Marianne Bronner and Scott Fraser for training and financial support during the first four years of my doctoral work; Debbie Marshall, Drs. I would also like to acknowledge the entire Bronner and Fraser laboratories during my tenure in the Fraser group for encouragement and friendship, and especially Drs. Funding was provided by the National Human Genome Research Institute (NHGRI) Center of Excellence in Genomic Science (CEGS) Grant: P50 HG004071, National Science Foundation (NSF) Human Frontiers Science Program (HFSP) Grant: RGP0028 to JLK for the paleomagnetic and FMR work and the Achievement Rewards to College Scientists (ARCS) Foundation for the ARCS Fellowship that enabled me to complete my doctoral work and be a mother.
I used two FT lines together with a modified version of in toto imaging to study sensory cells in the dorsal spinal cord. These unique transient chemo- and mechano-sensory cells have been well characterized in the dorsal spinal cord of lower vertebrates; however, the notion of persistent RBs contrasts with the dogma that suggests the entire population dies out during the early larval period. In the second part of my dissertation I describe my studies of biogenic magnetite, which has been discovered in a wide range of organisms, including magnetotactic bacteria, migratory fish and birds, invertebrates, and humans.
Introduction Introduction
The spinal cord in mammals, birds and zebrafish begins as the caudal extension of the rostral-caudal patterns of the neural tube (Appel, 2000). The downstream effects of these actions allow spatial constriction of the neuronal subtype despite the proximity of these progenitor domains to each other in the small space of the embryonic spinal cord. Progenitor cells are located medially in the ventricular zone and post-mitotic cells are located at the lateral margins of the spinal cord.
A time-lapse movie of the spinal cord in a ngn1::RFP embryo 32-42 hpf showed that RB neurons (indicated in white. I identified several transgenic lines as part of the FlipTrap (FT) screen in the Center of Excellence in Genomic Science (CEGS) subgroup of the Fraser laboratory Successful integration of the FT vector maintains endogenous control elements and the transgenic lines propagate stably.
Chromosome map of PKCα and the insertion point of the FT vector in ct 7a and 54a. Analysis of morphants revealed the expected reduction and later repopulation of the FT-tagged TG (data not shown) along with absent DRG and RB in single and double FT and ngn1::RFP transgenic lines (Figure 4A-D).
Touch assay score
Fluorescent protein expression phenotype
Beard neurons (RB neurons) are dogmatically defined as preterm primary sensory neurons that die at the end of the embryonic or larval period in most vertebrates, including zebrafish. In the present study, I show that PKCα-YFP-positive RB neurons constitute a subset of the overall population. Also, a morpholino against the fusion protein mRNA and native sequence may elucidate the contribution of the FT itself to the behavioral differences and which may be purely due to the reduction of PKCα.
This discrepancy invites an in vivo study of RB death together with reliable electron micrographic data of apoptotic RBs to shed more light on the cause and timing of the population's demise (Lamborghini, 1987; Reyes et al., 2004; Williams, 2000). Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo. Intramedullary sensory ganglion cells in the roof plate area of the embryonic human spinal cord.
Normal table of zebrafish postembryonic development: staging by externally visible anatomy of the live fish. A versatile gene trap to visualize and interrogate the function of the vertebrate proteome. (eds., pp. We report the unexpected presence of biogenic magnetite in the lateral line region of the genetically and physiologically tractable vertebrate model organism, Danio rerio.
However, all the behavioral evidence can be reasonably explained by the modulation of the magnetite-based receptor system by visual cues (Winklhofer and Kirschvink, 2010). The use of the term “map” has been the subject of debate (Bennett, 1996; Lohmann et al., 2007) because it can lead to misleading comparisons with human maps. The samples were allowed to equilibrate to subzero temperatures before measurement to prevent mechanical rotation of the ferromagnetic particles.
Kirschvink et al., (Kirschvink, Kopp, Raub, Baumgartner, & Holt, 2008), including gradual acquisition of anhysteretic remanent magnetization (ARM) in a peak 100 mT, 830 Hz, alternating field (Af) with a 0-1 mT DC bias, demagnetization of Af of this ARM peak, acquisition of isothermal residual magnetization (IRM) up to peak fields of 1000 mT, progressive demagnetization of Af of this IRM, and demagnetization of the last field of the IRM peak. Due to signal instability from the 77 K samples, only room temperature and 20 K data are reported. Small fragments of broken chiton teeth were located within the scan area but away from the sample to aid co-localization of the magnetic and optical images.
Normalized magnetic moment
Adult fish show changes in IRM indicative of the presence of ferromagnetic iron oxides such as magnetite. Also note that the z values are much more shifted in the frozen animal compared to the room temperature fish. Freezing helps keep the magnetite in a fixed position and prevents changes in orientation that would limit the crystal's ability to respond to the induced magnetic field.
A sample at room temperature is much more subject to thermal mixing and produces correspondingly less IRM signal. Rock magnetometry on whole (larvae) or freeze-dried samples showed measurable magnetic moments and curves characteristic of ferromagnetic material. A) Larva.
Trunks
We captured spectra of the ferromagnetic content of the freeze-dried powder from food-restricted adult zebrafish. When analyzed for biogenic magnetite, the hardness of ARM compared to IRM in Lowrie-Fuller curves points to the presence of single-domain magnetite. In the zebrafish, the demagnetization of ARM requires stronger peak-Af magnetic fields than saturation IRM for the same samples, indicating that the remanent magnetization of the sample is dominated by strongly interacting single-domain magnetic material.
Shown at the top, bottom and right side of the image is the reference strip of chiton toothpicks under non-magnetic Scotch tape. Projections of dorsal root ganglion (DRG), mature Rohon-Beard (RB) neurons, and the posterior lateral line innervate this region; RB neurons are the first to send axons throughout the trunk, followed by lateral line projections and finally the DRG. Moore and Riley reported the presence of single-domain, strongly interacting magnetite in an organic matrix associated with the lateral line of the Atlantic salmon (Moore et al., 1990); the residuals were high enough to indicate that the magneto-receptor system would be able to detect geomagnetic field direction.
Moore and Riley also found magnetite in the lateral line mandibular canal of the European eel, Anguilla anguilla (Moore and Riley, 2009); although migratory (silver) and non-migratory (yellow) stages showed measurable magnetite content, migratory eels had more than 5-fold SIRM per gram of tissue compared to non-migratory eels. The early embryonic and larval lateral line in zebrafish consists of anterior and posterior segments with prominent ganglia and neuromasts that appear to be highly conserved among teleosts (Ghysen and Dambly-Chaudiere, 2004). The lateral line neurons and their innervation are well structured to encounter magnetic stimuli from multiple directions even in young embryos.
The earliest iteration of the magnetosensory apparatus may have detected local geomagnetic anomalies during short-range movements. It is likely that in animals such as zebrafish that do not participate in extensive migrations, magnetite-based magnetoreceptors remained unchanged, while in migratory species such as eels and salmon, the magnetoreceptive capacity of the lateral line was selected for a specialized role in sensing large geomagnetic field differences. Zebrafish are an ideal candidate organism for studying magnetosensation in all its forms, from the neurophysiology and anatomy of magnetosensory structures to the genetics of magnetite biomineralization and the behavioral manifestations of the magnetic sense in vertebrates.
In addition, ADD would like to thank Scott Fraser for the use of the Biological Imaging Center (BIC) at Caltech and Drs.
Conclusion
Rohon-Beard neurons are present and functional throughout early zebrafish life and, as I showed in Chapter 3, seem to persist into adulthood. Given their location in the most dorsal part of the spinal cord, RBs can be easily visualized by a variety of microscopy techniques and are easily approached with microsurgical tools. The captured gene highlighted in the current work, protein kinase C-alpha (PKCα), may support RB neuron survival.
Each of these may be the mode of RB transdifferentiation and may be important in the mature function of RB neurons. Molecular evidence supporting a role for caspase-3-dependent programmed cell death (PCD) (Williams, 2000) is perhaps the strongest, but interestingly, activity is actually required for population elimination via Na+ currents in the Nav1.6- channel (Svoboda) , Linares and Ribera, 2001). The contribution of various developmentally important genes in the maintenance of this cell population, or until they decay, remains to be investigated.
For the reasons outlined above, the RB neuron and its sensory arbors are unique models for interrogating the developmental disorders in the CNS that can lead to adult disease. In addition, a comparison of the involvement of sodium and calcium channels, and studies on Ca2+ intracellular concentrations, ionic flux and tissue gradients in RB survival will shed light on how different ions acting in the same organ and circuits affect neuronal function and maturation, and CNS development in general. A homeodomain protein code specifies stem cell identity and neuronal fate in the ventral neural tube.
Avian magnetoreception: The elaborate iron mineral containing dendrites in the upper beak appear to be a common feature of birds. Developmental changes in the expression of alpha-, beta-, and gamma subunits of protein kinase C at synapses in the ventral horn of the embryonic and postnatal mouse spinal cord. Analysis of upstream elements in the HuC promoter leads to the creation of transgenic zebrafish with fluorescent neurons.
The Rohon beard cell : the formation, function and fate of a primary sensory system in the embryonic zebrafish, Danio rerio, vol.
