Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length.

Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. Given that individuals with CHARGE syndrome can experience visual impairment even in the absence of coloboma, a better understanding of CHD7 function in the retina is needed. In this study, we characterized the expression pattern of Chd7 in the developing zebrafish and mouse retina and documented ocular and retinal phenotypes in Chd7 loss-of-function mutants. Zebrafish Chd7 was expressed throughout the retinal neuroepithelium when retinal progenitor cells were actively proliferating, and later in subsets of newly post-mitotic retinal cells. At stages of retinal development when most retinal cell types had terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Intriguingly, strong expression of Chd7 was also observed in the outer nuclear layer where it was co-expressed with markers of post-mitotic cone and rod photoreceptors. Expression of mouse CHD7 displayed a similar pattern, including expression in the ganglion cells, subsets of inner nuclear layer cells, and in the distal outer nuclear layer as late as P15. Two different mutant chd7 zebrafish lines were characterized for ocular and retinal defects. These mutants displayed microphthalmia, reduced numbers of cone photoreceptors, and truncated rod and cone photoreceptor outer segments. Reduced cone photoreceptor number and abnormal outer segments were also observed in heterozygous Chd7 mutant mice. Taken together, our results in zebrafish and mouse reveal a conserved, previously undescribed role for Chd7 in retinal development and photoreceptor outer segment morphogenesis. Moreover, our work suggests an avenue of future investigation into the pathogenesis of visual system defects in CHARGE syndrome.

Eyes on CHARGE syndrome: Roles of CHD7 in ocular development.

The development of the vertebrate visual system involves complex morphogenetic interactions of cells derived from multiple embryonic lineages. Disruptions in this process are associated with structural birth defects such as microphthalmia, anophthalmia, and coloboma (collectively referred to as MAC), and inherited retinal degenerative diseases such as retinitis pigmentosa and allied dystrophies. MAC and retinal degeneration are also observed in systemic congenital malformation syndromes. One important example is CHARGE syndrome, a genetic disorder characterized by coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. Mutations in the gene encoding Chromodomain helicase DNA binding protein 7 (CHD7) cause the majority of CHARGE syndrome cases. However, the pathogenetic mechanisms that connect loss of CHD7 to the ocular complications observed in CHARGE syndrome have not been identified. In this review, we provide a general overview of ocular development and congenital disorders affecting the eye. This is followed by a comprehensive description of CHARGE syndrome, including discussion of the spectrum of ocular defects that have been described in this disorder. In addition, we discuss the current knowledge of CHD7 function and focus on its contributions to the development of ocular structures. Finally, we discuss outstanding gaps in our knowledge of the role of CHD7 in eye formation, and propose avenues of investigation to further our understanding of how CHD7 activity regulates ocular and retinal development.

Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing.

Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and developmental biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.

Variable Anterior Segment Dysgenesis and Cardiac Anomalies Caused by a Novel Truncating Variant of FOXC1.

Anterior segment dysgenesis (ASD) encompasses a wide spectrum of developmental abnormalities of the anterior ocular segment, including congenital cataract, iris hypoplasia, aniridia, iridocorneal synechiae, as well as Peters, Axenfeld, and Rieger anomalies. Here, we report a large five-generation Caucasian family exhibiting atypical syndromic ASD segregating with a novel truncating variant of FOXC1. The family history is consistent with highly variable autosomal dominant symptoms including isolated glaucoma, iris hypoplasia, aniridia, cataract, hypothyroidism, and congenital heart anomalies. Whole-exome sequencing revealed a novel variant [c.313_314insA; p.(Tyr105*)] in FOXC1 that disrupts the α-helical region of the DNA-binding forkhead box domain. In vitro studies using a heterologous cell system revealed aberrant cytoplasmic localization of FOXC1 harboring the Tyr105* variant, likely precluding downstream transcription function. Meta-analysis of the literature highlighted the intrafamilial variability related to FOXC1 truncating alleles. This study highlights the clinical variability in ASD and signifies the importance of combining both clinical and molecular analysis approaches to establish a complete diagnosis.

Detection and Establishment of Aedes notoscriptus (Diptera: Culicidae) Mosquitoes in Southern California, United States.

Aedes notoscriptus (Skuse), the Australian backyard mosquito, is a pestiferous daytime-biting species native to Australia and the surrounding southwestern Pacific region. It is suspected to play a role in the transmission of several arboviruses and is considered a competent vector of dog heartworm, Dirofilaria immitis (Leidy). This highly adaptable mosquito thrives in natural and artificial water-holding containers in both forested and urbanized areas, from tropical to temperate climates, and has benefitted from a close association with humans, increasing in abundance within its native range. It invaded and successfully established in New Zealand as well as in previously unoccupied temperate and arid regions of Australia. Ae. notoscriptus was discovered in Los Angeles County, CA, in 2014, marking the first time this species had been found outside the southwestern Pacific region. By the end of 2019, immature and adult mosquitoes had been collected from 364 unique locations within 44 cities spanning three southern California counties. The discovery, establishment, and rapid spread of this species in urban areas may signal the global movement and advent of a new invasive container-inhabiting species. The biting nuisance, public health, and veterinary health implications associated with the invasion of southern California by this mosquito are discussed.

Evolution of Wolbachia mutualism and reproductive parasitism: insight from two novel strains that co-infect cat fleas.

Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae, wCfeT and wCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA, USA). wCfeT is basal to nearly all described Wolbachia supergroups, while wCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution of Wolbachia host associations. wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across the Wolbachia tree, indicating parallel evolution towards mutualism. Alternately, wCfeJ harbors a toxin-antidote operon analogous to the wPip cinAB operon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies. wCfeJ cinB and three adjacent genes are collectively similar to large modular toxins encoded in CI-like operons of certain Wolbachia strains and Rickettsia species, signifying that CI toxins streamline by fission of large modular toxins. Remarkably, the C. felis genome itself contains two CI-like antidote genes, divergent from wCfeJ cinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongst C. felis colonies, though fleas in wild populations mostly harbor wCfeT alone. Collectively, genomes of wCfeT, wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.

Author Correction: Her9/Hes4 is required for retinal photoreceptor development, maintenance, and survival.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Her9/Hes4 is required for retinal photoreceptor development, maintenance, and survival.

The intrinsic and extrinsic factors that regulate vertebrate photoreceptor specification and differentiation are complex, and our understanding of all the players is far from complete. Her9, the zebrafish ortholog of human HES4, is a basic helix-loop-helix-orange transcriptional repressor that regulates neurogenesis in several developmental contexts. We have previously shown that her9 is upregulated during chronic rod photoreceptor degeneration and regeneration in adult zebrafish, but little is known about the role of her9 during retinal development. To better understand the function of Her9 in the retina, we generated zebrafish her9 CRISPR mutants. Her9 homozygous mutants displayed striking retinal phenotypes, including decreased numbers of rods and red/green cones, whereas blue and UV cones were relatively unaffected. The reduction in rods and red/green cones correlated with defects in photoreceptor subtype lineage specification. The remaining rods and double cones displayed abnormal outer segments, and elevated levels of apoptosis. In addition to the photoreceptor defects, her9 mutants also possessed a reduced proliferative ciliary marginal zone, and decreased and disorganized Müller glia. Mutation of her9 was larval lethal, with no mutants surviving past 13 days post fertilization. Our results reveal a previously undescribed role for Her9/Hes4 in photoreceptor differentiation, maintenance, and survival.

Rickettsial Infections among Cats and Cat Fleas in Riverside County, California.

Presently, few studies have investigated the role of domestic cats (Felis catus) in the recrudescence of flea-borne rickettsioses in California and the southern United States. In this study, we aimed to investigate the presence of Rickettsia typhi or Rickettisa felis in domestic cats (F. catus) and the fleas (primarily Ctenocephalides felis, the cat flea) associated with these cats in Riverside County, California. Thirty cats and 64 pools of fleas collected from these cats were investigated for rickettsial infections. Three cats and 17 flea pools (from 10 cats) tested positive for rickettsial infections. polymerase chain reaction and DNA sequencing indicated that one of the cats was positive for R. felis infections, whereas two were positive for Candidatus Rickettsia senegalensis infection. In addition, 12 of the flea pools were positive for R. felis, whereas five were positive for Ca. R. senegalensis. By contrast, no cats or their associated fleas tested positive for R. typhi. Finally, eight sera from these cats contained spotted fever group Rickettsia (SFGR) antibodies. The detection of R. felis and SFGR antibodies and the lack of R. typhi and TGR antibodies support R. felis as the main rickettsial species infecting cat fleas. The detection of Ca. R. senegalensis in both fleas and cats also provides additional evidence that cats and their associated fleas are infected with other R. felis-like organisms highlighting the potential risk for human infections with R. felis or R. felis-like organisms.

Rickettsial Infections among Ctenocephalides felis and Host Animals during a Flea-Borne Rickettsioses Outbreak in Orange County, California.

Due to a resurgence of flea-borne rickettsioses in Orange County, California, we investigated the etiologies of rickettsial infections of Ctenocephalides felis, the predominant fleas species obtained from opossums (Didelphis virginiana) and domestic cats (Felis catus), collected from case exposure sites and other areas in Orange County. In addition, we assessed the prevalence of IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) rickettsiae in opossum sera. Of the 597 flea specimens collected from opossums and cats, 37.2% tested positive for Rickettsia. PCR and sequencing of rickettsial genes obtained from C. felis flea DNA preparations revealed the presence of R. typhi (1.3%), R. felis (28.0%) and R. felis-like organisms (7.5%). Sera from opossums contained TGR-specific (40.84%), but not SFGR-specific antibodies. The detection of R. felis and R. typhi in the C. felis fleas in Orange County highlights the potential risk for human infection with either of these pathogens, and underscores the need for further investigations incorporating specimens from humans, animal hosts, and invertebrate vectors in endemic areas. Such studies will be essential for establishing a link in the ongoing flea-borne rickettsioses outbreaks.

A new frontonasal dysplasia syndrome associated with deletion of the SIX2 gene.

The frontonasal dysplasias are a group of craniofacial phenotypes characterized by hypertelorism, nasal clefting, frontal bossing, and abnormal hairline. These conditions are caused by recessive mutations in members of the aristaless gene family, resulting in abnormal cranial neural crest migration and differentiation. We report a family with a dominantly inherited craniofacial phenotype comprised of frontal bossing with high hairline, ptosis, hypertelorism, broad nasal tip, large anterior fontanelle, cranial base anomalies, and sagittal synostosis. Chromosomal microarray identified a heterozygous 108.3 kilobase deletion of chromosome 2p21 segregating with phenotype and limited to the sine oculis homeobox gene SIX2 and surrounding noncoding DNA. Similar to the human SIX2 deletion phenotype, one mouse model of frontonasal dysplasia, brachyrrhine, exhibits dominant inheritance and impaired cranial base chondrogenesis associated with reduced Six2 expression. We report the first human autosomal dominant frontonasal dysplasia syndrome associated with SIX2 deletion and with phenotypic similarities to murine models of Six2 Loss-of-function.

Nucleoside Reverse Transcriptase Inhibitors Suppress Laser-Induced Choroidal Neovascularization in Mice.

PURPOSE: To evaluate the efficacy of nucleoside reverse transcriptase inhibitors (NRTIs) in the laser-induced mouse model of choroidal neovascularization (CNV). METHODS: We evaluated the NRTIs lamivudine (3TC), zidovudine (AZT), and abacavir (ABC) and the P2X7 antagonist A438079. Choroidal neovascularization was induced by laser injury in C57BL/6J wild-type, Nlrp3-/-, and P2rx7-/- mice, and CNV volume was measured after 7 days by confocal microscopy. Drugs were administered by intravitreous injection immediately after the laser injury. Vascular endothelial growth factor-A in RPE-choroid lysates was measured 3 days after laser injury by ELISA. HEK293 cells expressing human and mouse P2X7 were exposed to the selective P2X7 receptor agonist 2', 3'-(benzoyl-4-benzoyl)-ATP (Bz-ATP) with or without 3TC, and VEGF-A levels in media were measured by ELISA. RESULTS: Intravitreous injection of 3TC, AZT, and ABC significantly suppressed laser-induced CNV in C57BL/6J wild-type and Nlrp3-/- mice (P < 0.05) but not in P2rx7-/- mice. Intravitreous injection of A438079 also suppressed the laser-induced CNV (P < 0.05). The NRTIs 3TC, AZT, and ABC blocked VEGF-A levels in the RPE/choroid after laser injury in wild-type (P < 0.05) but not P2rx7-/- mice. Moreover, there was no additive effect of 3TC on CNV inhibition when coadministered with a neutralizing VEGF-A antibody. Stimulation of human and mouse P2X7-expressing HEK293 cells with Bz-ATP increased VEGF secretion (P < 0.001), which was abrogated by 3TC (P < 0.001). Stimulation of primary human RPE cells with Bz-ATP increased VEGFA and IL6 mRNA levels, which were abrogated by 3TC. CONCLUSIONS: Multiple clinically relevant NRTIs suppressed laser-induced CNV and downregulated VEGF-A, via P2X7.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder.

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes.

BACKGROUND: Oliver-McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence-Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause. METHODS: Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines. RESULTS: Eight mutations in six families with Oliver-McFarlane or Laurence-Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver-McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver-McFarlane and Laurence-Moon syndromes revealed extensive cerebellar degeneration and atrophy. CONCLUSIONS: Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon-Holmes syndrome and Boucher-Neuhäuser syndromes. Discovery of these additional PNPLA6-opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance.

Arabidopsis thaliana WAPL is essential for the prophase removal of cohesin during meiosis.

Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes in mitosis and meiosis. The establishment of stable sister chromatid cohesion occurs during DNA replication and involves acetylation of the complex by the acetyltransferase CTF7. In higher eukaryotes, the majority of cohesin complexes are removed from chromosomes during prophase. Studies in fly and human have shown that this process involves the WAPL mediated opening of the cohesin ring at the junction between the SMC3 ATPase domain and the N-terminal domain of cohesin's α-kleisin subunit. We report here the isolation and detailed characterization of WAPL in Arabidopsis thaliana. We show that Arabidopsis contains two WAPL genes, which share overlapping functions. Plants in which both WAPL genes contain T-DNA insertions show relatively normal growth and development but exhibit a significant reduction in male and female fertility. The removal of cohesin from chromosomes during meiotic prophase is blocked in Atwapl mutants resulting in chromosome bridges, broken chromosomes and uneven chromosome segregation. In contrast, while subtle mitotic alterations are observed in some somatic cells, cohesin complexes appear to be removed normally. Finally, we show that mutations in AtWAPL suppress the lethality associated with inactivation of AtCTF7. Taken together our results demonstrate that WAPL plays a critical role in meiosis and raises the possibility that mechanisms involved in the prophase removal of cohesin may vary between mitosis and meiosis in plants.

Two pathogens and one disease: detection and identification of flea-borne Rickettsiae in areas endemic for murine typhus in California.

Results of an environmental assessment conducted in a newly emergent focus of murine typhus in southern California are described. Opossums, Didelphis virginiana Kerr, infested with cat fleas, Ctenocephalides felis Buché, in the suburban area were abundant. Animal and flea specimens were tested for the DNA of two flea-borne rickettsiae, Rickettsia typhi and Rickettsia felis. R. felis was commonly detected in fleas collected throughout this area while R. typhi was found at a much lower prevalence in the vicinity of just 7 of 14 case-patient homes identified. DNA of R. felis, but not R. typhi, was detected in renal, hepatic, and pulmonary tissues of opossums. In contrast, there were no hematologic polymerase chain reaction findings of R. felis or R. typhi in opossums, rats, and cats within the endemic area studied. Our data suggest a significant probability of human exposure to R. felis in the area studied; however, disease caused by this agent is not recognized by the medical community and may be misdiagnosed as murine typhus using nondiscriminatory serologic methods.

Urban focus of Rickettsia typhi and Rickettsia felis in Los Angeles, California.

Classic murine typhus, caused by Rickettsia typhi, is endemic in the continental United States in areas of Texas and southern California. We conducted an environmental investigation in an urban area of Los Angeles identified as the probable exposure site for a case of murine typhus. Four Rattus norvegicus heavily infested with Xenopsylla cheopis (average 32.5 fleas per animal, range 20-42) were trapped, and fleas, blood, and tissues were collected. DNAs from all specimens were tested for R. typhi and Rickettsia felis using a TaqMan assay targeting the rickettsial citrate synthase gene. Although rickettsiemia was not detected, DNA of R. felis was detected in at least one tissue from each rat. Tissues from 3 rats were also positive for R. typhi DNA. R. typhi and R. felis DNAs were detected in fleas collected from each animal with average minimal infection rates of 10% and 32.3%, respectively. Although R. typhi still circulates in urban Los Angeles in the classic Oriental flea-rat cycle, R. felis is more prevalent, even in this association.

Detection and identification of spotted fever group rickettsiae in Dermacentor species from southern California.

Dermacentor occidentalis Marx and Dermacentor variabilis (Say) commonly bite humans in California. These Dermacentor species may play a role in transmitting spotted fever group (SFG) rickettsiae to humans in many parts of the state where Dermacentor andersoni Stiles, a known vector for the etiologic agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is absent. However, the specific rickettsial agents present in these ticks and their current prevalence are poorly understood. In total, 365 D. occidentalis and 10 D. variabilis were collected by flagging vegetation at 16 sites in five counties of southern California. The presence of SFG rickettsial DNA in these ticks was detected with rOmpA and GltA gene polymerase chain reaction (PCR) assays. The rickettsial species were identified by sequencing PCR amplicons. Of 365 D. occidentalis, 90 (24.7%) contained R. rhipicephali DNA, 28 (7.7%) contained DNA of unclassified genotype 364D, two (0.55%) contained R. bellii DNA, and one (0.3%) contained R. rickettsii DNA. Of 10 D. variabilis, four (40%) contained only R. rhipicephali. Four new genotypes of R. rhipicephali were discovered. For the first time, we detected R. rickettsii in D. occidentalis. Our study provides the first molecular data on the prevalence and species identification of SFG rickettsiae circulating in populations of these California ticks. Because neither D. variabilis nor R. rickettsii were abundant, 364D should be evaluated further as a potential cause of human SFG rickettsioses in southern California.