Anne Hansen

Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements.

Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration

Inhalation of irritating substances leads to activation of the trigeminal nerve, triggering protective reflexes that include apnea or sneezing. Receptors for trigeminal irritants are generally assumed to be located exclusively on free nerve endings within the nasal epithelium, requiring that trigeminal irritants diffuse through the junctional barrier at the epithelial surface to activate receptors. We find, in both rats and mice, an extensive population of chemosensory cells that reach the surface of the nasal epithelium and form synaptic contacts with trigeminal afferent nerve fibers. These chemosensory cells express T2R “bitter-taste” receptors and α-gustducin, a G protein involved in chemosensory transduction. Functional studies indicate that bitter substances applied to the nasal epithelium activate the trigeminal nerve and evoke changes in respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory cells serve to expand the repertoire of compounds that can activate trigeminal protective reflexes. The trigeminal chemoreceptor cells are likely to be remnants of the phylogenetically ancient population of solitary chemoreceptor cells found in the epithelium of all anamniote aquatic vertebrates.

Correlation between Olfactory Receptor Cell Type and Function in the Channel Catfish

The olfactory epithelium of fish contains three intermingled types of olfactory receptor neurons (ORNs): ciliated, microvillous, and crypt. The present experiments were undertaken to test whether the different types of ORNs respond to different classes of odorants via different families of receptor molecules and G-proteins corresponding to the morphology of the ORN. In catfish, ciliated ORNs express OR-type receptors and Gαolf. Microvillous ORNs are heterogeneous, with many expressing Gαq/11, whereas crypt ORNs express Gαo. Retrograde tracing experiments show that ciliated ORNs project predominantly to regions of the olfactory bulb (OB) that respond to bile salts (medial) and amino acids (ventral) (Nikonov and Caprio, 2001). In contrast, microvillous ORNs project almost entirely to the dorsal surface of the OB, where responses to nucleotides (posterior OB) and amino acids (anterior OB) predominate. These anatomical findings are consistent with our pharmacological results showing that forskolin (which interferes with Gαolf/cAMP signaling) blocks responses to bile salts and markedly reduces responses to amino acids. Conversely, U-73122 and U-73343 (which interfere with Gαq/11/phospholipase C signaling) diminish amino acid responses but leave bile salt and nucleotide responses essentially unchanged. In summary, our results indicate that bile salt odorants are detected predominantly by ciliated ORNs relying on the Gαolf/cAMP transduction cascade. Nucleotides are detected by microvillous ORNs using neither Gαolf/cAMP nor Gαq/11/PLC cascades. Finally, amino acid odorants activate both ciliated and microvillous ORNs but via different transduction pathways in the two types of cells.

Differential distribution of olfactory receptor neurons in goldfish: structural and molecular correlates.

The olfactory system of many terrestrial vertebrates comprises a main olfactory organ and a vomeronasal organ each containing a morphologically distinct type of olfactory receptor neuron (ORN). The two cell types also differ in the expression of G‐proteins and odorant receptor molecules. Fish do not have a vomeronasal organ, and their olfactory neurons—three different morphological types—are contained in one epithelium. The olfactory organ of goldfish appears as a rosette, with the sensory epithelium lying along the proximal portion of each lamella, where it attaches to the midline raphe. Using immunocytochemistry, in situ hybridization, and electron microscopy, we tested whether a correlation exists between receptor cell morphology, distribution of cell type within the sensory epithelium, and expression of odorant receptors and G‐proteins. A strong correlation exists between ORN morphology, type of odorant receptor and G‐protein expressed and the distribution of sensory cells within the olfactory epithelium. The Buck and Axel type of odorant receptor and Gαolf are expressed in tall ciliated ORNs distributed homogenously across the entire sensory epithelium. In contrast, microvillous ORNs expressing V2R‐like receptors, and Gαo, Gαq, or Gαi‐3, and crypt type ORNs expressing Gαo and Gαq, are preferentially located along the dorsal margin of the epithelium and near the midline raphe. V2R‐ and OR‐type receptor molecules do not colocalize in one cell, and only crypt‐type ORNs express more than one G‐protein. J. Comp. Neurol. 477:347–359, 2004.

Taste bud development in the zebrafish, Danio rerio

Taste buds are chemosensory endorgans consisting of modified epithelial cells. Fish and other vertebrates use their taste bud cells to sample potential food, either selecting or rejecting substances according to their edibility. The adult gustatory system in fish has been studied thoroughly, including regeneration experiments. Taste buds occur in the epithelia of the lips, the mouth cavity, the oropharyngeal cavity, and also in the skin of the barbels, the head, and sometimes even all over the body surface. Despite its importance for feeding, little is known about the ontogeny of the fish taste system. We examined the development of taste buds in the zebrafish on the light microscopical and the scanning and transmission electron microscopical levels. Taste buds develop later than the olfactory organ and the solitary chemosensory cells, two other chemosensory systems in aquatic vertebrates. The first few taste bud primordia are visible within the epithelia of lips and gill arches 3 to 4 days after fertilization, and the first few taste buds with open receptor areas appear on the lips and simultaneously on the gill arches 4–5 days after fertilization, which coincides with the onset of feeding. Taste buds in the mouth cavity, on the head, and on the barbels are formed later in development. As seen in other fish, zebrafish taste buds contain elongate dark and light cells, termed according to their electron density. Dark cells with a cell apex of many short microvilli appear first, followed by the light cells with one large microvillus. In addition, the zebrafish has a third fusiform cell type, which appears last. This cell type is low in electron density and has a brush‐like apical ending with several small microvilli. This cell type has not been described previously. Furthermore, in zebrafish, the ontogenetic processes of taste bud formation differ from regenerative processes described in the literature.

Phyletic Distribution of Crypt-Type Olfactory Receptor Neurons in Fishes

The olfactory epithelium of teleost fishes contains ciliated and microvillous olfactory receptor neurons intermingled with supporting cells. Recently the crypt cell, a third type of olfactory receptor neuron (ORN), was described for two ostariophysans. This type of ORN bears apical microvilli as well as occult cilia extending into a crypt at the apex of the cell. The present study used scanning and transmission electron-microscopic methods to examine how widespread this cell type is in other groups of fish. We investigated the olfactory epithelia of 18 species, freshwater and marine, including various actinopterygian fish as well as 2 species of lungfishes belonging to the sarcopterygians. Crypt cells were detected in 13 species of actinopterygian fish, but in none of the sarcopterygian lungfishes. Crypt cells are present in basic as well as in highly derived actinopterygians. We conclude that crypt cells are a common feature of actinopterygian fish.

Ultrastructure of the Olfactory Organ in the Clawed Frog, Xenopus laevis, During Larval Development and Metamorphosis

Development of the olfactory epithelia of the African clawed frog, Xenopus laevis, was studied by scanning and transmission electron microscopy. Stages examined ranged from hatching through the end of metamorphosis. The larval olfactory organ consists of two chambers, the principal cavity and the vomeronasal organ (VNO). A third sensory chamber, the middle cavity, arises during metamorphosis. In larvae, the principal cavity is exposed to water‐borne odorants, but after metamorphosis it is exposed to airborne odorants. The middle cavity and the VNO are always exposed to waterborne odorants.

Early development of the olfactory organ in sturgeons of the genus Acipenser: a comparative and electron microscopic study

Formation and morphology of the olfactory organ of vertebrates has been intensely studied in some taxa for more than a century. As a functionally important and complex sensory organ, its ontogenetic development has often been a matter of debate on higher-level craniate evolution. However, sufficient knowledge of structure and development of the olfactory organ in the crucial taxa needed for a serious phylogenetic reasoning is generally not available. This study aims at this essential primary data source, the detailed structure, morphogenesis, and character definition of the olfactory organ in more basal clades of jawed vertebrates (Gnathostomata). Sturgeon fishes (Acipenseriformes) as recent basal actinopterygians are expected to provide insight into archaic characters and character combinations in bony fishes. Thus, the development of the olfactory placodes of the sterlet, Acipenser ruthenus, and the Siberian sturgeon, Acipenser baerii, was followed histologically, by semi-thin serial sections, and by scanning and transmission electron microscopy. Except for the timing, virtually no differences were observed between the two species. The olfactory placodes become two-layered early in embryonic development. Both the superficial epidermal and the subepidermal layer can easily be distinguished and their development followed by ultrastructural properties. There are three different types of receptor cells: ciliated, microvillous, and crypt cells. The development of the ciliated and the less abundant microvillous receptor cells from the subepidermal layer of the placode is demonstrated. The non-sensory cells of the differentiated olfactory epithelium, i.e. ciliated non-sensory cells and supporting cells, exclusively derive from the superficial epidermal layer. In this respect, acipenserids clearly demonstrate close resemblance to the morphogenetic process found in the tetrapod Xenopus (Anura). The only other adequately described mode found in the actinopterygian zebrafish (Danio rerio), is considered a derived character. In this case, all cells of the differentiated olfactory epithelium derive from one placodal cell layer. The mode of formation of the nasal sac and its ventilatory openings found in the acipenserids examined here, represents a widespread and probably a plesiomorphic condition of osteognathostomes. In both species, differentiation of the basic cellular composition of the olfactory epithelium is far advanced at the time of onset of extrinsic feeding.

Ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish, Carassius auratus.

The ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish was studied by scanning and transmission electron microscopy. A total of 58 adult goldfish of various survival times were examined to determine whether the different types of surgery—either olfactory nerve transection or bulbectomy—yielded differences in the extent or time course of cellular degeneration and renewal. Control animals were also examined in detail to elucidate previous controversial findings concerning the types of olfactory receptor neurons present in goldfish. We found that the intact olfactory epithelium of unoperated control goldfish contains the previously observed ciliated and microvillous receptor neurons, and the crypt cell, a cell type not yet seen in the goldfish but recently reported in other species of teleosts. Following either olfactory nerve transection or bulbectomy, the olfactory receptor neurons showed similar signs of degeneration and subsequent cell death, but, surprisingly, the thickness of the olfactory epithelium did not change significantly with either treatment. The time course of receptor cell renewal was different in axotomized and bulbectomized goldfish. In axotomized goldfish, the amount of receptor cells decreased continuously until 8–13 days after surgery, followed by rapid cell renewal. For bulbectomized goldfish, cell replacement began almost immediately after surgery, with degeneration and cell renewal occurring simultaneously. Six weeks after bulbectomy, cell death and cell proliferation reached a “steady state,” and the epithelia did not further improve. Microsc. Res. Tech. 45:325–338, 1999. 

High correlation between microvillous olfactory receptor cell abundance and sensitivity to pheromones in olfactory nerve-sectioned goldfish

Abstract 1. To determine whether microvillous olfactory receptor cells mediate responses to pheromonal cues, the olfactory nerves of mature male goldfish were axotomized and both the olfactory and behavioral sensitivity of these animals to olfactory stimuli investigated after which the histological condition of their olfactory epithelia was determined. 2. Behavioral responsiveness to food odor returned within 2 weeks but responsiveness to sexually-active females (pheromones) took 4–10 weeks to return. 3. Electro-olfactogram recordings from the olfactory epithelium of axotomized fish found that olfactory responsiveness to amino acids and pheromones changed little during the first week subsequent to axotomy. However, olfactory sensitivity decreased rapidly during the second week. During the course of the third week, electro-olfactogram sensitivity to amino acids remained while exposure to pheromones evoked no recordable electro-olfactogram. During week 4, sensitivity to amino acids increased further, and weak sensitivity to some pheromones became evident. Further recovery of electro-olfactogram sensitivity to all odorants was slow and erratic over the next 6 months, particularly to the pheromones. 4. Histological examination of the olfactory epithelia of axotomized fish demonstrated that while ciliated receptor cells were present within 2 weeks, microvillous receptor cells took approximately 4 weeks to regenerate. 5. Together these data suggest that microvillous receptor cells mediate responsiveness to pheromones in this species.