Publications by authors named "James D Lauderdale"

34 Publications

Ocular elongation and retraction in foveated reptiles.

Dev Dyn 2021 11 1;250(11):1584-1599. Epub 2021 May 1.

Department of Cellular Biology, The University of Georgia, Athens, Georgia, USA.

Background: Pronounced asymmetric changes in ocular globe size during eye development have been observed in a number of species ranging from humans to lizards. In contrast, largely symmetric changes in globe size have been described for other species like rodents. We propose that asymmetric changes in the three-dimensional structure of the developing eye correlate with the types of retinal remodeling needed to produce areas of high photoreceptor density. To test this idea, we systematically examined three-dimensional aspects of globe size as a function of eye development in the bifoveated brown anole, Anolis sagrei.

Results: During embryonic development, the anole eye undergoes dynamic changes in ocular shape. Initially spherical, the eye elongates in the presumptive foveal regions of the retina and then proceeds through a period of retraction that returns the eye to its spherical shape. During this period of retraction, pit formation and photoreceptor cell packing are observed. We found a similar pattern of elongation and retraction associated with the single fovea of the veiled chameleon, Chamaeleo calyptratus.

Conclusions: These results, together with those reported for other foveated species, support the idea that areas of high photoreceptor packing occur in regions where the ocular globe asymmetrically elongates and retracts during development.
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http://dx.doi.org/10.1002/dvdy.348DOI Listing
November 2021

Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy.

Science 2021 03;371(6536)

Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA, USA.

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species growing on aquatic vegetation, primarily the invasive Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production.
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http://dx.doi.org/10.1126/science.aax9050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8318203PMC
March 2021

Structural and functional consequences of PAX6 mutations in the brain: Implications for aniridia.

Brain Res 2021 04 28;1756:147283. Epub 2021 Jan 28.

Department of Cellular Biology, The University of Georgia, Athens, GA 30602, USA; Neuroscience Division of the Biomedical and Health Sciences Institute, The University of Georgia, Athens, GA 30602, USA. Electronic address:

The paired-box 6 (PAX6) gene encodes a highly conserved transcription factor essential for the proper development of the eye and brain. Heterozygous loss-of-function mutations in PAX6 are causal for a condition known as aniridia in humans and the Small eye phenotype in mice. Aniridia is characterized by iris hypoplasia and other ocular abnormalities, but recent evidence of neuroanatomical, sensory, and cognitive impairments in this population has emerged, indicating brain-related phenotypes as a prevalent feature of the disorder. Determining the neurophysiological origins of brain-related phenotypes in this disorder presents a substantial challenge, as the majority of extra-ocular traits in aniridia demonstrate a high degree of heterogeneity. Here, we summarize and integrate findings from human and rodent model studies, which have focused on neuroanatomical and functional consequences of PAX6 mutations. We highlight novel findings from PAX6 central nervous system studies in adult mammals, and integrate these findings into what we know about PAX6's role in development of the central nervous system. This review presents the current literature in the field in order to inform clinical application, discusses what is needed in future studies, and highlights PAX6 as a lens through which to understand genetic disorders affecting the human nervous system.
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http://dx.doi.org/10.1016/j.brainres.2021.147283DOI Listing
April 2021

Multiple roles for Pax2 in the embryonic mouse eye.

Dev Biol 2021 04 9;472:18-29. Epub 2021 Jan 9.

Department of Cell Biology & Human Anatomy, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA. Electronic address:

The vertebrate eye anlage grows out of the brain and folds into bilayered optic cups. The eye is patterned along multiple axes, precisely controlled by genetic programs, to delineate neural retina, pigment epithelium, and optic stalk tissues. Pax genes encode developmental regulators of key morphogenetic events, with Pax2 being essential for interpreting inductive signals, including in the eye. PAX2 mutations cause ocular coloboma, when the ventral optic fissure fails to close. Previous studies established that Pax2 is necessary for fissure closure and to maintain the neural retina -- glial optic stalk boundary. Using a Pax2 knock-in allele we discovered that the mutant optic nerve head (ONH) lacks molecular boundaries with the retina and RPE, rendering the ONH larger than normal. This was preceded by ventronasal cup mispatterning, a burst of overproliferation and followed by optic cup apoptosis. Our findings support the hypothesis that ONH cells are tripotential, requiring Pax2 to remain committed to glial fates. This work extends current models of ocular development, contributes to broader understanding of tissue boundary formation and informs the underlying mechanisms of human coloboma.
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http://dx.doi.org/10.1016/j.ydbio.2020.12.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956245PMC
April 2021

Taste buds are not derived from neural crest in mouse, chicken, and zebrafish.

Dev Biol 2021 03 14;471:76-88. Epub 2020 Dec 14.

Regenerative Bioscience Center, The University of Georgia, Athens, GA, USA; Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, The University of Georgia, Athens, GA, USA. Electronic address:

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreER/tdT mouse, GFP neural fold transplantation to GFP chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreER specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP/GFP chicken chimera model, GFP cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish.
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http://dx.doi.org/10.1016/j.ydbio.2020.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855483PMC
March 2021

Global and age-related neuroanatomical abnormalities in a Pax6-deficient mouse model of aniridia suggests a role for Pax6 in adult structural neuroplasticity.

Brain Res 2020 04 31;1732:146698. Epub 2020 Jan 31.

Department of Cellular Biology, University of Georgia, 250B Coverdell Center, 500 D.W. Brooks Drive, Athens, GA 30602, United States; Neuroscience Division of the Biomedical and Health Sciences Institute, The University of Georgia, Athens, GA 30602, United States. Electronic address:

PAX6 encodes a highly conserved transcription factor necessary for normal development of the eyes and central nervous system. Heterozygous loss-of-function mutations in PAX6 cause the disorder aniridia in humans and the Small eye trait in mice. Aniridia is a congenital and progressive disorder known for ocular phenotypes; however, recently, consequences of PAX6 haploinsufficiency in the brains of aniridia patients have been identified. These findings span structural and functional abnormalities, including deficits in cognitive and sensory processing. Furthermore, some of these abnormalities are accelerated as aniridia patients age. Although some functional abnormalities may be explained by structural changes, variability of results remain, and the effects of PAX6 heterozygous loss-of-function mutations on neuroanatomy, particularly with regard to aging, have yet to be resolved. Our study used high-resolution magnetic resonance imaging (MRI) and histology to investigate structural consequences of such mutations in the adult brain of our aniridia mouse model, Small eye Neuherberg allele (Pax6), at two adult age groups. Using both MRI and histology enables a direct comparison with human studies, while providing higher resolution for detection of more subtle changes. We show volumetric changes in major brain regions of the the Pax6 mouse compared to wild-type including genotype- and age-related olfactory bulb differences, age-related cerebellum differences, and genotype-related eye differences. We also show alterations in thickness of major interhemispheric commissures, particularly those anteriorly located within the brain including the optic chiasm, corpus callosum, and anterior commissure. Together, these genotype and age related changes to brain volumes and structures suggest a global decrease in adult brain structural plasticity in our Pax6 mice.
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http://dx.doi.org/10.1016/j.brainres.2020.146698DOI Listing
April 2020

CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes.

Cell Rep 2019 08;28(9):2288-2292.e3

Department of Genetics, University of Georgia, Athens, GA 30602, USA. Electronic address:

CRISPR-Cas9-mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are no reptiles with effective methods for targeted mutagenesis. Here, we demonstrate that the microinjection of immature oocytes within the ovaries of Anolis sagrei females enables the production of CRISPR-Cas9-induced mutations. This method is capable of producing F0 embryos and hatchlings with monoallelic or biallelic mutations. We demonstrate that these mutations can be transmitted through the germline to establish genetically modified strains of lizards. Direct tests of gene function can now be performed in Anolis lizards, an important model for studies of reptile evolution and development.
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http://dx.doi.org/10.1016/j.celrep.2019.07.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727204PMC
August 2019

A systematic study of injectable anesthetic agents in the brown anole lizard ( ).

Lab Anim 2020 Jun 26;54(3):281-294. Epub 2019 Jul 26.

Department of Genetics, University of Georgia, Athens, GA, USA.

lizards have served as important research models in fields ranging from evolution and ecology to physiology and biomechanics. However, anoles are also emerging as important models for studies of embryo development and tissue regeneration. The increased use of anoles in the laboratory has produced a need to establish effective methods of anesthesia, both for routine veterinary procedures and for research procedures. Therefore, we tested the efficacy of different anesthetic treatments in adult female . Alfaxalone, dexmedetomidine, hydromorphone, ketamine and tribromoethanol were administered subcutaneously (SC), either alone or combined at varying doses in a total of 64 female anoles. Drug induction time, duration, anesthesia level and adverse effects were assessed. Differences in anesthesia level were observed depending on injection site and drug combination. Alfaxalone/dexmedetomidine and tribromoethanol/dexmedetomidine were the most effective drug combinations for inducing a surgical plane of anesthesia in anoles. Brown anoles injected SC with alfaxalone (30 mg/kg) plus dexmedetomidine (0.1 mg/kg) or with tribromoethanol (400 mg/kg) plus dexmedetomidine (0.1 mg/kg) experienced mean durations of surgical anesthesia levels of 31.2 ± 5.3 and 87.5 ± 19.8 min with full recovery after another 10.9 ± 2.9 and 46.2 ± 41.8 min, respectively. Hydromorphone given with alfaxalone/dexmedetomidine resulted in deep anesthesia with respiratory depression, while ketamine/hydromorphone/dexmedetomidine produced only light to moderate sedation. We determined that alfaxalone/dexmedetomidine or tribromoethanol/dexmedetomidine combinations were sufficient to maintain a lizard under general anesthesia for coeliotomy. This study represents a significant step towards understanding the effects of anesthetic agents in anole lizards and will benefit both veterinary care and research on these animals.
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http://dx.doi.org/10.1177/0023677219862841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982557PMC
June 2020

Early and late auditory information processing show opposing deviations in aniridia.

Brain Res 2019 10 24;1720:146307. Epub 2019 Jun 24.

Departments of Psychology and Neuroscience, Bio-Imaging Research Center, University of Georgia, Athens, GA, United States.

Aniridia is a congenital disorder, predominantly caused by heterozygous mutations of the PAX6 gene. While ocular defects have been extensively characterized in this population, brain-related anatomical and functional abnormalities are emerging as a prominent feature of the disorder. Individuals with aniridia frequently exhibit auditory processing deficits despite normal audiograms. While previous studies have reported hypoplasia of the anterior commissure and corpus callosum in some of these individuals, the neurophysiological basis of these impairments remains unexplored. This study provides direct assessment of neural activity related to auditory processing in aniridia. Participants were presented with tones designed to elicit an auditory steady-state response (ASSR) at 22 Hz, 40 Hz, and 84 Hz, and infrequent broadband target tones to maintain attention during electroencephalography (EEG) recording. Persons with aniridia showed increased early cortical responses (P50 AEP) in response to all tones, and increased high-frequency oscillatory entrainment (84 Hz ASSR). In contrast, this group showed a decreased cortical integration response (P300 AEP to target tones) and reduced neural entrainment to cortical beta-band stimuli (22 Hz ASSR). Collectively, our results suggest that subcortical and early cortical auditory processing is augmented in aniridia, while functional cortical integration of auditory information is deficient in this population.
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http://dx.doi.org/10.1016/j.brainres.2019.146307DOI Listing
October 2019

Stripe artifact reduction for digital scanned structured illumination light sheet microscopy.

Opt Lett 2019 May;44(10):2510-2513

Light sheet microscopy is an important and widely used method for studying large and semi-opaque biological specimens. One drawback of the approach is that it often results in stripe artifacts due to absorption and scattering in the illumination path. Here we describe a new approach which will effectively mitigate the artifacts in digital scanned light sheet microscopy (DSLM) and digital scanned structured illumination light sheet microscopy (DSLM-SI). We further improve the results of DSLM-SI through a new reconstruction method which achieves clearer reconstructed images. We demonstrate the reduction of stripe artifacts by imaging 156 microns deep into the larval zebrafish central nervous system. The magnitude of stripe artifacts is reduced by an average of 20% across three datasets.
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http://dx.doi.org/10.1364/OL.44.002510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038152PMC
May 2019

Imaging neural events in zebrafish larvae with linear structured illumination light sheet fluorescence microscopy.

Neurophotonics 2019 Jan 5;6(1):015009. Epub 2019 Mar 5.

University of Georgia, College of Engineering, Athens, Georgia, United States.

Light sheet fluorescence microscopy (LSFM) is a powerful tool for investigating model organisms including zebrafish. However, due to scattering and refractive index variations within the sample, the resulting image often suffers from low contrast. Structured illumination (SI) has been combined with scanned LSFM to remove out-of-focus and scattered light using square-law detection. Here, we demonstrate that the combination of LSFM with linear reconstruction SI can further increase resolution and contrast in the vertical and axial directions compared to the widely adopted root-mean square reconstruction method while using the same input images. We apply this approach to imaging neural activity in 7-day postfertilization zebrafish larvae. We imaged two-dimensional sections of the zebrafish central nervous system in two colors at an effective frame rate of 7 frames per second.
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http://dx.doi.org/10.1117/1.NPh.6.1.015009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400141PMC
January 2019

Microstructural differences in visual white matter tracts in people with aniridia.

Neuroreport 2018 12;29(17):1473-1478

Neuroscience Division, Biomedical and Health Sciences Institute.

Aniridia is a panocular disorder characterized chiefly by iris hypoplasia. Most cases result from mutations of the PAX6 gene, which is important in both eye and brain development. In addition to ocular alterations, differences in global brain volume and functional connectivity have been reported in humans with aniridia. Understanding neural alterations in aniridia may require examination of possible differences in white matter structure, as few studies have assessed white matter in this population. The current study utilized diffusion-weighted imaging to assess white matter structure in 11 people with aniridia and 11 healthy comparison participants, matched for sex and age. A map of the local connectome was calculated to compare quantitative anisotropy (QA), an index of white matter tract density, in all white matter voxels, revealing subcomponents of white matter tracts with differing QA between people with aniridia and healthy comparisons. The analysis indicated that QA was lower for people with aniridia in portions of bilateral optic tract [t(20)=-4.23, P=0.001, d=-1.80], bilateral optic radiation [t(20)=-4.06, P=0.001, d=-1.73], forceps major [t(20)=-3.65, P=0.002, d=-1.55], bilateral superior longitudinal fasciculus [left: t(20)=-3.15, P=0.005, d=-1.34; right, t(20)=-4.28, P<0.001, d=-1.83], and right posterior corona radiata [t(20)=-3.19, P=0.006, d=-1.36]. These differences demonstrate that white matter structure is altered in people with aniridia in both visual tracts and associated posterior visual pathways.
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http://dx.doi.org/10.1097/WNR.0000000000001135DOI Listing
December 2018

Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino.

ACS Chem Neurosci 2019 01 24;10(1):266-278. Epub 2018 Sep 24.

New York University Abu Dhabi , PO Box 129188, Abu Dhabi , United Arab Emirates.

γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs, and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405 nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 day post fertilization (dpf), light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.
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http://dx.doi.org/10.1021/acschemneuro.8b00231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337688PMC
January 2019

A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish.

Biotechniques 2018 06;64(6):275-278

Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602-2607, USA.

The introduction of CRISPR-Cas9 technology for targeted mutagenesis has revolutionized reverse genetics and made genome editing a realistic option in many model organisms. One of the difficulties with this technique is screening for mutations in large numbers of samples. Many screening approaches for identifying CRISPR-Cas9 mutants have been published; however, in practice these methods are time consuming, expensive, or often yield false positives. This report describes a PCR-based screening approach using non-denaturing PAGE. This approach does not depend on the formation of heteroduplexes and reliably detects changes as small as 1 base-pair (bp) in nucleic acid length at the target site. This approach can be used to identify novel mutations and is also useful as a routine genotyping method.
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http://dx.doi.org/10.2144/btn-2018-0012DOI Listing
June 2018

Familial Limbal Stem Cell Deficiency: Clinical, Cytological and Genetic Characterization.

Stem Cell Rev Rep 2018 Feb;14(1):148-151

Research Unit for Rare Diseases; Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, Praha 2, 128 08, Prague, Czech Republic.

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http://dx.doi.org/10.1007/s12015-017-9780-yDOI Listing
February 2018

Structural brain abnormalities in 12 persons with aniridia.

F1000Res 2017 13;6:255. Epub 2017 Mar 13.

Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA.

Aniridia is a disorder predominately caused by heterozygous loss-of-function mutations of the gene, which is a transcriptional regulator necessary for normal eye and brain development.  The ocular abnormalities of aniridia have been well characterized, but mounting evidence has implicated brain-related phenotypes as a prominent feature of this disorder as well.  Investigations using neuroimaging in aniridia patients have shown reductions in discrete brain structures and changes in global grey and white matter.  However, limited sample sizes and substantive heterogeneity of structural phenotypes in the brain remain a challenge.  Here, we examined brain structure in a new population sample in an effort to add to the collective understanding of anatomical abnormalities in aniridia.  The current study used 3T magnetic resonance imaging to acquire high-resolution structural data in 12 persons with aniridia and 12 healthy demographically matched comparison subjects.  We examined five major structures: the anterior commissure, the posterior commissure, the pineal gland, the corpus callosum, and the optic chiasm.  The most consistent reductions were found in the anterior commissure and the pineal gland; however, abnormalities in all of the other structures examined were present in at least one individual.  Our results indicate that the anatomical abnormalities in aniridia are variable and largely individual-specific.  These findings suggest that future studies investigate this heterogeneity further, and that normal population variation should be considered when evaluating structural abnormalities.
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http://dx.doi.org/10.12688/f1000research.11063.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615777PMC
March 2017

A Multitaper, Causal Decomposition for Stochastic, Multivariate Time Series: Application to High-Frequency Calcium Imaging Data.

Conf Rec Asilomar Conf Signals Syst Comput 2016 Nov 6;2016:1056-1060. Epub 2017 Mar 6.

Department of Cellular Biology, University of Georgia, Athens, GA.

Neural data analysis has increasingly incorporated causal information to study circuit connectivity. Dimensional reduction forms the basis of most analyses of large multivariate time series. Here, we present a new, multitaper-based decomposition for stochastic, multivariate time series that acts on the covariance of the time series at all lags, (), as opposed to standard methods that decompose the time series, (), using only information at zero-lag. In both simulated and neural imaging examples, we demonstrate that methods that neglect the full causal structure may be discarding important dynamical information in a time series.
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http://dx.doi.org/10.1109/ACSSC.2016.7869531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479311PMC
November 2016

Cellular Stiffness as a Novel Stemness Marker in the Corneal Limbus.

Biophys J 2016 Oct;111(8):1761-1772

The G. W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia; The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia. Electronic address:

Healthy eyes contain a population of limbal stem cells (LSCs) that continuously renew the corneal epithelium. However, each year, 1 million Americans are afflicted with severely reduced visual acuity caused by corneal damage or disease, including LSC deficiency (LSCD). Recent advances in corneal transplant technology promise to repair the cornea by implanting healthy LSCs to encourage regeneration; however, success is limited to transplanted tissues that contain a sufficiently high percentage of LSCs. Attempts to screen limbal tissues for suitable implants using molecular stemness markers are confounded by the poorly understood signature of the LSC phenotype. For cells derived from the corneal limbus, we show that the performance of cell stiffness as a stemness indicator is on par with the performance of ΔNP63α, a common molecular marker. In combination with recent methods for sorting cells on a biophysical basis, the biomechanical stemness markers presented here may enable the rapid purification of LSCs from a heterogeneous population of corneal cells, thus potentially enabling clinicians and researchers to generate corneal transplants with sufficiently high fractions of LSCs, regardless of the LSC percentage in the donor tissue.
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http://dx.doi.org/10.1016/j.bpj.2016.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072985PMC
October 2016

Shared Enhancer Activity in the Limbs and Phallus and Functional Divergence of a Limb-Genital cis-Regulatory Element in Snakes.

Dev Cell 2015 Oct 1;35(1):107-19. Epub 2015 Oct 1.

Department of Genetics, University of Georgia, Athens, GA 30602, USA. Electronic address:

The amniote phallus and limbs differ dramatically in their morphologies but share patterns of signaling and gene expression in early development. Thus far, the extent to which genital and limb transcriptional networks also share cis-regulatory elements has remained unexplored. We show that many limb enhancers are retained in snake genomes, suggesting that these elements may function in non-limb tissues. Consistent with this, our analysis of cis-regulatory activity in mice and Anolis lizards reveals that patterns of enhancer activity in embryonic limbs and genitalia overlap heavily. In mice, deletion of HLEB, an enhancer of Tbx4, produces defects in hindlimbs and genitalia, establishing the importance of this limb-genital enhancer for development of these different appendages. Further analyses demonstrate that the HLEB of snakes has lost hindlimb enhancer function while retaining genital activity. Our findings identify roles for Tbx4 in genital development and highlight deep similarities in cis-regulatory activity between limbs and genitalia.
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http://dx.doi.org/10.1016/j.devcel.2015.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605891PMC
October 2015

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Dev Biol 2015 Dec 25;408(2):328-44. Epub 2015 Feb 25.

Department of Biology, University of Virginia, Charlottesville, VA 22904, USA; Department of Ophthalmology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. Electronic address:

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.
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http://dx.doi.org/10.1016/j.ydbio.2015.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549229PMC
December 2015

Keratopathy, cataract, and dry eye in a survey of aniridia subjects.

Clin Ophthalmol 2015 10;9:291-5. Epub 2015 Feb 10.

Department of Ophthalmology, University of Virginia School of Medicine, Charlottesville, VA, USA.

Purpose: To determine the prevalence of keratopathy, cataract, and dry eye in a group of individuals with aniridia.

Methods: We reviewed survey data from the Aniridia Foundation International (AFI) registry, which included 99 subjects who self-reported on corneal disease, cataract, and dry eye disease.

Results: The average age of respondents was 25.3±18.6 years, with a range of 0 to 67. Of 99 subjects, 46% stated they have corneal disease, 32% stated they did not, and 22% were unsure. The average age of diagnosis of keratopathy was 20.0±12.2 years. Keratolimbal allograft was reported in 20% and penetrating keratoplasty in 9%. Cataract was reported in 65%, with an average age of 9.4±14.0 years at time of diagnosis, and cataract surgery was reported in 32%. The average age of subjects at the time of cataract and corneal surgery was 28.4±13.7 and 33.5±11.4 years, respectively. Symptomatic dry eye was reported in 56% of subjects, with an average age at diagnosis of 23.8±13.3 years.

Conclusion: There is a high prevalence of corneal disease in aniridia, with diagnosis in late childhood or early adulthood in nearly one-half of subjects, often requiring corneal surgery. Cataract and dry eye are commonly associated with aniridia. Although aniridia subjects may have been aware of the diagnosis of cataract at an early age, they usually were treated for cataract and keratopathy as adults.
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http://dx.doi.org/10.2147/OPTH.S74648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334340PMC
February 2015

Increased functional connectivity in intrinsic neural networks in individuals with aniridia.

Front Hum Neurosci 2014 19;8:1013. Epub 2014 Dec 19.

Department of Psychology, University of Georgia Athens, GA, USA ; Department of Neuroscience, University of Georgia Athens, GA, USA.

Mutations affecting the PAX6 gene result in aniridia, a condition characterized by the lack of an iris and other panocular defects. Among humans with aniridia, structural abnormalities also have been reported within the brain. The current study examined the functional implications of these deficits through "resting state" or task-free functional magnetic resonance imaging (fMRI) in 12 individuals with aniridia and 12 healthy age- and gender-matched controls. Using independent components analysis (ICA) and dual regression, individual patterns of functional connectivity associated with three intrinsic connectivity networks (ICNs; executive control, primary visual, and default mode) were compared across groups. In all three analyses, the aniridia group exhibited regions of greater connectivity correlated with the network, while the controls did not show any such regions. These differences suggest that individuals with aniridia recruit additional neural regions to supplement function in critical intrinsic networks, possibly due to inherent structural or sensory abnormalities related to the disorder.
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http://dx.doi.org/10.3389/fnhum.2014.01013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271605PMC
January 2015

Mouse embryonic development in a serum-free whole embryo culture system.

J Vis Exp 2014 Mar 1(85). Epub 2014 Mar 1.

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

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.
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http://dx.doi.org/10.3791/50803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122383PMC
March 2014

Light-activated serotonin for exploring its action in biological systems.

Chem Biol 2013 Dec 12;20(12):1536-46. Epub 2013 Dec 12.

Department of Chemistry, University of Georgia, Athens, GA 30602, USA; New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, United Arab Emirates. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chembiol.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909739PMC
December 2013

Electrophysiological recording in the brain of intact adult zebrafish.

J Vis Exp 2013 Nov 19(81):e51065. Epub 2013 Nov 19.

Department of Genetics, University of Georgia.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.
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http://dx.doi.org/10.3791/51065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019770PMC
November 2013

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

Nat Protoc 2013 Jan 3;8(1):203-12. Epub 2013 Jan 3.

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.

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.
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http://dx.doi.org/10.1038/nprot.2012.156DOI Listing
January 2013

Ocular and systemic findings in a survey of aniridia subjects.

J AAPOS 2011 Dec;15(6):562-6

Department of Ophthalmology, University of Virginia School of Medicine, Charlottesville , Virginia 22908-0715, USA.

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

Methods: Survey forms developed by Aniridia Foundation International were sent to all members prior to the 2010 AFI member conference. An additional form was provided for completion by physicians caring for patients. Forms were then collected from all members who attended the meeting.

Results: A total of 155 surveys were distributed, of which 83 (53%) were completed. The mean age was 25.4 ± 18.4 years, with 65% sporadic and 35% familial cases, and 2.4% with WAGR (Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome. Ocular abnormalities included nystagmus (83%), cataract (71%), dry eye (53%), glaucoma (46%), keratopathy (45%), foveal hypoplasia (41%), strabismus (31%), and retinal disease (5%). The mean age at diagnosis of aniridia was 22.1 months (median, 1.5 months) and glaucoma was 13.6 years (median, 8.5 years). Of 38 subjects with aniridia and glaucoma, 76% were treated medically, and 58% had been treated surgically. In subjects with glaucoma, the mean number (± SD) of glaucoma medications was 1.8 ± 1.3, and number of surgical procedures was 1.7 ± 2.0. Developmental delay was reported in 17%. The mean body mass index and the prevalence of obesity in subjects with aniridia was significantly greater (P = 0.003) than in siblings without aniridia.

Conclusions: In this study, aniridia was associated with nystagmus and other motility problems, cataract, glaucoma, and keratopathy. Systemic abnormalities included increased average body mass index and obesity, which appeared to occur not only in WAGR syndrome but more broadly in aniridia.
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http://dx.doi.org/10.1016/j.jaapos.2011.07.009DOI Listing
December 2011

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

Vet Ophthalmol 2012 May 7;15(3):145-52. Epub 2011 Oct 7.

Department of Small Animal Medicine and Surgery, The University of Georgia College of Veterinary Medicine, 501 D.W. Brooks Drive, Athens, GA 30602, USA.

Objective: 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.

Procedure: Banked, Davidson's-fixed equine globes histopathologically diagnosed with ERU (n = 7) were compared immunohistochemically with healthy control globes (n = 7). Immunohistochemical staining was performed using a pan-Leptospira antibody and antibodies against IL-6, IL-17, and IL-23. Additionally, immunostaining was performed for T-cell (CD3) and B-cell (CD79α) markers. Specificity of immunoreactivity was confirmed by western blot analysis.

Results: Immunohistochemical staining was positive for IL-6, IL-17, and IL-23 within the cytoplasm of nonpigmented ciliary epithelial cells and mononuclear inflammatory cells infiltrating the iris, and ciliary body of ERU horses (n = 7) but negative in controls (n = 7). ERU-affected eyes were CD3 positive (n = 7) and CD79α negative (n = 7). Staining for Leptospira was negative in all ERU and control globes.

Conclusions: Strong immunoreactivity for IL-6, IL-17, and IL-23, in conjunction with the fact that T lymphocytes are the predominating inflammatory cells present in ERU, suggests that IL-17-secreting helper T-cells play a role in the pathogenesis of ERU. These findings suggest that horses with ERU may serve as a naturally occurring animal model for autoimmune uveitis.
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http://dx.doi.org/10.1111/j.1463-5224.2011.00951.xDOI Listing
May 2012

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

Front Neural Circuits 2011 22;5. Epub 2011 Feb 22.

Center for Bioinformatics, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University Beijing, China.

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.
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http://dx.doi.org/10.3389/fncir.2011.00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044448PMC
July 2011

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

J Opt Soc Am A Opt Image Sci Vis 2008 Sep;25(9):2185-94

Department of Mathematics and Faculty of Engineering, University of Georgia, AThens, GA 30602, USA.

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.
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http://dx.doi.org/10.1364/josaa.25.002185DOI Listing
September 2008
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