Impaired neural development in a zebrafish model for Lowe syndrome - PubMed (original) (raw)

. 2012 Apr 15;21(8):1744-59.

doi: 10.1093/hmg/ddr608. Epub 2011 Dec 30.

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Impaired neural development in a zebrafish model for Lowe syndrome

Irene Barinaga-Rementeria Ramirez et al. Hum Mol Genet. 2012.

Abstract

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.

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Figures

Figure 1.

Figure 1.

OCRL1 domain organization, subcellular localization, and tissue-specific splicing and expression are conserved in zebrafish. (A) Schematic view of human and zebrafish OCRL1 and Inpp5 showing the predicted PH, 5-phosphatase, ASH and RhoGAP-like domains. The table indicates amino acid identity between the proteins. (B) Immunofluorescence microscopy of zebrafish AB9 or PAC2 fibroblast cells expressing GFP-tagged OCRL1 isoform a or b (green) with or without mCherry-tagged clathrin light chain (CLC) and labelled with antibodies to Golgin84 or EEA1 (red). Arrows indicate colocalization of OCRL1a with EEA1 or mCherry-clathrin light chain. Scale bar, 10 µm. (C) RT–PCR of adult zebrafish tissues using primers against OCRL1 isoform a and b, or the control ZFPL1. B, brain; F, fin; G, gill; H, heart; I, intestine; K, kidney; L, liver; M, muscle; S, skin. (D) Western blot of zebrafish adult tissues using the indicated antibodies. K, kidney; L, liver; S, skin; B, brain; H, heart; G, gill. Ponceau staining of the membrane shows similar loading per lane.

Figure 2.

Figure 2.

Developmental expression of zebrafish OCRL1. (A) RT–PCR of cDNA prepared at different embryonic developmental time points using primers against total OCRL1 and the control ZFPL1. (B) Western blotting of protein extracts from zebrafish embryos at different developmental time points with the indicated antibodies. (C) Images of whole-mount ISH with OCRL1 probe antisense and sense (control) at the developmental time-points indicated. (D) Transverse (T), sagittal (S) and coronal (C) cryosections of whole-mount ISH with OCRL1 probe at the times of development indicated.

Figure 3.

Figure 3.

A zebrafish mutant deficient in OCRL1 has elevated PtdIns(4,5)_P_2 levels. (A) Left: Schematic diagram showing the location of the retroviral insertion upstream of the OCRL1 start codon. Primers used to amplify genomic DNA from zebrafish embryos are indicated. Right: Agarose gel analysis of DNA amplified from OCRL1−/−, +/− and +/+ embryos using the indicated primer pairs. (B) Western blotting of WT, OCRL1−/− and OCRL1+/+ zebrafish embryos at 2 dpf, or embryos injected with a translation-blocking OCRL1 morpholino (ATGMO). Representative blots and quantitation are shown. Results are expressed as the mean + SEM from five experiments. (C) Quantitation of PtdIns(4,5)_P_2 levels in WT and OCRL1−/− embryos at 2 dpf. Results are expressed as the mean + SEM from six experiments.

Figure 4.

Figure 4.

Electrographic seizure activity in OCRL1 mutant zebrafish. (A) Representative recoding taken from the forebrain of parental OCRL1+/+ and OCRL1−/− mutant zebrafish embryos at 6 dpf. (B) Plot of amplitude versus frequency of recordings taken from OCRL1+/+ and OCRL1−/− embryos. (C,D) Plot of average temperature at onset of seizure (C) and duration of seizure activity (D) in OCRL1+/+ and OCRL1−/− embryos. Data are presented as mean + SEM (n = 23–30). *P < 0.001.

Figure 5.

Figure 5.

White matter lesions in OCRL1 mutant zebrafish. (A) Transverse relaxation (T2)-weighted MRI images of the brains of WT and OCRL1−/− adult zebrafish. Sagittal images indicate the presence of a white matter anomaly (white arrow in insert) in OCRL−/−, which was not seen in WT. Coronal images show the presence of lesions adjacent to the ventricles (white arrow in insert). Immunohistochemistry was carried out on the same samples used for MRI. A higher number of GFAP-positive astrocytes are present in the periventricular lesion suggesting increased gliosis. (B) Examples of GFAP staining of WT and OCRL1−/− brain regions. Note the increased numbers of astrocytes concentrated in regions that correspond to periventricular lesions of OCRL1−/− embryos.

Figure 6.

Figure 6.

Impaired development of the brain and eyes in OCRL1-deficient embryos. (A) Representative bright field images of 26 hpf WT and OCRL1−/− uninjected embryos, or WT embryos injected with either mock or ATGMO morpholino. (B) Confocal slices of 1 dpf WT and OCRL1−/− embryos stained with DRAQ5 to label the nuclei. The overlay is false-coloured with WT staining in blue and OCRL1−/− in red. (C) Representative H&E-stained cryosections from the forebrain (top) or R5 hindbrain region (bottom) of 1 dpf WT and OCRL1−/− embryos. Arrows indicate the reduction in size of these regions in the OCRL mutant. Quantitation of neural tissue cross-sectional area of forebrain sections. Data are expressed as the mean + SEM (n = 10). (D) Quantitation of morphological phenotype. Results are expressed as the mean + SEM (n = 63–501 embryos from 4 to 13 experiments). The WT morphology is normal. Dysmorphic describes embryos with a smaller head and eyes and loss of defined MHB. This phenotype is seen in both OCRL1 morphant and mutant embryos. Other describes embryos with more severe anatomical defects not restricted to the brain and eyes that may arise from high-level over-expression of OCRL1a.

Figure 7.

Figure 7.

Decreased Akt signalling, cell survival and proliferation in OCRL1-deficient embryos. (A) Top: western blotting of 1–4 dpf WT and OCRL1−/− embryo extracts with the indicated antibodies. Representative blots and quantitation are shown. Results are expressed as the mean + SEM from five experiments. *P < 0.01. (A) Bottom: Extracts from 1 dpf WT, OCRL1−/− embryos or OCRL1−/− embryos expressing GFP–OCRL1 isoform a were analysed by western blotting with the indicated antibodies. Quantitation from two experiments is shown, with error bars indicating the range of values obtained. (B) WT, OCRL1−/− and OCRL1 −/− embryos expressing GFP–OCRL1 isoform a at 28 hpf were labelled with acridine orange (AO) to label apoptotic cells. Embryos with different levels of apoptosis were divided into four categories as indicated: from + to ++++. Embryos in each category were counted and data are presented as the mean + SEM (n = 37–96 embryos from 3 to 12 experiments). (C) WT, OCRL1−/− and OCRL1 −/− embryos expressing GFP–OCRL1 isoform a at 28 hpf were stained with anti-phosphohistone H3 (PH3) to label mitotic cells. Note the abundance of mitotic cells in the head region, where they align next to the ventricle boundary (arrows). Embryos were scored as having normal staining indicating WT levels of proliferation (+) or weak staining indicating clearly reduced proliferation (–). Embryos in each category were counted and data are presented as the mean + SEM (n = 35–89 embryos from 4 to 12 experiments).

Figure 8.

Figure 8.

Catalytic activity and binding to clathrin are required for OCRL1 function in brain development. (A) Representative bright field images of 26 hpf OCRL1−/− mutant embryos either uninjected (left) or injected with mRNA encoding GFP–OCRL1 D480A (right). The phenotypes were scored as dysmorphic (left) or severely dysmorphic (right), in which the brain size is further reduced when compared with the mutant alone. Quantitation of the morphological phenotypes obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 38–324 embryos from 2 to 18 experiments). (B) Representative AO staining of apoptosis in OCRL1−/− mutant embryos expressing GFP–OCRL1 D480A or GFP–OCRL1 ▵LIDLE. Note the increased apoptosis in the head region (left) and spinal cord (right), indicated by arrows. Quantitation of the AO staining obtained upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 29–136 embryos from 2 to 17 experiments). (C) Quantitation of proliferation as assessed by PH3 staining upon expression of the indicated constructs in OCRL1−/− mutant embryos. Results are expressed as the mean + SEM (n = 13–89 embryos from 2 to 12 experiments).

References

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