Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy.

Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.

N-Ethylmaleimide-Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium.

PURPOSE: Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE). METHODS: Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation. RESULTS: Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide-sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants. CONCLUSIONS: au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE.

L-Dopa and the albino riddle: content of L-Dopa in the developing retina of pigmented and albino mice.

BACKGROUND: The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown. METHODOLOGY/ PRINCIPAL FINDINGS: L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr(c2j) ) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses. CONCLUSIONS: L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.

Spatiotemporal features of early neuronogenesis differ in wild-type and albino mouse retina.

In albino mammals, lack of pigment in the retinal pigment epithelium is associated with retinal defects, including poor visual acuity from a photoreceptor deficit in the central retina and poor depth perception from a decrease in ipsilaterally projecting retinal fibers. Possible contributors to these abnormalities are reported delays in neuronogenesis (Ilia and Jeffery, 1996) and retinal maturation (Webster and Rowe, 1991). To further determine possible perturbations in neuronogenesis and/or differentiation, we used cell-specific markers and refined birth dating methods to examine these events during retinal ganglion cell (RGC) genesis in albino and pigmented mice from embryonic day 11 (E11) to E18. Our data indicate that relative to pigmented mice, more ganglion cells are born in the early stages of neuronogenesis in the albino retina, although the initiation of RGC genesis in the albino is unchanged. The cellular organization of the albino retina is perturbed as early as E12. In addition, cell cycle kinetics and output along the nasotemporal axis differ in retinas of albino and pigmented mice, both absolutely, with the temporal aspect of the retina expanded in albino, and relative to the position of the optic nerve head. Finally, blocking melanin synthesis in pigmented eyecups in culture leads to an increase in RGC differentiation, consistent with a role for melanin formation in regulating RGC neuronogenesis. These results point to spatiotemporal defects in neuronal production in the albino retina, which could perturb expression of genes that specify cell fate, number, and/or projection phenotype.

Retinal axon divergence in the optic chiasm: midline cells are unaffected by the albino mutation.

The visual pathway in albino animals is abnormal in that there is a smaller number of ipsilaterally projecting retinal ganglion cells. There are two possible sites of gene action that could result in such a defect. The first site is the retina where the amount of pigmentation in the retinal pigment epithelium is correlated with the degree of ipsilateral innervation (La Vail et al. (1978) J. Comp. Neurol. 182, 399-422). The second site is the optic chiasm, the site of retinal axon divergence. We investigated these two possibilities through a combination of in vivo and in vitro techniques. Our results demonstrate that the growth patterns of retinal axons and the cellular composition of the optic chiasm in albino mice are similar to those of normally pigmented mice, consistent with the albino mutation exerting its effects in the retina, and not on the cells from the chiasmatic midline. We directly tested whether the albino mutation affects the chiasm by studying 'chimeric' cultures of retinal explants and chiasm cells isolated from pigmented and albino mice. Crossed and uncrossed axons from pigmented or albino retinal explants display the same amount of differential growth when grown on either pigmented or albino chiasm cells, demonstrating that the albino mutation does not disrupt the signals for retinal axon divergence associated with the albino optic chiasm. Furthermore, in vitro, a greater proportion of albino retinal ganglion cells from ventrotemporal retina, origin of uncrossed axons, behave like crossed cells, suggesting that the albino mutation acts by respecifying the numbers of retinal ganglion cells that cross the chiasmatic midline.

Mouse albino-deletions: from genetics to genes in development.

Six essential genes located near the mouse albino locus have been identified as required during specific periods of development. Amongst these six, each is required either during the preimplantation stages of development, at specific times during gastrulation, within 12 hrs after birth or during juvenile development. These genes were identified as a result of extensive genetic complementation analysis using embryos homozygous for the albino deletions. Although, in principal, the associated developmental abnormalities could result from loss of multiple genes, the deletion phenotype in one case is identical to that induced by chemical mutagenesis. These results indicate that the abnormalities observed in deletion homozygotes may result from single gene loss. The deletions have proven useful not only as genetic tools to localize the position of the genes, but also as molecular entry points to the regions containing these genes. The current methodology being used to isolate candidate genes from the albino region is also reviewed here.

Molecular mapping of albino deletions associated with early embryonic lethality in the mouse.

The albino-deletion complex consists of more than 37 deletions that remove an area of mouse chromosome 7 including the albino coat-color locus. Previous genetic and embryological studies with five of these deletions (C11DSD, c5FR60Hg, c4FR60Hd, c2YPSj, c6H) defined at least two genes required for normal development of the embryonic and extraembryonic ectoderm of early postimplantation embryos. A molecular genetic analysis of this region has been initiated using palb18, a genomic clone that defines the D7TM18 locus that maps to a region of chromosome 7 removed by the c11DSD deletion but not by the c5FR60Hg, c4FR60Hd, c2YPSj, or c6H deletions. palb18 was obtained by chromosomal microdissection and microcloning of the wild-type albino region. A genomic clone isolated with palb18 contains a repeat sequence localized primarily to the proximal region of the five deletions. The repeat sequence hybridizes differentially to the five deletion DNAs. The patterns of hybridization associated with these DNAs were used to define the order of the proximal breakpoints as centromere-c11DSD-c2YPSj-(c5FR60Hg-c4FR60Hd)- c6H. This order was confirmed by isolation of additional single-copy sequences. The molecular probes described here should allow for identification and isolation of the deletion breakpoints and thus provide immediate access to the distal side of the deletions where the genes affecting the development of the embryonic and extraembryonic ectoderm are located.

Abnormal pigmentation and unusual morphogenesis of the optic stalk may be correlated with retinal axon misguidance in embryonic Siamese cats.

Studies of albino rodents have shown that an absence of pigment in the developing optic stalk may alter the position of the first retinal fibers that grow toward the brain, thereby disrupting the gross topographic relationship of fibers in the nerve (Silver and Sapiro: J. Comp. Neurol. 202:521-538, '81). The abnormalities associated with albinism are more extensive in the Siamese cat than-in previously studied species. Therefore, any abnormalities in differentiation of the stalk and axon guidance may be more readily detected. To investigate the guidance and/or misguidance of optic axons, light and electron microscope analyses were made of serial sections through the optic stalk in normally pigmented and Siamese fetal cats. On E20, before axons enter the optic stalk, the only clear morphological distinction between Siamese and normal cats is the distribution of pigment in the stalk. Pigment is found in the dorsal stalk cells of the normal cat for 200 microns from the optic disc. Although the retinal pigment epithelium of the Siamese optic stalk. By E23 axons invade the ventral optic stalk in both strains. Concurrent with the early stages of axonal exit from the retina, there is complete separation of the stalk's dorsal and ventral tiers. As the cleavage occurs, basal lamina invaginates into the zone of separation following along the plane of the old lumen. The ventral stalk fills with axons while the dorsal tier is shed gradually. In contrast, in the Siamese cat, dorsal stalk cells are not sloughed off properly and instead are incorporated ectopically into the nerve. Basal lamina invagination is irregular. Axons do not fill the Siamese stalk symmetrically but enter the region of ectopic cells, which in turn disrupts gross fiber position. Usually, in the mutant, axons originating from the retina temporal to the optic fissure are those that invade the dorsal tier of ectopic cells. The altered position of optic axons in the mutant stalk may provide an explanation for the chiasmatic misrouting of optic axons in this species.

The pigmentary system of developing axolotls. III. An analysis of the albino phenotype.

The albino mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analyses of pteridine pigments were also carried out, and the pattern of pteridines in albino animals was found to be more complex than, and quantitatively enhanced (at all developmental stages examined) over, the pattern observed in comparable wild-type axolotls. The golden colour of albino axolotls is due primarily to sepiapterin (a yellow pteridine) and secondarily to riboflavin (and other flavins). Coincident with enhanced levels of yellow pigments, xanthophore pigment organelles (pterinosomes) in albino skin reach a mature state earlier than they do in wild-type axolotl skin. This morphology is conserved throughout development in albino animals whereas it is gradually lost in the wild type. Unpigmented melanophores from albino axolotls are illustrated for the first time, and in larval albino axolotls the morphology of these cells is shown to be very similar to xanthophore morphology. In older animals xanthophores are easily distinguished from unpigmented melanophores. Iridophores seem to appear in albino skin at an earlier stage than they have been observed in wild-type skin. Morphologically, wild-type and albino iridophores are identical.

The early development of the optic nerve and chiasm in embryonic rat.

To establish the time course and major features of the development of the optic nerve and chiasm in the embryonic rat, the growth of axons from the retina to the brain has been studied by light and electron microscopy. On embryonic day 14 (E14), the first axons are generated by retinal ganglion cells. Fascicles of axons can be detected in the optic stalk at E14.5 and in the diencephalon by E15.0. In the vitreal retina and optic fissure, large extracellular spaces resemble the oriented channels previously described in the mouse. They form approximately 12 hours before the invasion of optic axons and contain hyaluronic acid. In the optic stalk and diencephalon of the rat, similar spaces are not present, but the timed autolysis of neuroepithelial cells could provide a pathway of minimal resistance for the earliest axons. Degenerating cells are prominent in the ventral stalk and rostral diencephalon prior to the arrival of the first optic axons that preferentially invade these regions. The role of pigment in the development of visual pathways is controversial. In one strain of rat, Manchester Hooded, the retinae are heavily pigmented, but little pigment is seen at any stage in the stalk; in albinos, pigment is absent from both retina and stalk. However, the distribution of axons within the developing optic stalk is very similar in both strains, suggesting that the reduction in size of the ipsilateral pathway observed in the albino rat compared with the Manchester Hooded is not due to a lack of pigment in the optic stalk early in development. Several factors previously reported to contribute to the development of retinotopic order in other species are also present in the rat. These include the sequence in which axons grow into the stalk, and fasciculation. Intermembranous contacts observed between growth cones and adjacent tissues suggest one mechanism by which fasciculation occurs. A small group of fascicles, which may represent the ipsilateral projection, diverges from the crossing fibers on E15.5, without evidence of being deflected by any glial or other structures.

Diploid gynogenesis in Xenopus laevis and the localization with respect to the centromere of the gene for periodic albinism ap.

Diploid gynogenetic Xenopus laevis were obtained by inseminating the eggs with u.v. irradiated spermatozoa, and treating them with hydrostatic pressure to inhibit the expulsion of the second polar body. A u.v. dose of 3000 ergs/mm2 genetically inactivates the spermatozoa without loss of their ability to activate egg development. The use of a genetic marker on very large samples of eggs made it possible to verify the efficiency of the methods employed. The comparison of the development of diploid gynogenetic embryos with that of haploid gynogenetic, triploid and diploid controls makes it very probable that the relatively high mortality or abnormal development obtained with the pressurized eggs is not due to partial homozygosity but rather to physical damage to the egg structure by this treatment. Altogether about 2500 developing eggs were used. In addition diploid gynogenetic reproduction from females heterozygous for a known mutation allows the mapping of the gene concerned relative to the centromere on the basis of the recombination rate. For this experiment we used the recessive mutation causing periodic albinism (ap) and found the position of this gene to be between 44 map units from the centromere and the end of the chromosome.

An analysis of pigment cell development in the periodic albino mutant of Xenopus.

The periodic al,bino mutant (apap) of Xenopus in which the development of melanophores is impaired, is further reported here to possess an aberrant pattern of iridophore differentiation. The development of mutant and wild-type neural crest explants isolated in vesicles derived from tissues from identical and different genotypes was examined to determine if the mutant effect resides in the pigment cells or is mediated by the environmental tissues. Mutant melanophores and iridophores cultured in either mutant or wild-type tissues exhibited mutant patterns of differentiation. Wild-type pigment cells cultured in both wild-type and mutant tissues exhibited wild-type patterns of differentiation. Hence the mutation affects the capacities of melanoblasts and iridoblasts to differentiate but not the ability of the environmental tissues to support pigment cell differentiation.

[Immunochemical study of the water-soluble lens proteins in the embryo of Xenopus laevis with the mutation of periodic albinism]. / Immunokhimicheskoe izuchenie vodorastvorimykh belkov khrustalika u zarodysha Xenopus laevis s mutatsiei periodicheskogo al'binizma.

The crystallins of ap mutants of Xenopus laevis have been studied in comparison with those of normal embryos and adults using the complex of immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoadsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The analysis was carried out with antisera to electrophoretic fractions of the mutant lens. 11 organ-specific antigens were found in the lens of both the normal and mutant animals. These proteins are heterogenous by electrophoretic mobility, isoelectrical point, antigenic and species specificity. Each class of crystallins contains antigens which are specific: a) for amphibians only, b) for lower vertebrates, c) for vertebrates in general. No qualitative differences were found between crystallins of the normal and mutant animals. Immunofluorescence analysis has shown that crystalins appear in the normal and mutant embryos practically at the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found (with various antisera). gamma-crystallins and, perhaps, a part of the primary lens fibers. Alpha-crystallins appear later. gamma-crystallins are first identified the synthesis of which manifests itself at the advanced developmental stages. The quantitative predominance of some beta--gamma-crystallins in the mutant lens detected by us (electrophoresis, isoelectrofocusing) is not related to their earlier synthesis in the embryogenesis.

Embryonic appearance of alpha, beta, and gamma crystallins in the periodic albinism (ap) mutant of Xenopus laevis.

The appearance of the crystallins during lens development in the periodic albinism (ap/ap) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, alpha + beta crystallins, and gamma crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to alpha + beta crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkas positive, however, for total lens crystallins. These results are at variance with earlier studies on lens development and the crystallins in wildtype (+/+) X. laevis, where a positive reaction for gamma and total crystallins could be detector total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.

Visual system anomalies in human ocular albinos.

Visually evoked potentials recorded from two types of human ocular albinos demonstrated significant hemispheric asymmetry following monocular stimulation. The asymmetry is indicative of disorganization of retinogeniculostriate projections similar to that reported for mammals with total albinism. Abnormal optic projections are associated with lack of ocular pigment and are not associated with any specific generalized pigment defect.

Ultrastructural analysis of gene interaction and melanosome differentiation in the retinal pigment cells of the albino goldfish.

The genotype of albino goldfish is represented by pp,cc, and the non-albino fish is PP,CC. The P gene begins to control the melanosome formation of retinal pigment cells at St-21; and the C, which is epistatic to P and p, governs melanosome formation in the dermal melanophores and in retinal pigment cells after St-22. Then in the pp,CC fish, between St-21 and St-22, melanosomes of the pigmented retina are albino in type, but after St-22 they become non-albino in type. From ultrastructural observations on the melanosomes of these late-melanizing fish, it is apparent that the imcompletely melanized albino pigment granules can differentiate into normal melanosomes under the control of the C gene. The processes of melanosome maturation in the late-melanizing goldfish are evident from these observations.

The development of animals homozygous for a mutation causing periodic albinism (ap) in Xenopus laevis.

This paper describes the development of a mutant strain associated with periodic albinism (ap) in the clawed toad Xenopus laevis. The most outstanding feature of this mutation is the instability of the albino state. In the course of the development there is a succession of three periods of pigment expression: (1) complete absence of melanin pigment, (2) appearance of melanin in the pigmented epithelium of the eyes and in small quantities in skin melanophores, (3) disappearance of most pigment granules. Repeated spawnings show that the mutant syndrome is inherited as a recessive trait. Possible ways of analysing pigment cell differentiation with the use of the mutation described are discussed.