An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease.

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.

Changes in brain architecture are consistent with altered fear processing in domestic rabbits.

The most characteristic feature of domestic animals is their change in behavior associated with selection for tameness. Here we show, using high-resolution brain magnetic resonance imaging in wild and domestic rabbits, that domestication reduced amygdala volume and enlarged medial prefrontal cortex volume, supporting that areas driving fear have lost volume while areas modulating negative affect have gained volume during domestication. In contrast to the localized gray matter alterations, white matter anisotropy was reduced in the corona radiata, corpus callosum, and the subcortical white matter. This suggests a compromised white matter structural integrity in projection and association fibers affecting both afferent and efferent neural flow, consistent with reduced neural processing. We propose that compared with their wild ancestors, domestic rabbits are less fearful and have an attenuated flight response because of these changes in brain architecture.

Aniridia with PAX6 mutations and narcolepsy.

PAX6 gene mutations cause a variety of eye and central nervous system (CNS) abnormalities. Aniridia is often accompanied by CNS abnormalities such as pineal gland atrophy or hypoplasia, leading to disturbed circadian rhythm and sleep disorders. Less is known on the coincidence of narcolepsy in this patient group. We aimed to find out whether the circadian rhythm or sleep-wake structure was affected in patients with aniridia. Four members of a family segregating with congenital aniridia in two generations were included in the study. The patients were subjected to genetic testing for a PAX6 mutation, multiple sleep latency test, whole-brain magnetic resonance imaging (MRI), hypocretin-1 in cerebrospinal fluid, and Human Leukocyte Antigen DQ beta1*06:02. All four members were heterozygous for the pathogenic c.959-1G>A mutation in the PAX6 gene. Sleep disturbance was observed in all family members. The index patient was diagnosed with narcolepsy. MRI showed a hypoplastic pineal gland in all members. We describe the first case of a patient with PAX6 haploinsufficiency, aniridia and pineal gland hypoplasia diagnosed with narcolepsy type-1, suggesting a complex sleep disorder pathogenesis.

The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A.

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.

Zinc finger gene nolz1 regulates the formation of retinal progenitor cells and suppresses the Lim3/Lhx3 phenotype of retinal bipolar cells in chicken retina.

BACKGROUND: The zinc-finger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the transcription factors Isl1, Lim1, and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal development. We, therefore, studied Nolz1 during retinal development. RESULTS: Nolz1 expression was seen in two waves during development: one early (peak at embryonic day 3-4.5) in retinal progenitors and one late (embryonic day 8) in newly differentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors, and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpression of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells. CONCLUSIONS: We conclude that Nolz1 has multiple functions during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progenitor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differentiation. Developmental Dynamics 247:630-641, 2018. © 2017 Wiley Periodicals, Inc.

A genomic duplication is associated with ectopic eomesodermin expression in the embryonic chicken comb and two duplex-comb phenotypes.

Duplex-comb (D) is one of three major loci affecting comb morphology in the domestic chicken. Here we show that the two Duplex-comb alleles, V-shaped (D*V) and Buttercup (D*C), are both associated with a 20 Kb tandem duplication containing several conserved putative regulatory elements located 200 Kb upstream of the eomesodermin gene (EOMES). EOMES is a T-box transcription factor that is involved in mesoderm specification during gastrulation. In D*V and D*C chicken embryos we find that EOMES is ectopically expressed in the ectoderm of the comb-developing region as compared to wild-type embryos. The confinement of the ectopic expression of EOMES to the ectoderm is in stark contrast to the causal mechanisms underlying the two other major comb loci in the chicken (Rose-comb and Pea-comb) in which the transcription factors MNR2 and SOX5 are ectopically expressed strictly in the mesenchyme. Interestingly, the causal mutations of all three major comb loci in the chicken are now known to be composed of large-scale structural genomic variants that each result in ectopic expression of transcription factors. The Duplex-comb locus also illustrates the evolution of alleles in domestic animals, which means that alleles evolve by the accumulation of two or more consecutive mutations affecting the phenotype. We do not yet know whether the V-shaped or Buttercup allele correspond to the second mutation that occurred on the haplotype of the original duplication event.

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PURPOSE: Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. METHODS: A 208 bp gene regulatory sequence from the chicken retinoid X receptor γgene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. RESULTS: Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. CONCLUSIONS: In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Whole-genome resequencing reveals loci under selection during chicken domestication.

Domestic animals are excellent models for genetic studies of phenotypic evolution. They have evolved genetic adaptations to a new environment, the farm, and have been subjected to strong human-driven selection leading to remarkable phenotypic changes in morphology, physiology and behaviour. Identifying the genetic changes underlying these developments provides new insight into general mechanisms by which genetic variation shapes phenotypic diversity. Here we describe the use of massively parallel sequencing to identify selective sweeps of favourable alleles and candidate mutations that have had a prominent role in the domestication of chickens (Gallus gallus domesticus) and their subsequent specialization into broiler (meat-producing) and layer (egg-producing) chickens. We have generated 44.5-fold coverage of the chicken genome using pools of genomic DNA representing eight different populations of domestic chickens as well as red jungle fowl (Gallus gallus), the major wild ancestor. We report more than 7,000,000 single nucleotide polymorphisms, almost 1,300 deletions and a number of putative selective sweeps. One of the most striking selective sweeps found in all domestic chickens occurred at the locus for thyroid stimulating hormone receptor (TSHR), which has a pivotal role in metabolic regulation and photoperiod control of reproduction in vertebrates. Several of the selective sweeps detected in broilers overlapped genes associated with growth, appetite and metabolic regulation. We found little evidence that selection for loss-of-function mutations had a prominent role in chicken domestication, but we detected two deletions in coding sequences that we suggest are functionally important. This study has direct application to animal breeding and enhances the importance of the domestic chicken as a model organism for biomedical research.

The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility.

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.

A complex genomic rearrangement involving the endothelin 3 locus causes dermal hyperpigmentation in the chicken.

Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals.

Inactivation of Pmel alters melanosome shape but has only a subtle effect on visible pigmentation.

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel⁻/⁻). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel⁻/⁻ melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour.

PURPOSE: Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken. METHODS: MYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells. RESULTS: The expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis. CONCLUSION: In conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.

Expression of carnitine palmitoyl-CoA transferase-1B is influenced by a cis-acting eQTL in two chicken lines selected for high and low body weight.

Carnitine palmitoyl-CoA transferase-1B is a mitochondrial enzyme in the fatty acid oxidation pathway. In a previous study, CPT1B was identified as differentially expressed in the hypothalamus of two lines of chickens established by long-term selection for high (HWS) or low (LWS) body weight. Mammals have three paralogs (CPT1a, b and c) while nonmammalian vertebrates only have two (CPT1A, B). CPT1A is expressed in liver and CPT1B in muscle. CPT1c is expressed in hypothalamus, where it regulates feeding and energy expenditure. We identified an intronic length polymorphism, fixed for different alleles in the two populations, and mapped the hitherto missing CPT1B locus in the chicken genome assembly, to the distal tip of chromosome 1p. Based on molecular phylogeny and gene synteny we suggest that chicken CPT1B is pro-orthologous of the mammalian CPT1c. Chicken CPT1B was differentially expressed in both muscle and hypothalamus but in opposite directions: higher levels in hypothalamus but lower levels in muscle in the HWS than in the LWS line. Using an advanced intercross population of the lines, we found CPT1B expression to be influenced by a cis-acting expression quantitative trait locus in muscle. The increased expression in hypothalamus and reduced expression in muscle is consistent with an increased food intake in the HWS line and at the same time reduced fatty acid oxidation in muscle yielding a net accumulation of energy intake and storage. The altered expression of CPT1B in hypothalamus and peripheral tissue is likely to be a mechanism contributing to the remarkable difference between lines.

Copy number variation in intron 1 of SOX5 causes the Pea-comb phenotype in chickens.

Pea-comb is a dominant mutation in chickens that drastically reduces the size of the comb and wattles. It is an adaptive trait in cold climates as it reduces heat loss and makes the chicken less susceptible to frost lesions. Here we report that Pea-comb is caused by a massive amplification of a duplicated sequence located near evolutionary conserved non-coding sequences in intron 1 of the gene encoding the SOX5 transcription factor. This must be the causative mutation since all other polymorphisms associated with the Pea-comb allele were excluded by genetic analysis. SOX5 controls cell fate and differentiation and is essential for skeletal development, chondrocyte differentiation, and extracellular matrix production. Immunostaining in early embryos demonstrated that Pea-comb is associated with ectopic expression of SOX5 in mesenchymal cells located just beneath the surface ectoderm where the comb and wattles will subsequently develop. The results imply that the duplication expansion interferes with the regulation of SOX5 expression during the differentiation of cells crucial for the development of comb and wattles. The study provides novel insight into the nature of mutations that contribute to phenotypic evolution and is the first description of a spontaneous and fully viable mutation in this developmentally important gene.

MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma.

Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We used the chicken retina, a well-established model for studying retinal neurogenesis, and established human embryonic stem cell-derived retinal organoids as model systems. We over-expressed MYCN by electroporation of piggyBac genome-integrating expression vectors. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human organoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7-9 weeks in chicken. Cells expressing MYCN could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for cone progenitors. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype.

Differentially expressed genes in hypothalamus in relation to genomic regions under selection in two chicken lines resulting from divergent selection for high or low body weight.

Long-term divergent selection for low or high body weight from the same founder population has generated two extremely divergent lines of chickens, the high- (HWS) and low-weight (LWS) selected lines. At selection age (56 days), the lines differ by more than nine times in body weight. The HWS line chickens are compulsive feeders, whereas in the LWS line, some individuals are anorexic and others have very low appetite. Previous studies have implicated the central nervous system and particularly the hypothalamus in these behavioural differences. Here, we compared the mRNA expression in hypothalamus tissue from chickens on day 4 post-hatch using oligonucleotide arrays and found that the divergent selection had resulted in minor but multiple expression differences. Differentially expressed genes were enriched in processes 'DNA metabolism, repair, induction of apoptosis and metabolism'. Several differentially expressed genes participate in the regulation of neuronal plasticity and development, including apoptosis, or are neurotransmittor receptor subtypes. Less change was seen when comparing hypothalamic neuropeptide mediators of appetite such as the melanocortin receptors. The genomic locations of these differentially expressed genes were then compared to the locations of growth QTLs and to a genome-wide map of chromosomal regions that have been under divergent selection between the lines. The results indicate which differentially expressed hypothalamic genes have responded to the divergent selection and that the results predict that it is more likely to find causative genes among these most differentially expressed genes. Because of such differential gene expression in hypothalamus, the lines may adapt behaviourally different particularly to the post-hatch situation when independent feeding to obtain energy is established.

Pax2 is expressed in a subpopulation of Müller cells in the central chick retina.

Müller cells in the chick retina are generally thought to be a homogeneous population. We show that the transcription factor Pax2 is expressed by Müller cells in the central chick retina and its expression was first observed at stage 32 (embryonic day [E] 7.5). Birth-dating indicated that the majority of Pax2-positive Müller cells are generated between stage 29 and 33 (E5.5-E8). At stage 42 (E16), several Müller cell markers, such as Sox2 and 2M6, had reached the peripheral retina, while the Pax2 labeling extended approximately half-way. A similar pattern was maintained in the 6-month-old chicken. Neither the Pax2-positive nor the Pax2-negative Müller cells could be specifically associated to proliferative responses in the retina induced by growth factors or N-methyl-D-aspartate. Pax2 was not detected in Müller cells in mouse, rat, guinea-pig, rabbit, or pig retinas; but the zebrafish retina displayed a similar pattern of central Pax2-expressing Müller cells.

Horizontal cell progenitors arrest in G2-phase and undergo terminal mitosis on the vitreal side of the chick retina.

We have addressed the question when horizontal cells in the chick retina are generated and undergo their terminal mitosis. Horizontal cell progenitors replicate their DNA early and migrate bi-directionally to the horizontal cell layer. It was hypothesized that the cells undergo mitosis directly after replication and migrate as post-mitotic transition cells before differentiating to horizontal cells. However, our results show that cells expressing markers for the axon-bearing and the axon-less subtypes of horizontal cells undergo terminal mitosis while residing on the vitreal side of the retina. By combining horizontal cell transcription factors Lim1, Isl1 and Prox1 labeling with phospho-histone H3, a marker for mitosis, we demonstrate that all or a clear majority of vitreal mitoses are undertaken by the horizontal cell committed progenitors. The pattern of cells that incorporated the thymidine analogue EdU implied that the progenitors replicated their genome while migrating towards the vitreal side. Upon arrival to the vitreal retina they become arrested for about two days prior to mitosis. Hence, cells expressing horizontal cell markers are arrested in G2-phase on the vitreal side of the retina. These results support the existence of committed progenitors that give rise to horizontal cells and that those cells become arrested in G2-phase before undergoing terminal mitosis on the vitreal side of the retina followed by migration to the horizontal cell layer. The results also indicate that the regulation of the transition from G2-phase to mitosis is important for the development of these committed progenitor cells.

Increased A-to-I RNA editing of the transcript for GABAA receptor subunit α3 during chick retinal development.

Adenosine-to-inosine (A-to-I) RNA editing is a cotranscriptional or posttranscriptional gene regulatory mechanism that increases the diversity of the proteome in the nervous system. Recently, the transcript for GABA type A receptor subunit α3 was found to be subjected to RNA editing. The aim of this study was to determine if editing of the chicken α3 subunit transcript occurs in the retina and if the editing is temporally regulated during development. We also raised the question if editing of the α3 transcript was temporally associated with the suggested developmental shift from excitation to inhibition in the GABA system. The editing frequency was studied by using Sanger and Pyrosequencing, and to monitor the temporal aspects, we studied the messenger RNA expression of the GABAA receptor subunits and chloride pumps, known to be involved in the switch. The results showed that the chick α3 subunit was subjected to RNA editing, and its expression was restricted to cells in the inner nuclear and ganglion cell layer in the retina. The extent of editing increased during development (after embryonic days 8-9) concomitantly with an increase of expression of the chloride pump KCC2. Expression of several GABAA receptor subunits known to mediate synaptic GABA actions was upregulated at this time. We conclude that editing of the chick GABAA subunit α3 transcript in chick retina gives rise to an amino acid change that may be of importance in the switch from excitatory to inhibitory receptors.

High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy.

Three-dimensional cell cultures are able to better mimic the physiology and cellular environments found in tissues in vivo compared to cells grown in two dimensions. In order to study the structure and function of cells in 3-D cultures, light microscopy is frequently used. The preparation of 3-D cell cultures for light microscopy is often destructive, including physical sectioning of the samples, which can result in the loss of 3-D information. In order to probe the structure of 3-D cell cultures at high resolution, we have explored the use of expansion microscopy and compared it to a simple immersion clearing protocol. We provide a practical method for the study of spheroids, organoids and tumor-infiltrating immune cells at high resolution without the loss of spatial organization. Expanded samples are highly transparent, enabling high-resolution imaging over extended volumes by significantly reducing light scatter and absorption. In addition, the hydrogel-like nature of expanded samples enables homogenous antibody labeling of dense epitopes throughout the sample volume. The improved labeling and image quality achieved in expanded samples revealed details in the center of the organoid which were previously only observable following serial sectioning. In comparison to chemically cleared spheroids, the improved signal-to-background ratio of expanded samples greatly improved subsequent methods for image segmentation and analysis.