Decreased GABA levels during development result in increased connectivity in the larval zebrafish tectum.

γ-aminobutyric acid (GABA) is an abundant neurotransmitter that plays multiple roles in the vertebrate central nervous system (CNS). In the early developing CNS, GABAergic signaling acts to depolarize cells. It mediates several aspects of neural development, including cell proliferation, neuronal migration, neurite growth, and synapse formation, as well as the development of critical periods. Later in CNS development, GABAergic signaling acts in an inhibitory manner when it becomes the predominant inhibitory neurotransmitter in the brain. This behavior switch occurs due to changes in chloride/cation transporter expression. Abnormalities of GABAergic signaling appear to underlie several human neurological conditions, including seizure disorders. However, the impact of reduced GABAergic signaling on brain development has been challenging to study in mammals. Here we take advantage of zebrafish and light sheet imaging to assess the impact of reduced GABAergic signaling on the functional circuitry in the larval zebrafish optic tectum. Zebrafish have three gad genes: two gad1 paralogs known as gad1a and gad1b, and gad2. The gad1b and gad2 genes are expressed in the developing optic tectum. Null mutations in gad1b significantly reduce GABA levels in the brain and increase electrophysiological activity in the optic tectum. Fast light sheet imaging of genetically encoded calcium indicator (GCaMP)-expressing gab1b null larval zebrafish revealed patterns of neural activity that were different than either gad1b-normal larvae or gad1b-normal larvae acutely exposed to pentylenetetrazole (PTZ). These results demonstrate that reduced GABAergic signaling during development increases functional connectivity and concomitantly hyper-synchronization of neuronal networks.

Dynamic changes in ocular shape during human development and its implications for retina fovea formation.

The human fovea is known for its distinctive pit-like appearance, which results from the displacement of retinal layers superficial to the photoreceptors cells. The photoreceptors are found at high density within the foveal region but not the surrounding retina. Efforts to elucidate the mechanisms responsible for these unique features have ruled out cell death as an explanation for pit formation and changes in cell proliferation as the cause of increased photoreceptor density. These findings have led to speculation that mechanical forces acting within and on the retina during development underly the formation of foveal architecture. Here we review eye morphogenesis and retinal remodeling in human embryonic development. Our meta-analysis of the literature suggests that fovea formation is a protracted process involving dynamic changes in ocular shape that start early and continue throughout most of human embryonic development. From these observations, we propose a new model for fovea development.

Maturation and refinement of the maculae and foveae in the Anolis sagrei lizard.

The fovea is a pit in the center of the macula, which is a region of the retina with a high concentration of photoreceptor cells, which accounts for a large degree of visual acuity in primates. The maturation of this primate visual acuity area is characterized by the shallowing and widening of the foveal pit, a decrease in the diameter of the rod-free zone, and an increase in photoreceptor cells packing after birth. Maturation occurs concurrently with progressing age, increasing eye size, and retinal length/area. These observations have led to the hypothesis that the maturation of the fovea might be a function of mechanical variables that remodel the retina. However, this has never been explored outside of primates. Here, we take advantage of the Anolis sagrei lizard, which has a bifoveated retina, to study maturation of the fovea and macula. Eyes were collected from male and female lizards-hatchling, 2-month, 4-month, 6-month, and adult. We found that Anolis maculae undergo a maturation process somewhat different than what has been observed in primates. Anole macular diameters actually increase in size and undergo minimal photoreceptor cell packing, possessing a near complete complement of these cells at the time of hatching. As the anole eye expands, foveal centers experience little change in overall retina cell density with most cell redistribution occurring at macular borders and peripheral retina areas. Gene editing technology has recently been developed in lizards; this study provides a baseline of normal retina maturation for future genetic manipulation studies in anoles.

Two NOTCH1 O-fucose sites have opposing functions in mouse retinal angiogenesis.

Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study, we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating 2 C57BL/6J mouse lines carrying NOTCH1 point mutations, which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f) retinas, we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increased JAG1 activation of NOTCH1 during co-expression with inhibitory Fringes. Although we predicted that the EGF8 O-fucose mutant (8f/8f) would not complete embryonic development due to the direct involvement of the O-fucose in engaging ligand, the 8f/8f mice were viable and fertile. In the 8f/8f retina, we measured increased vessel density consistent with established Notch1 hypomorphs. Overall, our data support the importance of NOTCH1 O-fucose residues for pathway function and confirms that single O-glycan sites are rich in signaling instructions for mammalian development.

Visual Acuity in Aniridia and WAGR Syndrome.

Purpose: 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.

Report on the 2021 Aniridia North America symposium on PAX6, aniridia, and beyond.

The inaugural Aniridia North America (ANA) Symposium was held on the first weekend in November 2021 in Charlottesville, VA, at the University of Virginia. The purpose of this meeting was to bring together an international group of scientists, physicians, patient advocacy groups, and individuals with aniridia to discuss recent advances in knowledge about aniridia and other congenital eye diseases and the development of potential treatments for congenital eye disorders using personalized medicine. Leaders in several areas of eye research and clinical treatment provided a broad perspective on new research advances that impact an understanding of the causes of the damage to the eye associated with aniridia and the development of novel treatments for this and related disorders. Here we summarize the research discussed at the symposium.

Volumetric light sheet imaging with adaptive optics correction.

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.

Piebaldism and chromatophore development in reptiles are linked to the tfec gene.

Reptiles display great diversity in color and pattern, yet much of what we know about vertebrate coloration comes from classic model species such as the mouse and zebrafish.1,2,3,4 Captive-bred ball pythons (Python regius) exhibit a remarkable degree of color and pattern variation. Despite the wide range of Mendelian color phenotypes available in the pet trade, ball pythons remain an overlooked species in pigmentation research. Here, we investigate the genetic basis of the recessive piebald phenotype, a pattern defect characterized by patches of unpigmented skin (leucoderma). We performed whole-genome sequencing and used a case-control approach to discover a nonsense mutation in the gene encoding the transcription factor tfec, implicating this gene in the leucodermic patches in ball pythons. We functionally validated tfec in a lizard model (Anolis sagrei) using the gene editing CRISPR/Cas9 system and TEM imaging of skin. Our findings show that reading frame mutations in tfec affect coloration and lead to a loss of iridophores in Anolis, indicating that tfec is required for chromatophore development. This study highlights the value of captive-bred ball pythons as a model species for accelerating discoveries on the genetic basis of vertebrate coloration.

Chromosome-scale genome assembly of the brown anole (Anolis sagrei), an emerging model species.

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.

Ocular elongation and retraction in foveated reptiles.

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.

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

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 Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production.

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

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.

Multiple roles for Pax2 in the embryonic mouse eye.

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 Pax2GFP/+ 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.