Microglia are CNS-resident macrophages that scavenge debris and regulate immune responses. Proliferation and development of macrophages, including microglia, requires Colony Stimulating Factor 1 ...Receptor (CSF1R), a gene previously associated with a dominant adult-onset neurological condition (adult-onset leukoencephalopathy with axonal spheroids and pigmented glia). Here, we report two unrelated individuals with homozygous CSF1R mutations whose presentation was distinct from ALSP. Post-mortem examination of an individual with a homozygous splice mutation (c.1754−1G>C) demonstrated several structural brain anomalies, including agenesis of corpus callosum. Immunostaining demonstrated almost complete absence of microglia within this brain, suggesting that it developed in the absence of microglia. The second individual had a homozygous missense mutation (c.1929C>A p.His643Gln) and presented with developmental delay and epilepsy in childhood. We analyzed a zebrafish model (csf1rDM) lacking Csf1r function and found that their brains also lacked microglia and had reduced levels of CUX1, a neuronal transcription factor. CUX1+ neurons were also reduced in sections of homozygous CSF1R mutant human brain, identifying an evolutionarily conserved role for CSF1R signaling in production or maintenance of CUX1+ neurons. Since a large fraction of CUX1+ neurons project callosal axons, we speculate that microglia deficiency may contribute to agenesis of the corpus callosum via reduction in CUX1+ neurons. Our results suggest that CSF1R is required for human brain development and establish the csf1rDM fish as a model for microgliopathies. In addition, our results exemplify an under-recognized form of phenotypic expansion, in which genes associated with well-recognized, dominant conditions produce different phenotypes when biallelically mutated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Sphingolipidoses are severe, mostly infantile lysosomal storage disorders (LSDs) caused by defective glycosphingolipid degradation. Two of these sphingolipidoses, Tay Sachs and Sandhoff diseases, are ...caused by β‐Hexosaminidase (HEXB) enzyme deficiency, resulting in ganglioside (GM2) accumulation and neuronal loss. The precise sequence of cellular events preceding, and leading to, neuropathology remains unclear, but likely involves inflammation and lysosomal accumulation of GM2 in multiple cell types. We aimed to determine the consequences of Hexb activity loss for different brain cell types using zebrafish. Hexb deficient zebrafish (hexb−/−) showed lysosomal abnormalities already early in development both in radial glia, which are the neuronal and glial progenitors, and in microglia. Additionally, at 5 days postfertilization, hexb−/− zebrafish showed reduced locomotor activity. Although specific oligosaccharides accumulate in the adult brain, hexb−/−) zebrafish are viable and apparently resistant to Hexb deficiency. In all, we identified cellular consequences of loss of Hexb enzyme activity during embryonic brain development, showing early effects on glia, which possibly underlie the behavioral aberrations. Hereby, we identified clues into the contribution of non‐neuronal lysosomal abnormalities in LSDs affecting the brain and provide a tool to further study what underlies the relative resistance to Hexb deficiency in vivo.
• hexb−/− zebrafish brain accumulates oligosaccharides associated with HEXB loss.
• hexb−/− zebrafish show early lysosomal abnormalities in radial glia and microglia.
• hexb−/− zebrafish are viable and fertile.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. ...Here, we analyzed
:GFP-expressing macrophages in
-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae,
+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in
-mutants, exclusively amoeboid
cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile
-deficient zebrafish exhibit systemic macrophage depletion. Thus,
deficiency disrupts embryonic to adult macrophage development. Zebrafish deficient for
are viable and permit analyzing the consequences of macrophage loss throughout life.
Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 ...individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential
UDP
-
glucose pyrophosphorylase
(
UGP2
) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in
UGP2
as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of ...messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Microglia are brain-resident macrophages with trophic and phagocytic functions. Dominant loss-of-function mutations in a key microglia regulator, colony-stimulating factor 1 receptor (CSF1R), cause ...adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a progressive white matter disorder. Because it remains unclear precisely how CSF1R mutations affect microglia, we generated an allelic series of csf1r mutants in zebrafish to identify csf1r-dependent microglia changes. We found that csf1r mutations led to aberrant microglia density and distribution and regional loss of microglia. The remaining microglia still had a microglia-specific gene expression signature, indicating that they had differentiated normally. Strikingly, we also observed lower microglia numbers and widespread microglia depletion in postmortem brain tissue of ALSP patients. Both in zebrafish and in human disease, local microglia loss also presented in regions without obvious pathology. Together, this implies that CSF1R mainly regulates microglia density and that early loss of microglia may contribute to ALSP pathogenesis.
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•csf1ra and csf1rb together regulate microglia density in the adult zebrafish brain•csf1r haploinsufficient microglia are normally differentiated and show normal signature•CSF1R haploinsufficiency causes reduced microglia density and widespread depletion•Microglia loss may be an early pathogenic event contributing to leukodystrophy
Oosterhof et al. show that colony-stimulating factor 1 receptor (CSF1R) primarily regulates microglia density and not their normal differentiation. In addition, they find widespread depletion of microglia in CSF1R-haploinsufficient zebrafish and leukodystrophy patients, also in the absence of pathology, indicating that microglia depletion may contribute to loss of white matter.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a ...substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.
IDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.
In total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity.
We have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Microglia are brain resident macrophages important for brain development, connectivity, homeostasis and disease. However, it is still largely unclear how microglia functions and their identity are ...regulated at the molecular level. Although recent transcriptomic studies have identified genes specifically expressed in microglia, the function of most of these genes in microglia is still unknown. Here, we performed RNA sequencing on microglia acutely isolated from healthy and neurodegenerative zebrafish brains. We found that a large fraction of the mouse microglial signature is conserved in the zebrafish, corroborating the use of zebrafish to help understand microglial genetics in mammals in addition to studying basic microglia biology. Second, our transcriptome analysis of microglia following neuronal ablation suggested primarily a proliferative response of microglia, which we confirmed by immunohistochemistry and in vivo imaging. Together with the recent improvements in genome editing technology in zebrafish, these data offer opportunities to facilitate functional genetic research on microglia in vivo in the healthy as well as in the diseased brain. GLIA 2016;65:138–149
Main Points
We identified the zebrafish microglia transcriptome.
Microglial gene expression in mammals is conserved in zebrafish.
Microglia following acute neuronal cell death are characterized mainly by proliferation.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Microglia are brain-resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive ...the migration of yolk sac macrophages (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based
reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, by Cas9/gRNA complex injections, followed by Neutral-Red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in microglia development
were ambiguous. Our data, and a concurrent paper, show that, in zebrafish,
is required during the earliest seeding of the brain by microglia. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified
as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.
Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, ...hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (
FLII
) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific
FLII
variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.
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