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.

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.

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.

Deletion in the Bardet-Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs.

In golden retriever dogs, a 1 bp deletion in the canine TTC8 gene has been shown to cause progressive retinal atrophy (PRA), the canine equivalent of retinitis pigmentosa. In humans, TTC8 is also implicated in Bardet-Biedl syndrome (BBS). To investigate if the affected dogs only exhibit a non-syndromic PRA or develop a syndromic ciliopathy similar to human BBS, we recruited 10 affected dogs to the study. The progression of PRA for two of the dogs was followed for 2 years, and a rigorous clinical characterization allowed a careful comparison with primary and secondary characteristics of human BBS. In addition to PRA, the dogs showed a spectrum of clinical and morphological signs similar to primary and secondary characteristics of human BBS patients, such as obesity, renal anomalies, sperm defects, and anosmia. We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-length TTC8 transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet-Biedl syndrome with heterogeneous clinical signs.

Brain Transcriptomics of Wild and Domestic Rabbits Suggests That Changes in Dopamine Signaling and Ciliary Function Contributed to Evolution of Tameness.

Domestication has resulted in immense phenotypic changes in animals despite their relatively short evolutionary history. The European rabbit is one of the most recently domesticated animals, but exhibits distinct morphological, physiological, and behavioral differences from their wild conspecifics. A previous study revealed that sequence variants with striking allele frequency differences between wild and domestic rabbits were enriched in conserved noncoding regions, in the vicinity of genes involved in nervous system development. This suggests that a large proportion of the genetic changes targeted by selection during domestication might affect gene regulation. Here, we generated RNA-sequencing data for four brain regions (amygdala, hypothalamus, hippocampus, and parietal/temporal cortex) sampled at birth and revealed hundreds of differentially expressed genes (DEGs) between wild and domestic rabbits. DEGs in amygdala were significantly enriched for genes associated with dopaminergic function and all 12 DEGs in this category showed higher expression in domestic rabbits. DEGs in hippocampus were enriched for genes associated with ciliary function, all 21 genes in this category showed lower expression in domestic rabbits. These results indicate an important role of dopamine signaling and ciliary function in the evolution of tameness during rabbit domestication. Our study shows that gene expression in specific pathways has been profoundly altered during domestication, but that the majority of genes showing differential expression in this study have not been the direct targets of selection.

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.

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.

Alpha 2-Adrenergic Receptor Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in the Human MIO-M1 Müller Cell Line.

PURPOSE: Alpha 2-adrenergic receptor (α2-ADR) agonists are used clinically for a range of indications including reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Animal experiments show that α2-ADR agonists attenuate the injury-induced Müller cell dedifferentiation by a mechanism that involves activation and regulation of extracellular signal-regulated kinase (ERK) 1/2 leading to transactivation of epidermal growth factor receptors (EGFRs). The purpose of this study was to study and corroborate the activation of this system in human cells. MATERIAL AND METHODS: The human Müller cell line MIO-M1 was treated with the α2A-ADR agonist brimonidine in combination with inhibitors for Src-kinase, EGFR-kinase, matrix metalloproteinase (MMP) as well as small interfering RNAs (siRNAs) for the EGFR. The cells were analyzed using immunocytochemistry, quantitative PCR and western blot techniques. RESULTS: Our results show that human MIO-M1 cells express α2A-ADRs and that stimulation of these receptors caused a robust increase of ERK1/2 and protein kinase B (PKB/AKT) (Thr-308) phosphorylation in MIO-M1 cells. P-ERK1/2 and P-AKT (Thr-308) signaling was mediated by Src-kinase and associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173). In addition, the agonist caused activation of MMPs. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478), EGFR-siRNA and a MMP inhibitor (GM6001). CONCLUSION: The results confirm that this human Müller cell line responds to ADR stimulation with phosphorylation of ERK and AKT, which suggests that it is possible to pharmacologically target ADR to modulate the early events in human Müller cell dedifferentiation in a similar fashion as been shown for chicken Müller cells. ABBREVIATIONS: CRALBP: cellular retinaldehyde binding protein; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1/2; GS: glutamine synthetase; GPCR: G protein-coupled receptor; IR: immunoreactivity; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; P-ERK1/2: phospho-ERK1/2; qRT-PCR: quantitative reverse transcriptase PCR.

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.

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.

Heterogeneity in retinoblastoma: a tale of molecules and models.

Retinoblastoma, an intraocular pediatric cancer, develops in the embryonic retina following biallelic loss of RB1. However, there is a wide range of genetic and epigenetic changes that can affect RB1 resulting in different clinical outcomes. In addition, other transformations, such as MYCN amplification, generate particularly aggressive tumors, which may or may not be RB1 independent. Recognizing the cellular characteristics required for tumor development, by identifying the elusive cell-of-origin for retinoblastoma, would help us understand the development of these tumors. In this review we summarize the heterogeneity reported in retinoblastoma on a molecular, cellular and tissue level. We also discuss the challenging heterogeneity in current retinoblastoma models and suggest future platforms that could contribute to improved understanding of tumor initiation, progression and metastasis in retinoblastoma, which may ultimately lead to more patient-specific treatments.

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.

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.

Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors.

Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173) EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-inhibitor (AG1478), EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001), consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in which endothelins among several other functions, serve as an injury-signal that regulate the gliotic response of Müller cells.

Neuroprotection by α2-Adrenergic Receptor Stimulation after Excitotoxic Retinal Injury: A Study of the Total Population of Retinal Ganglion Cells and Their Distribution in the Chicken Retina.

We have studied the effect of α2-adrenergic receptor stimulation on the total excitotoxically injured chicken retinal ganglion cell population. N-methyl-D-aspartate (NMDA) was intraocularly injected at embryonic day 18 and Brn3a positive retinal ganglion cells (Brn3a+ RGCs) were counted in flat-mounted retinas using automated routines. The number and distribution of the Brn3a+ RGCs were analyzed in series of normal retinas from embryonic day 8 to post-hatch day 11 retinas and in retinas 7 or 14 days post NMDA lesion. The total number of Brn3a+ RGCs in the post-hatch retina was approximately 1.9x106 with a density of approximately 9.2x103 cells/mm2. The isodensity maps of normal retina showed that the density decreased with age as the retinal size increased. In contrast to previous studies, we did not find any specific region with increased RGC density, rather the Brn3a+ RGCs were homogeneously distributed over the central retina with decreasing density in the periphery and in the region of the pecten oculli. Injection of 5-10 µg NMDA caused 30-50% loss of Brn3a+ cells and the loss was more severe in the dorsal than in the ventral retina. Pretreatment with brimonidine reduced the loss of Brn3a+ cells both 7 and 14 days post lesion and the protective effect was higher in the dorsal than in the ventral retina. We conclude that α2-adrenergic receptor stimulation reduced the impact of the excitotoxic injury in chicken similarly to what has been shown in mammals. Furthermore, the data show that the RGCs are evenly distributed over in the retina, which challenges previous results that indicate the presence of specific high RGC-density regions of the chicken retina.

Horizontal Cells, the Odd Ones Out in the Retina, Give Insights into Development and Disease.

Thorough investigation of a neuronal population can help reveal key aspects regarding the nervous system and its development. The retinal horizontal cells have several extraordinary features making them particularly interesting for addressing questions regarding fate assignment and subtype specification. In this review we discuss and summarize data concerning the formation and diversity of horizontal cells, how morphology is correlated to molecular markers, and how fate assignment separates the horizontal lineage from the lineages of other retinal cell types. We discuss the novel and unique features of the final cell cycle of horizontal cell progenitors and how they may relate to retinoblastoma carcinogenesis.

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments.

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high. This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

Alpha2-Adrenergic-Agonist Brimonidine Stimulates Negative Feedback and Attenuates Injury-Induced Phospho-ERK and Dedifferentiation of Chicken Müller Cells.

PURPOSE: Retinal injury induces Müller cell dedifferentiation by activating extracellular signal-regulated kinase (ERK) signaling. Stimulation of α2-adrenergic receptors protects against injury but also activates ERK in Müller cells. The purpose of this work was to study the effect of α2-adrenergic signaling on injury-induced ERK and Müller cell dedifferentiation. We tested the hypothesis that α2-stimulation triggers negative feedback regulation of the injury-induced ERK pathway that attenuates Müller cell dedifferentiation. METHODS: Chicken retina injured by N-methyl-D-aspartate and cultured primary Müller cells were stimulated by the α2-adrenergic agonist brimonidine. Immunostaining, quantitative RT-PCR, and Western blot techniques in combination with receptor blockers were used for analysis of the cellular responses. RESULTS: Alpha2-adrenergic receptor stimulation attenuated injury-induced ERK activation and dedifferentiation of Müller cells as seen by decreased phospho-ERK, expression of transitin, and retinal progenitor cell genes. The attenuation was concomitant with a synergistic upregulation of several negative ERK-signal feedback regulators including ERK-phosphatases, Raf1-, and growth factor receptor-binding proteins. The results were also seen in cultures of primary Müller cells. CONCLUSIONS: Alpha2-adrenergic signaling on Müller cells elicits an intracellular attenuation of the injury response that comprises negative ERK-signaling feedback leading to attenuated Müller cell dedifferentiation. The implications of this study are that adrenergic stress signals may directly modulate glial function in retina and that α2-adrenergic receptor pharmacology may be used to control glial injury response.

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.

Specific spatial distribution of caspase-3 in normal lenses.

PURPOSE: To determine the distribution of active caspase-3 in rat eye lens epithelium. METHODS: In total, 120 sagittal sections from forty rats were assessed for active caspase-3 labelling using immunohistochemistry. Lens epithelial cells were counted, and the fraction of active caspase-3 labelled cells and their relative positions were identified in each section. RESULTS: Active caspase-3 is present in normal lens epithelium. The active caspase-3 expression was higher in the anterior pole of the lens. Probability of radial spatial distribution of labelling was fitted with a logistic model. The increase rate and the inflection point were estimated as CI (0.95) to 23 ± 3 cells and 114 ± 3 cells, respectively. CONCLUSION: The gradually decreasing active caspase-3 labelling from the anterior pole to the periphery suggests that active caspase-3 may be involved in normal protein turnover caused by, for example, incident light.