Polarity and Competition in the Development of the Calyx of Held Terminal in the Medial Nucleus of the Trapezoid Body in the Mouse

In the auditory brainstem, the connection between globular bushy cells of the anteroventral cochlear nucleus and principal cells (PCs) of the medial nucleus of the trapezoid body (MNTB) is created by one of the largest nerve terminals in the central nervous system, the calyx of Held (CH). The characteristics of the CH:MNTB connection—a short developmental period (48-72 hours), accessibility for recording from pre- and postsynaptic components, and clear monoinnervated end point—make this system an ideal model system for studying nervous system development. Model systems undergo stereotyped stages of development, including exuberant overinnervation, competition between terminals, and a refinement of innervation through removal of weak inputs. However, unlike other similar model systems (climbing fiber:Purkinje cell, retinal ganglion cells:dorsolateral geniculate nucleus), it has been a long-standing question whether the CH:MNTB system undergoes competition. We investigated the innervation state of PCs using the novel technique of segmentation and 3D reconstruction of PCs and their associated inputs across important developmental timepoints (postnatal days (P)2,3,4,6,9,30). This was accomplished by application of serial block-face scanning electron microscopy (SBEM), a method of serial section electron microscopy providing high spatial resolution (~4-10nm) and a high degree of alignment between images with very little section loss. Applying this technique, we show early exuberant innervation of PCs (P2), establish that competition is a common process, and pinpoint the 24-hour period from P3-P4 as a uniquely active day in CH growth during which terminal contact with PCs increases at a rate exceeding 200 µm2/day. Common morphological characteristics of the CH:MNTB connection also became qualitatively evident based on 3D reconstructions, particularly an eccentric PC nucleus and preference for polarized terminal growth. Based on these observations, we undertook a quantitative study of polarity in CH:MNTB development using our 3D reconstructions. The results of this investigation demonstrate a novel polarity in development of both the CH and PC; developing PCs are characterized by eccentrically placed nuclei that establish an “intrasomatic polarity” that persists through young adulthood (P30). This polarity appears to define a unique territory opposite of the nuclear location that is amenable to growth of the calyx, is enriched in dendrites, and is selectively enlarged as the principal cell matures to create glia-free surface area for innervation. To our knowledge, this is the first report of a polarity program in the coordinated pre- and postsynaptic development of a non-laminar brain region. Additionally, our findings have codified a progression of dendritic pruning in the maturation of principal cells that may influence and be influenced by the developmental state of the cell. Taken in totality, these results indicate a highly polarized, systemic competitive process in the MNTB during the development of the calyx of Held and suggests potential mediators of competition that deserve further study

Developmental Toxicity of Microplastic Leachates on Marine Larvae

Marine plastic pollution is now considered a diverse contaminant suite, differing in product origin, polymer composition, size, morphology, colour, additives and environmental co-contaminants. The environmental hazards associated with marine plastic pollution have been widely documented, however much of the existing research has yet to document developmental abnormalities observed when biota develop in plastic contaminated systems. The effect of microplastic leachate exposure on two marine echinoderms early developmental stages were investigated. Psammechinus milaris and Paracentrotus lividus embryonic and larval cultures were exposed to leachates derived from industrial or environmental exposed plastic pellets to investigate the effect of polymer additives and environmental contaminants. Toxicity was evaluated morphologically using images of live embryos and larvae, along with immunostaining of key developmental tissue groups to determine the extent of impact on a physiological level. This body of work suggests that leachates from pellets exposed to environmental contaminates (biobead and pre-production nurdle pellets) and highly plasticised industrial pellets (polyvinyl chloride) elicit severe, consistent and treatment-specific phenotypes in P. lividus embryonic and larval developmental stages, with impacts on morphogenic processes. Key differences in larvae morphology were documented between plastic types and environmental exposure. Industrial polyvinyl chloride pellets elicited the most pronounced abnormalities from the wild type at 24 hours post-fertilisation. However, leachates from un-plasticised industrial polyethylene pellets showed little differences from the wildtype with regards to developmental timing and abnormalities. Leachates from environmental sourced pellets elicited the most severe developmental delays and abnormalities at 48 hours post-fertilisation. Preliminary chemical analysis was also performed on industrial and beached pellet leachates, to investigate compound composition and to determine possible contributors to the developmental defects. To summarise, the findings suggest industrial and environmental microplastic leachate exposure elicits morphological malformations and specific abnormalities in neural, cilia and muscle tissues groups in both embryonic and larval stages of marine larvae of P. lividus. However, more research and investigation are needed to draw conclusive data

H+/K+ ATPase activity is required for biomineralization in sea urchin embryos

AbstractThe bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H+/K+ ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles

Molecular Techniques Reveal Wide Phyletic Diversity of Heterotrophic Microbes Associated with Discodermia spp. (Porifera: Demospongiae)

Sponges are well known to harbor large numbers of heterotrophic microbes within their mesohyl. Studies to determine the diversity of these associated microbes have been attempted for only a few shallow water species. We cultured various microorganisms from several species of Discodermia collected from deep water using the \u27Johnson-Sea-Link\u27 manned submersibles, and characterised them by standard microbiological identification methods. Characterisation of a small proportion (ca. 10%) of the total and potential eubacterial isolate collection with molecular systematics techniques revealed a wide diversity of microbes. Phylogenetic analyses of 32 small subunit (SSU) 16S-like rRNA gene sequences from different micorbes indicated high levels of taxonomic diversity assoiated with this genus of sponge. For example, bacteria from at least five cubacterial subdivisions - gamma, alpha, beta, Cytophaga and Gram positive - were isolated from the mesohyl of Discodermia. Several strains were unidentifiable from current sequence databases. No overlap was found between sequences of 24 isolates and 8 sequences obtained by PCR and cloning directly from sponge samples. The abundance and diversity of microbes associated with sponges such as Discodermia suggest that they may play important roles in marine microbial ecology, dispersal and evolution

Bioelectrical dynamics are required for normal development of the sea urchin embryo

Bioelectricity refers to differential membrane voltage and cytoplasmic ion concentrations in tissues or cells which persist over long periods of time. Differences in these steady-state ionic conditions are responsible for large-scale axial patterning and morphogenesis in developing embryos. The sea urchin embryo is an excellent model organism for studying embryonic development, yet a comprehensive study of bioelectricity in sea urchin development has not been reported. Differential ion channel activity is a primary means by which bioelectricity is controlled; thus, we hypothesized that disrupting ion channel activity would reveal the requirements for bioelectricity in the sea urchin embryo. We performed a screen of ion channel inhibitors and discovered that their activities are required for many processes in sea urchin development. We chose two interesting phenotypes to investigate further. First, we demonstrate that H+/K+ ATPase (HKA) activity is required for biomineralization of the sea urchin larval skeleton. We determined that embryos raised with HKA inhibitors initially exhibit voltage and pH changes, then revert to normal voltage and pH during biomineralization via compensatory changes in sodium and chloride ions; it is likely that these compensatory changes lead to defects in transport of carbonate ions, that in turn, inhibit biomineralization of the calcium carbonate skeleton. We hypothesize that similar mechanisms are at play in human patients on long-term HKA inhibitors to treat acid reflux, in whom biomineralization is also decreased. Next, we demonstrate that V-type H+ ATPase (VHA) activity is required for specification of the dorsal-ventral (DV) axis, for the normal inactivation of p38 MAPK in the presumptive dorsal region, and for the subsequent asymmetric onset of expression of the TGFβ family member Nodal, that locally specifies the ventral territory. Embryos treated with VHA inhibitors exhibit global p38 MAPK activity and Nodal expression, and are ventralized. We describe previously unknown gradients of voltage and pH across the DV axis, the sharpness of which requires VHA activity. We propose that the voltage and pH gradients encode spatial information which confers asymmetry on p38 MAPK activity. Overall, we demonstrate that bioelectrical changes are essential for development of the sea urchin embryo, specifically via roles in biomineralization and DV axis specification.2019-01-2

Lack of Chemical Defense in Two Species of Stalked Crinoids: Support for the Predation Hypothesis for Mesozoic Bathymetric Restriction

Methanol/dichloromethane extracts of (1) the arms and pinnules, and (2) the stalk and cirri of the deep water stalked crinoids Endoxocrinus parrae (Gervais) and Neocrinus decorus (Carpenter) were imbedded at ecologically relevant volumetric concentrations in alginate food pellets containing 2% krill as a feeding stimulant and presented in situ to an assemblage of shallow-water reef fish. Experimental pellets were highly palatable to reef fish; no significant differences in pellet consumption occurred between experimental pellets containing extracts from either species of stalked crinoid or control pellets. Small pieces of cirri, stalks, calyx, arms and pinnules of both species were also tested in in situ feeding assays. While immediate consumption by fish was not apparent, Blue Headed Wrasse (Thalassoma bifasciatum (Block)) and Dusky Damselfish (Stegastes fuscus (Cuvier)) bit at pieces of each body component. Similar fish biting behaviors were also observed when two living Endoxocrinus parrae were deployed on the shallow reef. Observations indicate that neither species of stalked crinoid is chemically defended from predation by a natural assemblage of reef fish. This supports the predation hypothesis that restriction of stalked crinoids in deep-water habitats may have resulted from the Mesozoic radiation of durophagous fishes in shallow seas, resulting in a reduction of stalked crinoids from shallow water

PhD

dissertationThe mammalian retina is comprised of 55-60 cell types mediating transduction of photic information through visual preprocessing channels. These cell types fall into six major cell superclasses including photoreceptors, horizontal, amacrine, Muller and ganglion cells. Through computational molecular phenotyping, using amino acids as discriminands, this dissertation shows that the major cellular superclasses of the murine retina are subdivisible into the following natural classes; 1 retinal pigment epithelium class, 2 photoreceptor, 2 bipolar cell, 1 horizontal cell, 15 amacrine cell, 1 Muller cell, and 7 ganglion cell classes. Retinal degenerative diseases like retinitis pigmentosa result in loss of photoreceptors, which constitutes deafferentation of the neural retina. This deafferentation, when complete, is followed by retinal remodeling, which is the common fate of all retinal degenerations that trigger photoreceptor loss. The same strategy used to visualize cell classes in wild type murine retina was applied to examples of retinal degenerative disease in human tissues and naturally and genetically engineered models, examining all cell types in 17 human cases of retinitis pigmentosa (RP) and 85 cases of rodent retinal degenerations encompassing 13 different genetic models. Computational molecular phenotyping concurrently visualized glial transformations, neuronal translocations, and the emergence of novel synaptic complexes, achievements not possible with any other method. The fusion of phenotyping and anatomy at the ultrastructure level also enabled the modeling of synaptic connections, illustrating that the degenerating retina produces new synapses with vigor with the possibility that this phenomenon might be exploited to rescue vision. However, this circuitry is likely corruptive of visual processing and reflects, we believe, attempts by neurons to find synaptic excitation, demonstrating that even minor rewiring seriously corrupts signal processing in retinal pathways leaving many current approaches to bionic and biological retinal rescue unsustainable. The ultimate conclusion is that the sequelae of retinal degenerative disease are far more complex than previously believed, and schemes to rescue vision via bionic implants or stem/engineered cells are based on presumed beliefs in preservation of normal wiring and cell population patterning after photoreceptor death. Those beliefs are incorrect: retinal neurons die, migrate, and create new circuitries. Vision rescue strategies will need to be refined

PhD

dissertationThe mammalian retina is comprised of 55-60 cell types mediating transduction of photic information through visual preprocessing channels. These cell types fall into six major cell superclasses including photoreceptors, horizontal, amacrine, Muller and ganglion cells. Through computational molecular phenotyping, using amino acids as discriminands, this dissertation shows that the major cellular superclasses of the murine retina are subdivisible into the following natural classes; 1 retinal pigment epithelium class, 2 photoreceptor, 2 bipolar cell, 1 horizontal cell, 15 amacrine cell, 1 Muller cell, and 7 ganglion cell classes. Retinal degenerative diseases like retinitis pigmentosa result in loss of photoreceptors, which constitutes deafferentation of the neural retina. This deafferentation, when complete, is followed by retinal remodeling, which is the common fate of all retinal degenerations that trigger photoreceptor loss. The same strategy used to visualize cell classes in wild type murine retina was applied to examples of retinal degenerative disease in human tissues and naturally and genetically engineered models, examining all cell types in 17 human cases of retinitis pigmentosa (RP) and 85 cases of rodent retinal degenerations encompassing 13 different genetic models. Computational molecular phenotyping concurrently visualized glial transformations, neuronal translocations, and the emergence of novel synaptic complexes, achievements not possible with any other method. The fusion of phenotyping and anatomy at the ultrastructure level also enabled the modeling of synaptic connections, illustrating that the degenerating retina produces new synapses with vigor with the possibility that this phenomenon might be exploited to rescue vision. However, this circuitry is likely corruptive of visual processing and reflects, we believe, attempts by neurons to find synaptic excitation, demonstrating that even minor rewiring seriously corrupts signal processing in retinal pathways leaving many current approaches to bionic and biological retinal rescue unsustainable. The ultimate conclusion is that the sequelae of retinal degenerative disease are far more complex than previously believed, and schemes to rescue vision via bionic implants or stem/engineered cells are based on presumed beliefs in preservation of normal wiring and cell population patterning after photoreceptor death. Those beliefs are incorrect: retinal neurons die, migrate, and create new circuitries. Vision rescue strategies will need to be refined

A GAP in Form and Function: The Rac-GAP Alpha2-Chimaerin Regulates Dendrite Morphogenesis and Synaptic Plasticity.

A remarkable feature of the central nervous system is the highly choreographed wiring of neuronal circuits, which provides the structural and functional framework for cognitive abilities in humans. One critical parameter in this process is the elaboration of axonal and dendritic arbors as their size dictates the number of synaptic inputs a neuron can form. Such exquisite connectivity relies on the morphological complexity of the individual neuron. In neurons, cytoskeletal regulators dictate the extent of axonal and dendritic arborization. At the synapse, molecular mechanisms alter cytoskeletal dynamics in response to extracellular cues, thus linking neuronal morphological plasticity to synaptic activity. A central goal in Neuroscience is to elucidate the molecular mechanisms that regulate intracellular cytoskeletal rearrangement, which mediates morphological transformations in dendritic spines. Importantly, aberrant dendritic spine morphogenesis and synaptic function are unifying features in neurodevelopmental, neuropsychiatric and neurodegenerative diseases. Defects in EphR/ephrin signaling and Rac1 function at the synapse are implicated in the etiology of neurological disorders that exhibit dendritic spine defects. My dissertation contains three independent studies that focus on the molecular players that link structural and functional mechanisms at the synapse. First, I identified that the Rac-GAP, α2-chimaerin, is a key regulator of convergent signaling pathways required for normal dendritic spine morphogenesis and synaptic function. Second, I demonstrate that the loss of α2-chimaerin alters proliferation in the adult dentate gyrus that may correlate to a reduction in basal anxiety in rodents. Third, I characterized an excitatory ionotropic GABA receptor in the nematode Caenorhabditis elegans, and determined it plays a role in extrasynaptic spillover transmission. Together, these data shed new insight into the molecular mechanisms that regulate dendritic spine morphogenesis and synaptic function, and demonstrate that α2-chimaerin is poised at the nexus of critical signaling pathways to transduce extracellular synaptic signaling to intracellular regulation of dendritic spines and synapses.PhDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/120893/1/cmvaldez_1.pd