Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. In particular, ...genetic tools to analyze the role of Irf8 in zebrafish macrophage development at larval and adult stages are lacking. We generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils and excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.
Arthrogryposis multiplex congenita is defined by the presence of contractures across two or more major joints and results from reduced or absent fetal movement. Here, we present three consanguineous ...families affected by lethal arthrogryposis multiplex congenita. By whole-exome or targeted exome sequencing, it was shown that the probands each harbored a different homozygous mutation (one missense, one nonsense, and one frameshift mutation) in GPR126. GPR126 encodes G-protein-coupled receptor 126, which has been shown to be essential for myelination of axons in the peripheral nervous system in fish and mice. A previous study reported that Gpr126−/− mice have a lethal arthrogryposis phenotype. We have shown that the peripheral nerves in affected individuals from one family lack myelin basic protein, suggesting that this disease in affected individuals is due to defective myelination of the peripheral axons during fetal development. Previous work has suggested that autoproteolytic cleavage is important for activating GPR126 signaling, and our biochemical assays indicated that the missense substitution (p.Val769Glu c.2306T>A) impairs autoproteolytic cleavage of GPR126. Our data indicate that GPR126 is critical for myelination of peripheral nerves in humans. This study adds to the literature implicating defective axoglial function as a key cause of severe arthrogryposis multiplex congenita and suggests that GPR126 mutations should be investigated in individuals affected by this disorder.
In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether ...they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.
Myelination by oligodendrocytes is critical for fast axonal conduction and for the support and survival of neurons in the central nervous system. Recent studies have emphasized that myelination is ...plastic and that new myelin is formed throughout life. Nonetheless, the mechanisms that regulate the number, length, and location of myelin sheaths formed by individual oligodendrocytes are incompletely understood. Previous work showed that the lysosomal transcription factor TFEB represses myelination by oligodendrocytes and that the RagA GTPase inhibits TFEB, but the step or steps of myelination in which TFEB plays a role have remained unclear. Here, we show that TFEB regulates oligodendrocyte differentiation and also controls the length of myelin sheaths formed by individual oligodendrocytes. In the dorsal spinal cord of tfeb mutants, individual oligodendrocytes produce myelin sheaths that are longer than those produced by wildtype cells. Transmission electron microscopy shows that there are more myelinated axons in the dorsal spinal cord of tfeb mutants than in wildtype animals, but no significant change in axon diameter. In contrast to tfeb mutants, oligodendrocytes in rraga mutants produce shorter myelin sheaths. The sheath length in rraga; tfeb double mutants is not significantly different from wildtype, consistent with the antagonistic interaction between RagA and TFEB. Finally, we find that the GTPase activating protein Flcn and the RagCa and RagCb GTPases are also necessary for myelination by oligodendrocytes. These findings demonstrate that TFEB coordinates myelin sheath length and number during myelin formation in the central nervous system.
Main Points
Myelin sheath length is decreased in rraga mutants, increased in tfeb mutants, and normal in rraga; tfeb double mutants.
Antagonism between RagA and TFEB regulates myelin sheath length in the CNS.
Flcn, RagCa, and RagCb are necessary for CNS myelination.
Myelin allows for fast and efficient axonal conduction, but much remains to be determined about the mechanisms that regulate myelin formation. To investigate the genetic basis of myelination, we ...carried out a genetic screen using zebrafish. Here, we show that the lysosomal G protein RagA is essential for CNS myelination. In rraga
mutant oligodendrocytes, target genes of the lysosomal transcription factor Tfeb are upregulated, consistent with previous evidence that RagA represses Tfeb activity. Loss of Tfeb function is sufficient to restore myelination in RagA mutants, indicating that hyperactive Tfeb represses myelination. Conversely, tfeb
single mutants exhibit ectopic myelin, further indicating that Tfeb represses myelination during development. In a mouse model of de- and remyelination, TFEB expression is increased in oligodendrocytes, but the protein is localized to the cytoplasm, and hence inactive, especially during remyelination. These results define essential regulators of myelination and may advance approaches to therapeutic remyelination.
Pathogenic variants in ACTA2, encoding smooth muscle α‐actin, predispose to thoracic aortic aneurysms and dissections. ACTA2 variants altering arginine 179 predispose to a more severe, multisystemic ...disease termed smooth muscle dysfunction syndrome (SMDS; OMIM 613834). Vascular complications of SMDS include patent ductus arteriosus (PDA) or aortopulmonary window, early‐onset thoracic aortic disease (TAD), moyamoya‐like cerebrovascular disease, and primary pulmonary hypertension. Patients also have dysfunction of other smooth muscle‐dependent systems, including congenital mydriasis, hypotonic bladder, and gut hypoperistalsis. Here, we describe five patients with novel heterozygous ACTA2 missense variants, p.Arg179Gly, p.Met46Arg, p.Thr204Ile, p.Arg39Cys, and p.Ile66Asn, who have clinical complications that align or overlap with SMDS. Patients with the ACTA2 p.Arg179Gly and p.Thr204Ile variants display classic features of SMDS. The patient with the ACTA2 p.Met46Arg variant exhibits exclusively vascular complications of SMDS, including early‐onset TAD, PDA, and moyamoya‐like cerebrovascular disease. The patient with the ACTA2 p.Ile66Asn variant has an unusual vascular complication, a large fusiform internal carotid artery aneurysm. The patient with the ACTA2 p.Arg39Cys variant has pulmonary, gastrointestinal, and genitourinary complications of SMDS but no vascular manifestations. Identifying pathogenic ACTA2 variants associated with features of SMDS is critical for aggressive surveillance and management of vascular and nonvascular complications and delineating the molecular pathogenesis of SMDS.
We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice‐site variant (NM_001267550.1:c.39974‐11T>G), inherited in trans with a second ...pathogenic TTN variant. Muscle‐derived RNA studies of three individuals confirmed mis‐splicing induced by the c.39974‐11T>G variant; in‐frame exon 214 skipping or use of a cryptic 3′ splice‐site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213‐217 within the described skeletal muscle TTN N2A isoform. However, RNA‐sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213‐217 in TTN transcripts (inclusion rate ≥66%). Further, RNA‐sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213‐217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript‐only exons.
Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic‐ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the ...clinical presentation. Mutations in ADD3 (encoding γ‐adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). We studied a 16‐year‐old male with spastic diplegia. Several investigations including neurometabolic testing, brain and spine magnetic resonance imaging (MRI) and CGH‐Array were normal. Further, clinical genetics assessment and whole exome sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ‐adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G > A, p.(Gly367Asp). Mutations in this gene have been described as an ultra‐rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ‐adducin and is therefore expected to result in a decreased level of functional protein. Pan‐neuronal over‐expression or knock‐down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.
Mutations in ADD3 (encoding γ‐adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568).Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ‐adducin and is therefore expected to result in a decreased level of functional protein. Pan‐neuronal over‐expression or knock‐down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease.
Muconic acid is a potential platform chemical for the production of nylon, polyurethanes, and terephthalic acid. It is also an attractive functional copolymer in plastics due to its two double bonds. ...At this time, no economically viable process for the production of muconic acid exists. To harness novel genetic targets for improved production of
-muconic acid (CCM) in the yeast
, we employed a CCM-biosensor coupled to GFP expression with a broad dynamic response to screen UV-mutagenesis libraries of CCM-producing yeast. Via fluorescence activated cell sorting we identified a clone Mut131 with a 49.7% higher CCM titer and 164% higher titer of biosynthetic intermediate-protocatechuic acid (PCA). Genome resequencing of the Mut131 and reverse engineering identified seven causal missense mutations of the native genes (
,
,
,
,
,
, and
) and a duplication of two CCM biosynthetic genes, encoding dehydroshikimate dehydratase and catechol 1,2-dioxygenase, which were not recognized as flux controlling before. The Mut131 strain was further rationally engineered by overexpression of the genes encoding for PCA decarboxylase and AROM protein without shikimate dehydrogenase domain (Aro1p
), and by restoring
prototrophy. The resulting engineered strain produced 20.8 g/L CCM in controlled fed-batch fermentation, with a yield of 66.2 mg/g glucose and a productivity of 139 mg/L/h, representing the highest reported performance metrics in a yeast for
CCM production to date and the highest production of an aromatic compound in yeast. The study illustrates the benefit of biosensor-based selection and brings closer the prospect of biobased muconic acid.
Phosphate concentration is tightly regulated at the cellular and organismal levels. The first metazoan phosphate exporter, XPR1, was recently identified, but its in vivo function remains unknown. In ...a genetic screen, we identified a mutation in a zebrafish ortholog of human XPR1, xpr1b. xpr1b mutants lack microglia, the specialized macrophages that reside in the brain, and also displayed an osteopetrotic phenotype characteristic of defects in osteoclast function. Transgenic expression studies indicated that xpr1b acts autonomously in developing macrophages. xpr1b mutants display no gross developmental defects that may arise from phosphate imbalance. We constructed a targeted mutation of xpr1a, a duplicate of xpr1b in the zebrafish genome, to determine whether Xpr1a and Xpr1b have redundant functions. Single mutants for xpr1a were viable, and double mutants for xpr1b;xpr1a were similar to xpr1b single mutants. Our genetic analysis reveals a specific role for the phosphate exporter Xpr1 in the differentiation of tissue macrophages.
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•xpr1b phosphate exporter is required for tissue macrophage differentiation in vivo•xpr1b acts autonomously in macrophages•Xpr1 zygotic function is dispensable for viability of the embryo and early larva
XPR1 is the only known metazoan exporter for inorganic phosphate, a crucial ion necessary for bone mineralization and diverse cellular activities. Meireles et al. now identify null mutations in xpr1a and xpr1b, the zebrafish orthologs of human XPR1. Zebrafish xpr1b mutants lack microglia and other tissue-resident macrophage populations but display no gross developmental defects, and xpr1a;xpr1b double mutants have a similar phenotype. This genetic analysis reveals a specific role for Xpr1b in the differentiation of tissue macrophages.
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