Browsing by Author "Linney, Elwood A"
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Item Open Access Intercellular Signaling Pathways in the Initiation of Mammalian Forebrain Development(2007-05-03T18:54:17Z) Yang, Yu-PingThe Spemann organizer in amphibians gives rise to the anterior mesendoderm (AME) and is capable of inducing neural tissues. This inductive activity is thought to occur largely via the antagonism of Bone Morphogenetic Protein (BMP) signaling in the organizer. In the mouse, BMP antagonists Chordin and Noggin function redundantly in the AME and are required during forebrain maintenance. However, the timing of forebrain initiation and the function of BMP antagonism in forebrain initiation remained unclear prior to this study. In addition, the Transforming Growth Factor β (TGFβ) ligand Nodal patterns the forebrain via its function in the anterior primitive streak (APS), the precursor tissue of the AME. Whether BMP and Nodal signaling pathways interact has not been previously investigated. The goal of this dissertation was to investigate the cellular and molecular mechanisms involved in early mammalian forebrain establishment by embryonic and genetic manipulations. This study determined that forebrain initiation occurs during early gastrulation and requires signals from the AVE and AME. The AVE was identified as a source of active BMP antagonism in vivo, and the BMP antagonism supplied by exogenous tissues was capable to promote forebrain initiation and maintenance in the murine ectoderm. It is likely that BMP antagonism enhances forebrain gene expression via inhibiting posteriorization. This study further identified a possible crosstalk between BMP and Nodal signaling. Loss of Chordin or Noggin in combination with heterozygosity for Nodal or Smad3 results in holoprosencephaly. Molecular analyses suggest that the BMP-Nodal interaction occurs in the APS and/or the AME. Failure of this interaction results in an imbalance of BMP and Nodal signal levels that devastate APS and AME patterning during early forebrain establishment, ultimately leading to holoprosencephaly in mid-gestation. This interaction is likely to occur extracellularly, possibly by formation of a BMP-Nodal heteromeric complex. Furthermore, the spatiotemporal expression of phospho-Smad1/5/8, an effector of BMP signaling pathway, was characterized during early mouse embryogenesis. Distribution of phospho-Smad1/5/8 serves as a faithful readout of BMP signaling activity and helps to better understand how BMPs are involved in patterning early embryos. The implication of phospho-Smad1/5/8 expression in both wildtype and mutant embryos is also discussed.Item Open Access Organophosphorus Pesticide Exposure Effects on Neurobehavioral Development of Zebrafish and Characterization of Developmental Switch Between Two Cation-chloride Cotransporters (zfnkcc1 and zfkcc2)(2012) Yen, JerryOrganophosphorus pesticides (OPs) are neuroactive compounds that can affect the development of the nervous system and behavior. Using zebrafish, we compared the effectiveness of three different OPs, chlorpyrifos (CPF), diazinon (DZN) and parathion (PA) on survival, AChE inhibition and larval motility. We conclude that at equimolar concentrations CPF is the most effective OP of the three tested. 300nM CPF inhibited AChE when measured at 5 days post fertilization (dpf) by 80%, which correlated to a 35% decrease in larval motility when measured at 6 dpf.
We isolated the cDNA of zfkcc2, which encodes for an important cation-chloride cotransporter responsible for establishing a chloride gradient that allows for the neurotransmitter GABA to become inhibitory for a subset of neurons. By isolating the cDNA of zfkcc2, we were able to characterize its expression pattern up to 4 dpf. Using qRT-PCR, we showed that the expression of zfkcc2 rises from 2 to 96 hours post fertilization (hpf). The expression of zfkcc2 is found at 1 dpf in the forebrain, midbrain and hindbrain in a distinct pattern. By 3 dpf, zfkcc2 expression is found throughout most of the developing zebrafish brain. We were then able to characterize the expression pattern of zfnkcc1, which encodes for a cation-chloride cotransporter that works opposite to zfkcc2 and establishes a chloride gradient allowing GABA to be excitatory during development. Expression of zfnkcc1 is found at 1 dpf throughout most of the developing zebrafish head including in some non-neural tissue, but appears concentrated to the otic vesicle at 3 dpf. Using double in situ hybridization, we investigated the nature of the developmental switch from zfnkcc1 to zfkcc2 by looking at regions where there was an interaction between both genes' expression patterns in the developing zebrafish nervous system.
We preliminarily examined how affecting nicotinic signaling with exposures to nicotinic agonists/antagonists can affect the expression of zfnkcc2 and zfkcc2. While there were no apparent changes in the expression pattern from any of the exposures at 2 dpf, we found that by 3 dpf nicotine and CPF exposures resulted in an incomplete expression pattern for zfkcc2 that seemed to be delayed. However, the expression of zfnkcc1 at the otic vesicle was unaffected by exposure.
Our results suggest that disrupting nicotinic signaling during development can affect the expression of an important cation-chloride cotransporter, zfkcc2, that plays a major role in establishing the necessary chloride gradient for the proper functioning of GABA during development. Future studies should focus on investigating the nature of the developmental switch between zfnkcc1 and zfkcc2 to see if the possibility of their regulation being coupled is a target for exposures.