes are a result of a duplication event in the lineage leading to amphioxus. We discuss the relevance of our results to the evolution of the cholinesterases in the chordates. Previously, we reported that amphioxus contained a single cholinesterase activity with properties intermediate to AChE and BuChE (Pezzementi et al. [1991] In: Cholinesterases: Structure, Function, Mechanism, Genetics and Cell Biology. J. Massoulié et al., eds. ACS: Washington, D.C., pp. 24-31).

Ciliary specializations in branchial stigmatal cells of protochordates

Tissues from the pharynx of five representative species of the protochordates (subphylum Tunicata, the three classes Ascidiacea, Thaliacea and Appendicularia, and subphylum Cephalochordata) were examined in both thin sections and freeze-fracture replicas. In all species, the stigmatal cilia of the branchial chamber are neatly arranged and move continuously to propel sea-water in a fixed direction for respiration and feeding of the organism. A number of specializations are found in the basal region of these cilia and are represented by: a) bridges connecting axonemal doublets numbers 5 and 6; b) dense fibrous material linking the doublet microtubules of the axoneme to the ciliary membrane, sometimes in the shape of longitudinal strands or as clusters of filaments; c) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters evenly aligned along the ciliary shaft in relation to the underlying axonemal doublets. Ciliary specializations are distributed along the plane of the effective stroke of the beat in both the ascidian Botryllus schlosseri and in the thaliacean Pyrosoma atlanticum and the amphioxus Branchiostoma lanceolatum, whereas in the thaliacean Doliolum nationalis and the appendicularian Oikopleura dioica a more uniform distribution of these specializations all around the basal portion of the cilia is observed. Whatever the disposition of the ciliary specializations in all the examined species, they are always present at the base of the water-propelling cilia. Some morphological evidence suggests that these specializations play a mechanical function in tethering the ciliary membrane to the axoneme. We propose that they help maintain the orientation of the cilia during beating, enhance their stiffness and improve their efficiency.

Amino acid sequences of four isoforms of amphioxus sarcoplasmic calcium‐binding proteins

The protochordate amphioxus (Branchiostoma lanceolatum) contains different isoforms of sarcoplasmic, high-affinity Ca2(+)-binding proteins (SCP). The amino acid sequences of the two major isoforms SCP I and II, reported previously [Takagi, T., Konishi, K. & Cox, J.A. (1986) Biochemistry 25, 3585-3592], have been corrected and differ from each other by seven amino acid substitutions in a 17-residue-long segment (positions 20-36). We also report on the isolation and amino acid sequence determination of two minor isoforms, i.e. amphioxus SCP III and IV. Although they behave very differently from the major forms with respect to net charge, they differ from SCP I by only one amino acid: SCP III has Met at position 20 (Tyr in SCP I) and SCP IV has Asn at position 23 (Asp in SCP I). Together the sequence data on amphioxus SCP suggest that, in contrast with SCP of other invertebrate phyla, the isoforms are generated by alternative splicing of the primary RNA transcript with a mutually exclusive pattern.

Electron-microscopic studies of iodine-binding and peroxidase activity in the endostyle of the larval amphioxus (Branchiostoma lanceolatum)

The asymmetric endostyle in the larval amphioxus (Branchiostoma lanceolatum) was examined by light-and electron-microscopic cytochemistry (peroxidase; incubation in diaminobenzidine) and autoradiography (incubation in 125I-). Compared to the adult the same cellular zones were also found in the larval endostyle, with the exception of zone 1, which was absent. The corresponding adult and larval zones had a similar morphology. All cells in zones 5a, 5b, and 6 were reactive for peroxidase. A reaction product was also present in the lateral 2 to 3 cell rows of zone 3. The dense reaction product was located on the inner surface of membranes of the rough endoplasmatic reticulum, Golgi apparatus and vesicles, and multivesicular bodies as well as on the outer surface of the luminal plasma membrane. An incomplete row of granule-containing, peroxidase-negative cells was located between zones 5b and 6. After incubation of larvae in sea water containing 125I-, autoradiographic grains were selectively concentrated over the lumen at the apical surface of all peroxidase-positive zones. The highest grain density occurred in relation to zone 5a, which in the adult has been recognized as the iodination center. Few grains were located over the cytoplasm. Methimazole, an inhibitor of peroxidase, abolished the cytochemical reaction and the appearance of autoradiographic grains. The observations indicate that iodination in the larval endostyle takes place extracellularly and is catalyzed by peroxidase bound in the plasma membrane.

In vitro conversion of androgen to estrogen in amphioxus gonadal tissues

The ability to convert androgen to estrogen (aromatization) is a constant feature of gonadal and neural tissues in all major vertebrate groups. In experiments reported here, the existence of this pathway was investigated in the protochordate amphioxus ( Branchiostoma lanceolatum). Following incubation with [ 3H]19-hydroxyandrostenedione, gonadal homogenates contained authentic estrone and estradiol-17β, as determined by derivative formation and recrystallization to constant specific activity. Cephalic (“brain”) and other segments were aromatase negative. The results indicate that a potential for estrogen biosynthesis in the gonads predates that in other tissues and arises prior to the evolution of true vertebrates.

Thyroid hormone formation in thyroglobulin synthesized in the Amphioxus ( Branchiostoma lanceolatum pallas)

1. 1. Thyroglobulin (Tg) biosynthesis in the endostyles of Amphioxus ( Branchiostoma lanceolatum Pallas) has been demonstrated. 2. 2. The protein molecule shows characteristics similar to those of Tg from ammocoetes of fresh-water lampreys, sedimenting with a coefficient of 17-19S and incorporating in vivo 125I. 3. 3. Iodothyronine synthesis occurs in thyroglobulin in animals incorporating 125I both after in vivo and after in vitro iodination with lactoperoxidase in presence of labelled iodide. 4. 4. Labelled T3 and T4 are present in the Tg molecule of Amphioxus as in control mammalian Tg and practically in the same molecular rates. 5. 5. Thus thyroid hormones are present in thyroglobulin included in the cells of endostyles. 6. 6. It is shown that in evolution of Chordates the synthesis of thyroglobulin, as the site of formation of thyroid hormones, preceeds the morphological differentiation of thyroid cells and the folliclular organization out (fishes) or into the thyroid glands of higher vertebrates.

The Circulatory System of Amphioxus (Branchiostoma lanceolatum(Pallas))

The morphology of the circulatory system of Amphioxus (Branchiostoma lanceolatum (Pallas)) has been investigated using a new intravascular injection technique. A survey of the vessels of Amphioxus using this technique is given. The dorsal arteries and their ramifications are described in detail. The new injection technique brought to light myoseptal plexi, supplied from the dorsal arteries, between every two myomeres. Also the ventral parietal arteries have a much more complicated course than hitherto accepted. They are connected with an atrial plexus which is a continuous net of small vessels in the whole length of the dorso-lateral wall of the atrial cavity. It is postulated that this plexus has a supplementary function in respiration. Plexi of minute vessels in the gonads and a real blood circulation with afferent and efferent gonadal vessels have been demonstrated. Two vessels connecting the liver plexus with the cardinal vein (or the atrial plexi) have been noticed, the v. communicans accessoria anterior mentioned in 1900 by Burchardt, and a so-called oblique vessel never described before. The vessels of the caudal region are analyzed completely and also here a real blood circulation appears possible.

Ciliary rootlet-fibres as tail fin-rays in larval amphioxus ( Branchiostoma lanceolatum, pallas)

In 1 mm long and two weeks old larvae of amphioxus ( Branchiostoma lanceolatum ) a prominent epithelial tail fin is present. Birefringent tappered rods with a maximal diameter of 0.8 μ m and a total length of 10 to 25 μ m radiate from the base to the apex of the fin. In the transmission electron microscope these fin-rays reveal their true nature as rootlets related to basal bodies and perhaps cilia. One rootlet is found within each cell of the epithelium. Each rootlet is made up of 5 nm thick filaments organized in a cross-striated pattern with a periodicity of 78 nm and several substriations. Filament free longitudinal channels are present within each rootlet, and microtubules are intimately associated with their surface. Microtubules of two distinct diameters, 13 nm and 21 nm, were observed in both transverse and longitudinal sections. It is concluded that the primary function of these rootlets is to give the tail fin an elastic resistance against bending forces rather than to support the cilia of the tail.

The connection between spinal cord and notochord in Amphioxus (Branchiostoma lanceolatum).

Silver impregnation of nerves, histochemical reactions for acetylcholinesterase, and electron microscopy reveal an efferent innervation of the notochord in amphioxus. Extensions of the notochordal lamellae end in groups (the “notochordal horns”) just below the ventro-lateral surface of the spinal cord where they are opposed to large nerve terminals originating as short collaterals of axons running longitudinally in the nerve cord. This neurochordal junction resembles an ordinary neuro-muscular junction in several respects and is interpreted as a part of the muscular system found in the notochord itself.

Paramyosin muscle in the notochord of Amphioxus.

As is evident from every textbook of zoology, the internal skeleton or notochord of amphioxus (Branchiostoma lanceolatum) has been considered as a passive supporting organ by all but a very few investigators. Our examination, using modern techniques, however, supports the view of Ebner1 and Joseph2, who proposed a muscular nature of the notochord (Fig. 1).

The Early Development of the Nephridia in Amphioxus: Introduction and Part I, Hatschek’s Nephridium

ABSTRACT It is now some thirty years since I first undertook the study of “the development of the excretory organs of Amphioxus (Branchiostoma lanceolatum, Pallas). When it was discovered (Goodrich, 1902) that Amphioxus is provided with nephridia of essentially the same structure as the solenocytebearing protonephridia of various Polychaetes and other Invertebrates it became a matter of interest to find out how they are developed. Yet, so far as I am aware, only one author has attempted to give a detailed account of their early development in two short papers (R. Legros, 1909 and 1910). In 1909 I described the nephridia in larval stages, and since then from time to time, as opportunity offered, I have tried to make out their early development and their first origin. The investigator of this problem is confronted with many difficulties. The material is hard to obtain, difficult to preserve satisfactorily, and particularly difficult to stain well. After trying various well-known fixatives, a mixture of corrosive-acetic and picro-nitric for embryonic and Bouin’s fluid for larval stages were found to give the best results. It is essential to anaesthetize the later larvae before fixation to prevent muscular contraction. Borax-carmine and picro-nigrosin, Heidenhain’s iron-haematoxylin and eosin or light-green, and Mann’s methyl-blue and eosin are the most satisfactory stains. The cells are so small and their derivation so difficult to make out that sections of more than about 3 or 4 p in thickness are almost useless. The observations recorded below on embryonic stages were made on material provided by the Zoological Station at Naples for which I here express my gratitude. The larval material was preserved by me at Faro, at Naples, and in Heligoland. I have also to thank the authorities of the Biologisches Anstalt in Heligoland for sending me preserved larvae by post.