Ron Koss

GDF6, a novel locus for a spectrum of ocular developmental anomalies

Colobomata represent visually impairing ocular closure defects that are associated with a diverse range of developmental anomalies. Characterization of a chromosome 8q21.2-q22.1 segmental deletion in a patient with chorioretinal coloboma revealed elements of nonallelic homologous recombination and nonhomologous end joining. This genomic architecture extends the range of chromosomal rearrangements associated with human disease and indicates that a broader spectrum of human chromosomal rearrangements may use coupled homologous and nonhomologous mechanisms. We also demonstrate that the segmental deletion encompasses GDF6, encoding a member of the bone-morphogenetic protein family, and that inhibition of gdf6a in a model organism accurately recapitulates the probands phenotype. The spectrum of disorders generated by morpholino inhibition and the more severe defects (microphthalmia and anophthalmia) observed at higher doses illustrate the key role of GDF6 in ocular development. These results underscore the value of integrated clinical and molecular investigation of patients with chromosomal anomalies.

Fine structural studies of the nervous system and the apical organ in the planula larva of the sea anemone Anthopleura elegantissima

The nervous system of the planula larva of Anthopleura elegantissima consists of an apical organ, one type of endodermal receptor cell, two types of ectodermal receptor cells, central neurons and nerve plexus. Both interneural and neuromuscular synapses are found in the nerve plexus.

Fine structure of the cephalic sensory organ in the larva of the nudibranch Rostanga pulchra (Mollusca, Opisthobranchia, Nudibranchia)

SummaryThe cephalic sensory organ in the veliger larva of Rostanga pulchra is situated dorsally between the rhinophores, emerging as a tuft of cilia. This organ is made up of three types of sensory cells, and based on their morphology have been termed ampullary, parampullary and ciliary tuft cells. The cell bodies of the organ originate in the cerebral commissure, and their dendrites pass to the epidermis as three tracts. Dendrites terminate in the epidermis to form a sectorial field. Axons of these cells run into the mass of neurites in the cerebral commissure but no synapses were observed in this area. Morphological evidence suggests that the cephalic sensory organ may function in chemoreception and mechanoreception related to substrate selection at settlement, feeding, or other behaviors.

Early development of an identified serotonergic neuron in Helisoma trivolvis embryos: Serotonin expression, de-expression, and uptake

In early-stage embryos of Helisoma trivolvis, a bilateral pair of identified neurons (ENC1) express serotonin and project primary descending neurites that ramify in the pedal region of the embryo prior to the formation of central ganglia. Pharmacological studies suggest that serotonin released from ENC1 acts in an autoregulatory pathway to regulate its own neurite branching and in a paracrine or synaptic pathway to regulate the activity of pedal ciliary cells. In the present study, several key features of early ENC1 development were characterized as a necessary foundation for further experimental studies on the mechanisms underlying ENC1 development and its physiological role during embryogenesis. ENC1 morphology was determined by confocal microscopy of serotonin-immunostained embryos and by differential-interference contrast (DIC) microscopy of live embryos. The soma was located at an anteriolateral superficial position and contained several distinguishing features, including a large spherical nucleus with prominent central nucleolus, large granules in the apical cytoplasm, a broad apical dendrite ending in a sensory-like structure at the embryonic surface, and a ventral neurite. ENC1 first expressed serotonin immunoreactivity around stage E13, followed immediately by the appearance of an immunoreactive neurite (stage E14). Both the intensity of immunoreactivity and primary neurite length were consistently greater in the right ENC1 at early stages. Serotonin uptake, as indicated by 5,7-dihydroxytryptamine-induced fluorescence, first occurred between stages E18 and E25. At later stages of embryogenesis (after stage E65), serotonin immunoreactivity disappeared, whereas serotonin uptake and normal cell morphology were retained.

Induction of Settlement and Metamorphosis of the Veliger Larvae of the Nudibranch, Onchidoris bilamellata

Summary Onchidoris bilamellata veligers were reared in the laboratory on a combination of phytonagellates and diatoms. They attained metamorphic competence after a period of 28 to 32 days at 11°C, or 60 to 80 days at a temperature averaging 7.5° C. Experimental evidence suggests that settlement is stimulated by a diffusible chemical emanating from living barnacles, whereas metamorphosis is induced by a chemical or mechanochemical cue, which is also associated with barnacles. Settlement and metamorphosis are considered to be separable events in O. bilamellata. The settlement response is reversible and can be repeated; it involves a characteristic behavioral repertoire including descent to the bottom, foot contortions and crawling on the pedal sole. Settlement occurs only in seawater that contains, or had previously contained, living barnacles. Metamorphosis is irreversible and involves the resorption of the velum, loss of the larval shell, and incorporation of the visceral mass into the cephalopedal mass. ...

Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells

SUMMARY Embryos of the pond snail Helisoma trivolvis express three known subtypes of ciliary cells on the surface of the embryo early in development: pedal, dorsolateral and scattered single ciliary cells (SSCCs). The pedal and dorsolateral ciliary cells are innervated by a pair of serotonergic sensory-motor neurons and are responsible for generating the earliest whole-animal behavior, rotation within the egg capsule. Previous cell culture studies on unidentified ciliary cells revealed that serotonin (5-hydroxytryptamine; 5-HT) produces a significant increase in the ciliary beat frequency (CBF) in a large proportion of ciliary cells. Both Ca2+ influx and a unique isoform of protein kinase C (PKC) were implicated in the signal transduction pathway underlying the cilio-excitatory response to 5-HT. The goal of the present study was to characterize the anatomical and physiological differences between the three known populations of superficial ciliary cells. The pedal and dorsolateral ciliary cells shared common structural characteristics, including flat morphology, dense cilia and lateral accessory ciliary rootlets. By contrast, the SSCCs had a cuboidal morphology, reduced number of cilia, increased ciliary length and absence of lateral accessory rootlets. In cultures containing unidentified ciliary cells, the calcium/calmodulin-dependent enzyme inhibitor calmidazolium (2 μmol l–1) blocked the stimulatory effect of 5-HT (100 μmol l–1) on CBF. In addition, 50% of unidentified cultured cells responded to 5-HT (100 μmol l–1) with an increase in [Ca2+]i. To facilitate the functional analyses of the individual populations, we developed a method to culture identified ciliary subtypes and characterized their ciliary and calcium responses to 5-HT. In cultures containing either pedal or dorsolateral ciliary cells, 5-HT (100μ mol l–1) produced a rapid increase in CBF and a slower increase in [Ca2+]i in all cells examined. By contrast, the CBF and [Ca2+]i of SSCCs were not affected by 100μ mol l–1 5-HT. Immunohistochemistry for two putative 5-HT receptors recently cloned from Helisoma revealed that pedal and dorsolateral ciliary cells consistently express the 5-HT1Hel protein. Intense 5-HT7Hel immunoreactivity was observed in only a subset of pedal and dorsolateral ciliary cells. Cells neighboring the SSCCs, but not the ciliary cells themselves, expressed 5-HT1Hel and 5-HT7Hel immunoreactivity. These data suggest that the pedal and dorsolateral ciliary cells, but not the SSCCs are a homogeneous physiological subtype that will be useful for elucidating the signal transduction mechanisms underlying 5-HT induced cilio-excitation.

Pbx homeodomain proteins pattern both the zebrafish retina and tectum

BackgroundPbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined.ResultsAnalysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth.ConclusionThese data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.

Fine structure of the larval rhinophores of the nudibranch, Rostanga pulchra, with emphasis on the sensory receptor cells

SummaryThe rhinophores of the veliger larva of Rostanga pulchra are located in the intravelar field near the base of the velar lobes. Each rhinophore is a cylindrical structure, tapering distally, and covered with a dense meshwork of microvilli. A conspicuous row of ciliary tufts runs along each side of the rhinophore and several stiffer tufts, composed of fewer cilia, are positioned around the tip or at the base. The rhinophoral epithelium consists of supporting cells, ciliated cells (giving rise to the ciliary rows), dendritic terminals (giving rise to the tufts around the apex), and sinuses containing occasional amebocytes. The lumen of the rhinophore is occupied by the rhinophoral ganglion and muscle cells that are oriented in two perpendicular planes. Cell bodies of the dendritic endings are located within the rhinophoral ganglion, which in turn joins into the optic and cerebral ganglia. Rhinophoral ganglionic neurons do not synapse with each other, but numerous neuromuscular synapses are found in the lumen of the rhinophore.Morphological evidence suggests that the dendritic endings are chemoreceptors and the ciliated cells are possibly mechanoreceptors but are not functional at this stage in development. The functional role of the rhinophores is discussed in relation to larval behavior at settlement and metamorphosis.

Fine structural study of the statocysts in the veliger larva of the nudibranch, Rostanga pulchra

SummaryThe two statocysts of the veliger larva of Rostanga pulchra are positioned within the base of the foot. They are spherical, fluid-filled capsule that contain a large, calcareous statolith and several smaller concretions. The epithelium of the statocyst is composed of 10 ciliated sensory cells (hair cells) and 11 accessory cells. The latter group stains darkly and includes 2 microvillous cells, 7 supporting cells, and 2 glial cells. The hair cells stain lightly and each gives rise to an axon; two types can be distinguished. The first type, in which a minimum of 3 cilia are randomly positioned on the apical cell membrane, is restricted to the upper portion of the statocyst. The second type, in which 9 to 11 cilia are arranged in a slightly curved row, is found exclusively around the base of the statocyst. Each statocyst is connected dorso-laterally to the ipsilateral cerebral ganglion by a short static nerve, formed by axons arising from the hair cells. Ganglionic neurons synapse with these axons as the static nerve enters the cerebral ganglion. The lumen of the statocyst is continuous with a blind constricted canal located beneath the static nerve.A diagram showing the structure of the statocyst and its association with the nervous system is presented. Possible functions of the statocyst in relation to larval behavior are discussed.

A fine structural study of metamorphosis of the hydrozoan Mitrocomella polydiademata

This report is a comprehensive fine structural analysis of the morphological changes occurring during metamorphosis of the marine hydrozoan Mitrocomella polydiademata. Five stages are recognized during metamorphosis: planulae just prior to settlement, ball and filiform stages, immature polyps, and primary feeding polyps. Settlement and metamorphosis of cnidarian planulae involve such changes as ciliary arrest, discharge of nematocytes, secretion of glandular cells, differentiation of cells, and changes in cell and body shape.