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Michael A Krause,1 Kelly L Trout,2 James D Lauderdale,3 Peter A Netland1 1Department of Ophthalmology, University of Virginia School of Medicine, Charlottesville, VA, USA; 2International WAGR Syndrome Association, Montgomery Village, MD, USA; 3Department of Cellular Biology, University of Georgia, Athens, GA, USACorrespondence: Peter A Netland, Department of Ophthalmology, University of Virginia School of Medicine, 1300 Jefferson Park Avenue, P.O. Box 800715, Charlottesville, VA, 22908-0715, USA, Tel +1 434-982-1086, Email pnetland@virginia.eduPurpose: Our purpose was to evaluate visual acuity in aniridia subjects and the more severely affected phenotype in WAGR syndrome subjects, and to assess potential impact on visual function.Materials and Methods: This was a retrospective comparative study of 25 aniridia subjects with nonsense mutations of PAX6 (50 eyes) and 25 WAGR syndrome subjects with large deletion mutations involving PAX6 (50 eyes). Aniridia subjects were age- and gender-matched with WAGR syndrome subjects in the Coordination of Rare Diseases at Sanford (CoRDS) database. Best-corrected ETDRS visual acuity measurements were converted to LogMAR visual acuity values, which were used to perform statistical analyses.Results: The age and gender distribution of the subjects was not statistically significantly different. The mean LogMAR values in aniridia and WAGR syndrome subjects were 0.95± 0.53 and 1.51± 0.99, respectively (P< 0.001). In the better-seeing eye, mean LogMAR values were 0.78± 0.15 in aniridia subjects and 1.40± 0.88 in WAGR syndrome subjects (P=0.001). The mean LogMAR values for the better-seeing eye corresponded to Snellen visual acuity of 20/125 in aniridia subjects and 20/500 in WAGR syndrome subjects. This average visual acuity was worse than the threshold for profound visual impairment (WHO criteria) and legal blindness (AAO criteria) in WAGR syndrome but not in aniridia subjects. In analysis of both eyes, the visual efficiency was 34% in aniridia subjects and 2% in WAGR syndrome subjects.Conclusion: Visual acuity was significantly worse in WAGR subjects with multi-gene deletion mutations compared with aniridia subjects with nonsense mutations, which corresponded to differences in standard visual function thresholds. Our results suggest that visual acuity may indicate severity of ocular involvement and variability of phenotype in aniridia and WAGR syndrome.Keywords: aniridia, WAGR syndrome, WAGR spectrum, PAX6, visual impairment, blindness

Light sheet microscopy has developed quickly over the past decades and become a popular method for imaging live model organisms and other thick biological tissues. For rapid volumetric imaging, an electrically tunable lens can be used to rapidly change the imaging plane in the sample. For larger fields of view and higher NA objectives, the electrically tunable lens introduces aberrations in the system, particularly away from the nominal focus and off-axis. Here, we describe a system that employs an electrically tunable lens and adaptive optics to image over a volume of 499 × 499 × 192 μm3 with close to diffraction-limited resolution. Compared to the system without adaptive optics, the performance shows an increase in signal to background ratio by a factor of 3.5. While the system currently requires 7s/volume, it should be straightforward to increase the imaging speed to under 1s per volume.

We present a light sheet microscope incorporating an electrically tunable lens and adaptive optics for volumetric imaging of zebrafish larvae, optimized for an extended field of view with a high-NA objective lens.

Rapid technological improvements are democratizing access to high quality, chromosome-scale genome assemblies. No longer the domain of only the most highly studied model organisms, now non-traditional and emerging model species can be genome-enabled using a combination of sequencing technologies and assembly software. Consequently, old ideas built on sparse sampling across the tree of life have recently been amended in the face of genomic data drawn from a growing number of high-quality reference genomes. Arguably the most valuable are those long-studied species for which much is already known about their biology; what many term emerging model species. Here, we report a highly complete chromosome-scale genome assembly for the brown anole, Anolis sagrei - a lizard species widely studied across a variety of disciplines and for which a high-quality reference genome was long overdue. This assembly exceeds the vast majority of existing reptile and snake genomes in contiguity (N50 = 253.6 Mb) and annotation completeness. Through the analysis of this genome and population resequence data, we examine the history of repetitive element accumulation, identify the X chromosome, and propose a hypothesis for the evolutionary history of fusions between autosomes and the X that led to the sex chromosomes of A. sagrei.

BackgroundThe fovea, a pit in the retina, is believed to be important for high-acuity vision and is a feature found in the eyes of humans and a limited number of vertebrate species that include certain primates, birds, lizards, and fish. At present, model systems currently used for ocular research lack a foveated retina and studies investigating fovea development have largely been limited to histological and molecular studies in primates. As a result, progress towards understanding the mechanisms involved in regulating fovea development in humans is limited and is completely lacking in other, non-primate, vertebrates. To address this knowledge gap, we provide here a detailed histological atlas of retina and fovea development in the bifoveated Anolis sagrei lizard, a novel reptile model for fovea research. We also further test the hypothesis that retinal remodeling, which leads to fovea formation and photoreceptor cell packing, is related to asymmetric changes in eye shape.ResultsAnole retina development follows the conventional spatiotemporal patterning observed in most vertebrates, where retina neurogenesis begins within the central retina, progresses throughout the temporal retina, and concludes in the nasal retina. One exception to this general rule is that areas that give rise to the fovea undergo retina differentiation prior to the rest of the retina. We find that retina thickness changes dynamically during periods of ocular elongation and retraction. During periods of ocular elongation, the retina thins, while during retraction it becomes thicker. Ganglion cell layer mounding is also observed in the temporal fovea region just prior to pit formation.ConclusionsAnole retina development parallels that of humans, including the onset and progression of retinal neurogenesis followed by changes in ocular shape and retinal remodeling that leads to pit formation in the retina. We propose that anoles are an excellent model system for fovea development research.Key FindingsRetina mounding that occurs in foveal areas prior to retinal differentiation progressively disappear as foveal regions of the eye elongate.The central and temporal foveal areas undergo retina differentiation before the rest of the retina.GCL mounding prior to pit formation occurs in the area of the temporal fovea but not the central fovea.When the eye is experiencing ocular retraction, photoreceptor cell packing, and pit formation are observed within foveal regions.

Rapid technological improvements are democratizing access to high quality, chromosome-scale genome assemblies. No longer the domain of only the most highly studied model organisms, now non-traditional and emerging model species can be genome-enabled using a combination of sequencing technologies and assembly software. Consequently, old ideas built on sparse sampling across the tree of life have recently been amended in the face of genomic data drawn from a growing number of high-quality reference genomes. Arguably the most valuable are those long-studied species for which much is already known about their biology; what many term emerging model species. Here, we report a new, highly complete chromosome-scale genome assembly for the brown anole, Anolis sagrei – a lizard species widely studied across a variety of disciplines and for which a high-quality reference genome was long overdue.

Anolis 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 Anolis sagrei. 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.

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.

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(GFP/+) 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.

Light Sheet Microscopy has many advantages for imaging living model organisms. Its optical sectioning capability and high volumetric imaging speed over a large field of view make it especially favorable for recording highly dynamic biological events, such as neural signaling. The combination of an electrical tunable lens (ETL) and a scanning light sheet allows us to record image stacks at high speed without moving the sample or the detection objective. The performance of the light sheet microscope is affected by aberrations from the sample mounting and the sample itself as well as aberrations introduced by The ETL which limit the usable field of view and focusing range of the system. Here, we present the development of a light sheet microscope optimized for volumetric imaging of zebrafish larvae with adaptive optics correction for extended focusing range and increased image quality at a speed of 0.6Hz over 400 × 400 × 100μm3 using an electrical tunable lens.

BackgroundAnterior eye development has been explored in different vertebrate species ranging from fish to mammals. However, missing from this diverse group is a representative of reptiles. A promising candidate to fill this void is the brown anole, Anolis sagrei, which is easily raised in the laboratory and for which genome editing techniques exist. Here we provide a detailed histological analysis of the development of the anterior structures of the eye in A. sagrei, which include the cornea, iris, ciliary body, lens, trabecular meshwork, and sclera ossicles.ResultsDevelopment of the anterior segment in Anoles proceeds as for other vertebrates with the lens forming first followed by the cornea, then the iris, ciliary body, trabecular meshwork, and sclera ossicles. The onset of these latter structures occurs first temporally than nasally. Unlike the eyes of mammals and birds, anoles possess a remarkably thin cornea, flat ciliary body, and a trabecular meshwork that lacks an obvious Schlemm’s canal.ConclusionsThis study highlights several features present in anoles and represents an important step towards understanding reptile eye development.Key FindingsThe anole cornea epithelium is thin, composed mainly of a single basal cell layer.The ciliary body lacks a ciliary process.Iris and ciliary body formation occur in a spatiotemporal fashion, developing first temporally then nasally.The anole trabecular meshwork is composed of a spongiform tissue and lacks a Schlemm’s canal.

BackgroundPronounced 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.ResultsDuring 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.ConclusionsThese 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.Key FindingsThe eyes of the brown anole,Anolis sagrei, and veiled chameleon,Chamaeleo calyptratusundergo dynamic asymmetrical changes in ocular shape during development.In both species, asymmetric elongation and retraction of the ocular globe is associated with fovea morphogenesis.Pit formation and photoreceptor cell packing in the foveal area occur when the corresponding region of the ocular globe is retracting relative to adjacent regions.

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-iCreERT2/tdT mouse, GFP+ neural fold transplantation to GFP− chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreERT2 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.

INTRODUCTION Vacuolar myelinopathy (VM) is a neurological disease characterized by widespread vacuolization in the white matter of the brain. First diagnosed in 1994 in bald eagles, it has since spread throughout the southeastern United States. In addition to avian species such as waterfowl and birds of prey, VM has also been found to affect amphibians, reptiles, and fish. Despite intense research efforts, the cause of this mysterious disease has been elusive. Neither contagious agents nor xenobiotics were detected in deceased animals, but field and laboratory studies demonstrated that VM can be transferred through the food chain from herbivorous fish and wildlife to birds of prey. RATIONALE Occurrence of VM has been linked to a cyanobacterium (Aetokthonos hydrillicola) growing on an invasive plant (Hydrilla verticillata) in man-made water bodies. Cyanobacteria are known to produce potent toxins, so we hypothesized that a neurotoxin produced by the epiphytic cyanobacterium causes VM. RESULTS Field studies in the southeastern United States confirmed that H. verticillata was colonized with A. hydrillicola in more than half of the watersheds. Wildlife VM deaths occurred only in reservoirs with dense H. verticillata and A. hydrillicola populations. Laboratory bioassays confirmed the neurotoxicity of crude extracts of A. hydrillicola–H. verticillata biomass collected during VM outbreaks, but neurotoxicity was not detected in samples from VM-free sites. Laboratory cultures of the cyanobacterium, however, did not elicit VM. A. hydrillicola growing on H. verticillata collected at VM-positive reservoirs was then analyzed by mass spectrometry imaging, which revealed that cyanobacterial colonies were colocalized with a brominated metabolite. Supplementation of an A. hydrillicola laboratory culture with potassium bromide resulted in pronounced biosynthesis of this metabolite. H. verticillata hyperaccumulates bromide from the environment, potentially supplying the cyanobacterium with this biosynthesis precursor. Isolation and structure elucidation of the metabolite revealed a structurally unusual pentabrominated biindole alkaloid, which we called aetokthonotoxin (AETX). Genome sequencing of A. hydrillicola allowed the identification of the AETX biosynthetic gene cluster. Biochemical characterization of a halogenase detected in the cluster demonstrated that it brominates tryptophan with the expected substitution pattern. AETX is highly toxic to the nematode Caenorhabditis elegans [median lethal concentration (LC50) 40 nM] and zebrafish (Danio rerio; LC50 275 nM). Leghorn chickens (Gallus gallus) gavaged with AETX developed brain lesions characteristic of VM, whereas no lesions were observed in control chickens. VM diagnosis in treated chickens was verified using transmission electron microscopy of brain tissue. CONCLUSION We confirmed that AETX is the causative agent of VM. AETX biosynthesis relies on the availability of bromide. Seasonal environmental conditions promoting toxin production of A. hydrillicola are watershed specific. The consequences of elevated bromide from geologic and anthropogenic sources (e.g., water treatment and power plants) on VM should be further investigated. Notably, integrated chemical plant management plans to control H. verticillata should avoid the use of bromide-containing chemicals (e.g., diquat dibromide). AETX is lipophilic with the potential for bioaccumulation during transfer through food webs, so mammals may also be at risk. Increased monitoring and public awareness should be implemented for A. hydrillicola and AETX to protect both wildlife and human health.

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.

Aniridia is a congenital condition characterized by absence of iris and is caused by a semidominant mutation in the transcription factor encoded by thePAX6gene. Although ocular phenotypes of this disorder are well characterized, recent studies report that individuals with aniridia have a higher propensity for obesity, infertility, polycystic ovarian disease, and severe eczema compared to theirPax6-normal siblings. These symptoms collectively suggest an underlying endocrine disturbance related to haploinsufficient levels ofPax6.In mice, during development,Pax6expression in the pituitary gland begins at E9.0 in the primordial anterior pituitary gland (Rathke’s Pouch). This expression becomes restricted to the dorsal anterior pituitary by E11.5, but is expressed throughout the anterior lobe by E14.5, and remains through adulthood. It is possible that a reduction inPax6could result in a change in pituitary hormone levels or cell numbers, which may explain symptoms experienced by aniridics. Using theSmall eyemouse model, we find thatPax6reduction results in a decrease in GH-producing cells and an increase in TSH-producing cells in neonate mice, with the TSH increase continuing into adulthood. AdultPax6haploinsufficient mice also have an increase in anterior pituitary volume and weigh significantly less than their wild-type littermates. Furthermore, we show that the increase in TSH-producing cells leads to an increase in thyroxin (T4) in mutant mice, although tri-iodothyronine (T3) levels remain unchanged. These findings present a new role forPax6in the endocrine system, which serves to refine our current understanding ofPax6in endocrine development and maintenance and provides new avenues for investigating endocrine-related symptomatology in aniridia.

Light Sheet Microscopy has developed rapidly over the past decade and is the ideal approach for imaging model organisms such as zebrafish and other thick tissue specimens. Despite the superior optical sectioning capability, high imaging speed, and large field of view, the performance of light sheet microscopy still suffers from optical aberrations. We have implemented a scene-based Shack-Hartmann wavefront sensor for directly measuring the optical aberrations on the emission side of the light-sheet microscope. In this work, we show that our system is capable of AO correction using sensor based and sensorless based approaches. We demonstrate correction up-to one hundred microns deep in zebrafish and fruitfly embryos.

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.

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 (Pax6SeyNeu/+), 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 Pax6SeyNeu/+ 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 Pax6SeyNeu/+ mice.

Anolis 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 Anolis sagrei. 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.

Summary: 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. : The reproductive biology of reptiles makes accessing single-cell embryos difficult and presents a major barrier for deploying gene-editing technologies in these species. Rasys et al. report that the microinjection of Cas9 RNP into immature lizard oocytes enables the production of lizards with targeted mutations. Keywords: Anolis, lizard, CRISPR, Cas9, gene editing, reptile, oocyte, tyrosinase

Zebrafish are an important vertebrate model used to view the mechanisms underlying seizure disorders. Due to their relatively small size and transparency, larval zebrafish are an excellent model through which to view the occurence of seizure-like neural activity in vivo using light sheet fluorescence microscopy (LSFM). Although LSFM possesses good optical sectioning capability and high speed, the resolution and contrast degrade as the imaging plane is moved deeper into the sample due to refractive index variations. We have developed a system that combines a structured illumination light sheet microscope with adaptive optics in the emission path to correct optical aberrations and increase the resolution when imaging deep into the sample. We show that our system can record neural activity fast enough to capture seizure events, and is able to correct optical aberrations throughout the sample.

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

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.

Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

Light Sheet Microscopy is well suited for imaging model organisms and tissue sections that are hundreds of microns thick. Adaptive Optics is needed to correct aberrations in these samples. Here we describe our design of a scene based Shack Hartmann Wavefront Sensor for Light Sheet Microscopy.

We report on PAX6 alleles associated with a clinical diagnosis of classical aniridia in 81 affected individuals representing 66 families. Allelic variants expected to affect PAX6 function were identified in 61 families (76 individuals). Ten cases of sporadic aniridia (10 families) had complete (8 cases) or partial (2 cases) deletion of the PAX6 gene. Sequence changes that introduced a premature termination codon into the open reading frame of PAX6 occurred in 47 families (62 individuals). Three individuals with sporadic aniridia (three families) had sequence changes (one deletion, two run-on mutations) expected to result in a C-terminal extension. An intronic deletion of unknown functional significance was detected in one case of sporadic aniridia (one family), but not in unaffected relatives. Within these 61 families, single nucleotide substitutions accounted for 30/61 (49%), indels for 23/61 (38%), and complete deletion of the PAX6 locus for 8/61 (13%). In five cases of sporadic aniridia (five families), no disease-causing mutation in the coding region was detected. In total, 23 unique variants were identified that have not been reported in the Leiden Open Variation Database (LOVD) database. Within the group assessed, 92% had sequence changes expected to reduce PAX6 function, confirming the primacy of PAX6 haploinsufficiency as causal for aniridia.

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.

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, $C(\tau)$, as opposed to standard methods that decompose the time series, $\mathbf{X}(t)$, 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., Comment: This invited paper was presented at the Asilomar 50th Conference on Signals, Systems, and Computers

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.

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.

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.

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.

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.

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.