Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

University of Georgia

Pax6, a member of the paired-family of transcription factors, exhibits restricted expression and essential functions in the developing eye, olfactory system, central nervous system, and pancreas. To understand Pax6 function, which critically depends on induction of proper expression levels during development, it is necessary to elucidate the molecular mechanisms governing Pax6 transcription. Although previous studies using classic transgenic approaches have provided a wealth of information about the distribution and types of regulatory elements involved in Pax6 regulation, genetic studies in both humans and mice indicate that these enhancers alone are not sufficient for fully regulated Pax6 expression. We report here our analysis of mice transgenic for a 160 kb mouse Pax6 BAC transgene, which was generated as a necessary first step towards testing the long-range control of Pax6 expression in vivo. We show that this BAC transgene replicates Pax6 expression in the eye. This is the first time that a reporter transgene has been expressed in a normal Pax6-like pattern in all of the tissues of the eye and defines an eye regulatory region within the Pax6 downstream regulatory region (DRR). Second, we show that this BAC transgene contains all of the cis regulatory elements required for normal Pax6 expression within the developing embryo, except for within the diencephalon and olfactory bulb. Third, we show that this transgene is subject to Pax6 autoregulation. Lastly, we identify, for the first time in mammals, an isoform of the Pax6 protein lacking the paired domain. This isoform is expressed in the developing olfactory bulb and eye. Over-expression of Pax6DeltaPD causes a microphthalmic phenotype in both Pax6(+/+) mice and Pax6(+/-) mice. These results demonstrate a role for Pax6DeltaPD in eye development, which appears to be different than that ascribed to either canonical Pax6 or Pax6(5a).

Analysis of Pax6 expression using a BAC transgene reveals the presence of a paired-less isoform of Pax6 in the eye and olfactory bulb.

The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.

Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals.

Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)-based Ca(2+) indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.

New statistical methods enhance imaging of cameleon fluorescence resonance energy transfer in cultured zebrafish spinal neurons.

The Pax6 transcription factor is required for multiple aspects of vertebrate eye development. The Pax6 gene encodes isoforms that either contain (Pax6+PD) or lack (Pax6DeltaPD) the N-terminal paired-box DNA-binding domain, in addition to the homeodomain. Alternative promoters control the expression of Pax6+PD and Pax6DeltaPD in the eye. Using a modified bacterial artificial chromosome (BAC) transgene that specifically expresses Pax6DeltaPD, but not paired-containing Pax6, in the normal endogenous pattern, we show that overexpression of Pax6DeltaPD causes a severe microphthalmic phenotype in both wild-type and Pax6-deficient (Sey(/+)) mice in a dosage-dependent manner. The microphthalmic phenotype is due to lens degeneration during embryonic development. Lens development initiates correctly, but cells in the lens undergo apoptotic cell death between E12 and E13. Concomitantly, in these mice, changes in Bmp4, Msx1, and Wnt2b expression were observed in the mesenchymal cells of the developing cornea. To visualize Pax6DeltaPD expression, we developed a dual-reporter Pax6 BAC transgene in which EGFP and DsRed demonstrate paired-containing and pairedless transcripts, respectively. In BAC transgenic mice, DsRed is predominantly expressed in the peripheral neural retina during early eye development, but not in the developing lens or cornea. Later DsRed is strongly expressed in the developing ciliary body, but not in the iris. We suggest that the ratio of Pax6+PD and Pax6DeltaPD isoforms in the distal retina is important for both cornea and lens development, either directly by controlling transcription of necessary growth factors or indirectly by controlling development of the distal neural retina.

Overexpression of pairedless Pax6 in the retina disrupts corneal development and affects lens cell survival.

Ratiometric fluorescent indicators are used for making quantitative measurements of a variety of physiological variables. Their utility is often limited by noise. This is the second in a series of papers describing statistical methods for denoising ratiometric data with the aim of obtaining improved quantitative estimates of variables of interest. Here, we outline a statistical optimization method that is designed for the analysis of ratiometric imaging data in which multiple measurements have been taken of systems responding to the same stimulation protocol. This method takes advantage of correlated information across multiple datasets for objectively detecting and estimating ratiometric signals. We demonstrate our method by showing results of its application on multiple, ratiometric calcium imaging experiments.

Estimating weak ratiometric signals in imaging data. II. Meta-analysis with multiple, dual-channel datasets.

The ability to map functional connectivity is necessary for the study of the flow of activity in neuronal circuits. Optical imaging of calcium indicators, including FRET-based genetically encoded indicators and extrinsic dyes, is an important adjunct to electrophysiology and is widely used to visualize neuronal activity. However, techniques for mapping functional connectivities with calcium imaging data have been lacking. We present a procedure to compute reduced functional couplings between neuronal ensembles undergoing seizure activity from ratiometric calcium imaging data in three steps: (1) calculation of calcium concentrations and neuronal firing rates from ratiometric data; (2) identification of putative neuronal populations from spatio-temporal time-series of neural bursting activity; and then, (3) derivation of reduced connectivity matrices that represent neuronal population interactions. We apply our method to the larval zebrafish central nervous system undergoing chemoconvulsant-induced seizures. These seizures generate propagating, central nervous system-wide neural activity from which population connectivities may be calculated. This automatic functional connectivity mapping procedure provides a practical and user-independent means for summarizing the flow of activity between neuronal ensembles.

Mapping Functional Connectivity between Neuronal Ensembles with Larval Zebrafish Transgenic for a Ratiometric Calcium Indicator.

Equine recurrent uveitis (ERU) is a spontaneous disease that is the most common cause of blindness in horses, affecting up to 15% of the horse population. Th17 cells are a major cell population driving the pathogenesis in several mouse models of autoimmune inflammation, including experimental autoimmune uveitis. The purpose of this study is to investigate the role a Th17 cell-mediated response plays in the pathogenesis of ERU.

Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis.

To determine the prevalence of ocular and systemic abnormalities in a group of subjects with aniridia.

Ocular and systemic findings in a survey of aniridia subjects.

Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ~15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.

Directed differentiation of human pluripotent cells to neural crest stem cells.

Serotonin (5-HT) is a neuromodulator involved in regulating mood, appetite, memory, learning, pain, and establishment of left-right (LR) asymmetry in embryonic development. To explore the role of 5-HT in physiology, we have created two forms of "caged" 5-HT, BHQ-O-5HT and BHQ-N-5HT. When exposed to 365 or 740 nm light, BHQ-O-5HT releases 5-HT through one- or two-photon excitation, respectively. BHQ-O-5HT mediated changes in neural activity in cultured mouse primary sensory neurons and the trigeminal ganglion and optic tectum of intact zebrafish larvae in the form of high-amplitude spiking in response to light. In Xenopus laevis embryos, light-activated 5-HT increased the occurrence of LR patterning defects. Maximal rates of LR defects were observed when 5-HT was released at stage 5 compared with stage 8. These experiments show the potential for BHQ-caged serotonins in studying 5-HT-regulated physiological processes.

James D. Lauderdale

Neuroscience Division of the Biomedical & Health Sciences Institute,
Department of Cellular Biology

University of Georgia

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

James D. Lauderdale

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Neuroscience Division of the Biomedical & Health Sciences Institute,Department of Cellular Biology

University of Georgia

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Neuroscience Division of the Biomedical & Health Sciences Institute,
Department of Cellular Biology