Patterning of the vertebrate ventral spinal cord

We review investigations that have lead to a model of how the ventral spinal cord of higher vertebrate embryos is patterned during development. Central to this model is the secreted morphogen protein, Sonic hedgehog. There is now considerable evidence that this molecule acts in a concentration-dependent manner to direct the development of the spinal cord. Recent studies have suggested that two classes of homeodomain proteins are induced by threshold concentrations of Sonic hedgehog. Reciprocal inhibition between the two classes acts to convert the continuous gradient of Sonic hedgehog into defined domains of transcription factor expression. However, a number of aspects of ventral spinal cord patterning remain to be elucidated. Some issues currently under investigation involve temporal aspects of Shh-signalling, the role of other signals in ventral patterning and the characterisation of ventral interneurons. In this review, we discuss the current state of knowledge of these issues and present some preliminary studies aimed at furthering understanding of these processes in spinal cord patterning.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

The distribution and morphological characteristics of serotonergic cells in the brain of monotremes

The distribution and cellular morphology of serotonergic neurons in the brain of two species of monotremes are described. Three clusters of serotonergic neurons were found: a hypothalamic cluster, a cluster in the rostral brainstem and a cluster in the caudal brainstem. Those in the hypothalamus consisted of two groups, the periventricular hypothalamic organ and the infundibular recess, that were intimately associated with the ependymal wall of the third ventricle. Within the rostral brainstem cluster, three distinct divisions were found: the dorsal raphe nucleus (with four subdivisions), the median raphe nucleus and the cells of the supralemniscal region. The dorsal raphe was within and adjacent to the periaqueductal gray matter, the median raphe was associated with the midline ventral to the dorsal raphe, and the cells of the supralemniscal region were in the tegmentum lateral to the median raphe and ventral to the dorsal raphe. The caudal cluster consisted of three divisions: the raphe obscurus nucleus, the raphe pallidus nucleus and the raphe magnus nucleus. The raphe obscurus nucleus was associated with the dorsal midline at the caudal-most part of the medulla oblongata. The raphe pallidus nucleus was found at the ventral midline of the medulla around the inferior olive. Raphe magnus was associated with the midline of the medulla and was found rostral to both the raphe obscurus and raphe pallidus. The results of our study are compared in an evolutionary context with those reported for other mammals and reptiles. Copyright (C) 2002 S. Karger AG, Basel.

Adaptation of rainbow fish to lake and stream habitats

Fish occupy a range of hydrological habitats that exert different demands on locomotor performance. We examined replicate natural populations of the rainbow fishes Melanotaenia eachamensis and M. duboulayi to determine if colonization of low-velocity (lake) habitats by fish from high-velocity (stream) habitats resulted in adaptation of locomotor morphology and performance. Relative to stream conspecifics, lake fish had more posteriorly positioned first dorsal and pelvic fins, and shorter second dorsal fin bases. Habitat dimorphism observed between wild-caught fish was determined to be heritable as it was retained in M. eachamensis offspring raised in a common garden. Repeated evolution of the same heritable phenotype in independently derived populations indicated body shape divergence was a consequence of natural selection. Morphological divergence between hydrological habitats did not support a priori expectations of deeper bodies and caudal peduncles in lake fish. However, observed divergence in fin positioning was consistent with a family-wide association between habitat and morphology, and with empirical studies on other fish species. As predicted, decreased demand for sustained swimming in takes resulted in a reduction in caudal red muscle area of lake fish relative to their stream counterparts. Melanotaenia duboulayi lake fish also had slower sustained swimming speeds (U-crit) than stream conspecifics. In M. eachamensis, habitat affected U-crit of males and females differently. Specifically, females exhibited the pattern observed in M. duboulayi (lake fish had faster U-crit than stream fish), but the opposite association was observed in males (stream males had slower Ucrit than lake males). Stream M. eachamensis also exhibited a reversed pattern of sexual dimorphism in U-crit (males slower than females) relative to all other groups (males faster than females). We suggest that M. eachamensis males from streams responded to factors other than water velocity. Although replication of muscle and U,,it phenotypes across same habitat populations within and/or among species was suggestive of adaptation, the common garden experiment did not confirm a genetic basis to these associations. Kinematic studies should consider the effect of the position and base length of dorsal fins.

Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85-90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re-evaluated to determine the true taxonomic and phylogenetic position of this species.

The embryonic development of the bodywall and nervous system of the cestode flatworm Hymenolepis diminuta

Cestodes (tapeworms) are a derived, parasitic clade of the phylum Platyhelminthes (flatworms). The cestode body wall represents an adaptation to its endoparasitic lifestyle. The epidermis forms a nonciliated syncytium, and both muscular and nervous system are reduced. Morphological differences between cestodes and free-living flatworms become apparent already during early embryogenesis. Cestodes have a complex life cycle that begins with an infectious larva, called the oncosphere. In regard to cell number, cestode oncospheres are among the simplest multicellular organisms, containing in the order of 50-100 cells. As part of our continuing effort to analyze embryonic development in flatworms, we describe here the staining pattern obtained with acTub in embryos and larvae of the cestode Hymenolepis diminuta and, briefly, the monogenean Neoheterocotyle rhinobatidis. In addition, we labeled the embryonic musculature of Hymenolepis with phalloidin. In Hymenolepis embryos, two different cell types that we interpret as neurons and epidermal gland cells express acTub. There exist only two neurons that develop close to the midline at the anterior pole of the embryo. The axons of these two neurons project posteriorly into the center of the oncosphere, where they innervate the complex of muscles that is attached to the booklets. In addition to neurons, acTub labels a small and invariant set of epidermal gland cells that develop at superficial positions, anteriorly adjacent to the neurons, in the dorsal midline, and around the posteriorly located hooklets. During late stages of embryogenesis they spread and form a complete covering of the embryo. We discuss these data in the broader context of platyhelminth embryology.

Immunohistochemical localization of fibromodulin in the periodontium during cementogenesis and root formation in the rat molar

Background: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, fibromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. Objective: The aim of this study was to examine the distribution of fibromodulin during cementogenesis and root formation. Methods: A standard indirect immunoperoxidase technique was employed, using an antifibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. Results: Immunoreactivity to fibromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament fibers insert into the alveolar bone and where the Sharpey's fibers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antifibromodulin antibody increased proportionally with increasing tissue maturation. Conclusion: The results from this study suggest that fibromodulin is a significant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hard-soft tissue interface during cementogenesis.

In ovo electroporation of Crim1 in the developing chick spinal cord

The novel mammalian gene Crim1 encodes a transmembrane bound protein with similarity to the secreted bone morphogenetic protein (BMP) antagonists, vertebrate Chordin, and its Drosophila homologue short gastrulation. Crim1 is expressed in the neural tube in mouse in a restricted pattern, but its function in central nervous system development is largely unknown. We isolated the chicken Crim1 orthologue and analyzed its expression in the developing neural tube. Chicken CRIM1 shares strong homology to human/mouse CRIM1 and C. elegans CRIM1-like proteins. Crim1 is expressed in a similar but not identical pattern to that in the developing spinal cord of mouse, including the notochord, floor plate, motor neurons, and the roof plate. Unlike follistatin, a secreted inhibitor of BMPs, in ovo electroporation of CRIM1, as a full-length transmembrane bound or secreted ectodomain was not sufficient to disrupt early patterning of the neural tube. However, ectodomain CRIM1 overexpression leads to an approximate 50% decrease in populations of specific ventral neuronal populations, including ISL-1(+) motor neurons, CHX-10(+) V1, and EN-1(+) V2 interneurons.

Occlusable corneas in toadfishes: light transmission, movement and ultrastruture of pigment during light- and dark-adaptation

The toadfishes Tetractenos hamiltoni and Torquigener pleurogramma (Tetraodontidae) possess occlusable yellow corneas. We examine the light transmission and location of the yellow/orange pigment throughout the cornea, the temporal properties of pigment migration and the ultrastructure of the pigmented processes during light- and dark-adaptation. Each species was dark-adapted during the day and light-adapted during the night and then exposed to either sun illumination or darkness for different lengths of time (0-70 min). Movement of corneal pigment could be induced in both species regardless of time of day or night. The pigment was able to migrate in a dorsal or ventral direction and changed from minimal to maximal pigmentation within 60 min. Three types of transmission curves were found with varying degrees of transmission in the 400-500 nm waveband, indicating that the pigment distribution is not uniform across the cornea; some areas of the cornea transmit near UV light, while others absorb blue light. The gradual change of the transmission characteristics in different areas of the cornea indicates the presence of different concentrations of a single type of pigment. Ultrastructural examination of the corneas showed that the layer containing the pigment is situated within the scleral cornea either surrounding (T. pleurogramma) or abutting (T. hamiltoni) an iridescent layer. Long sheet-like processes or chromatophores extending centrally from dorsal and ventral reservoirs are filled with pigment during the light-adapted state but empty in the dark-adapted state.

Description of Paeon asymboli n. sp. (Copepoda : Sphyriidae), parasitic on Asymbolus spp. (catsharks) and a new host record for P-australis Kabata, 1993.

Paeon asymboli n. sp. ( Copepoda: Sphyriidae) is described and illustrated from two female specimens taken from the gills of a grey spotted catshark Asymbolus analis ( Ogilby) and an orange spotted catshark A. rubiginosus ( Last, Gomon & Gledhill) ( Scyliorhinidae) from off southeastern Queensland, Australia. The key features for identification are: a pear- shaped trunk, longer than it is wide, along with a cephalothorax characterised by two large ventral papillae, projecting laterally and supporting a number of secondary lobes; a single mid- line, subconical papilla located antero- dorsal to the ventral papillae; an anterior surface bearing two prominent stalked papillae; and an absence of posterolateral lobular processes. P. australis Kabata, 1993 is recorded for the first time from the eastern shovelnose ray Aptychotrema rostrata ( Shaw & Nodder) ( Rhinobatidae).

Trichostome ciliates from Australian marsupials. I. Bandia gen nov (Litostomatea : Amylovoracidae)

A new genus of amylovoracid ciliates, Bandia gen.nov., is described. They are endosymbiotic/endocommensal in the stomachs of macropodid marsupials. Six new species, B. beveridgei, B. equimontanensis, B. tammar, B. deveneyi, B. cribbi and B. smalesae, are described from Setonix brachyurus, Petrogale assimilis, Macropus eugenii, M. robustus, M. parryi and M. agilis respectively. The gross morphology of Bandia is similar to that of Bitricha, with holotrichous somatic ciliation in two fields, longitudinal dorsal and oblique ventral. The somatic kineties are arranged in groups between non-ciliated. major interkinetal ridges; the groups of kineties thus give the cell a banded appearance. Several species are bimorphic, one form holotrichous and the other with a glabrous right body groove which appears to be derived from an ingrowth of one of the major interkinetal ridges. The groove may function in attachment either in sequestration or conjugation. The ultrastructure of the somatic kineties and the oral structures is similar to that of Amylovorax. Bandia also has unique ultrastructural features associated with the major interkinetal ridges, right body groove and a karyophore. Morphological evolution within the Amylovoracidae may have proceeded from simple forms such as Amylovorax via a process of cellular torsion and/or oral migration to forms similar to Bitricha and by further torsion and cellular elaboration to Bandia.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and