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Impaired neural development in a zebrafish model for Lowe syndrome.

Ramirez IB, Pietka G, Jones DR, Divecha N, Alia A, Baraban SC, Hurlstone AF, Lowe M - Hum. Mol. Genet. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Manchester, The Michael Smith Building, Manchester M13 9PT, UK.

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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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.
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DDR608F6: 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.

Mentions: The presence of neurological abnormalities, namely increased seizure susceptibility, in OCRL1-deficient zebrafish embryos suggests that OCRL1 may play a role in neural development during embryogenesis. We therefore analysed the OCRL1-deficient mutant embryos in more detail. The morphology of mutant embryos was assessed at different stages of development. The most striking phenotype is the reduction in the size of the brain and eyes of OCRL1 mutant embryos, which is most obvious at 1 dpf (Fig. 6A). The reduced size of these tissues does not simply reflect smaller overall size of the mutant embryos, as this was the same as WT embryos at 1 dpf (data not shown). The midbrain–hindbrain boundary is less defined when compared with controls at this stage, and the eyes are underdeveloped. There are however no obvious cataracts or opacities in the lens of mutant embryos. The mutant embryos also have a reduction in pigmentation that is also seen at later stages (Supplementary Material, Fig. S4A and data not shown). To better assess the reduction in brain size in the mutants, we performed confocal microscopy of the head region of whole-mount embryos, and histology on transverse sections taken through different regions of the head. Both approaches revealed a significant reduction in neural tissue in the fore-, mid- and hindbrain regions of mutants at 1 dpf (Fig. 6B and C). Importantly, these differences are not due to a general developmental delay. Embryos were confirmed to be at the same stage of development according to somite number (Supplementary Material, Fig. S7). Moreover, time-lapse microscopy showed that the OCRL1-deficient embryos progress through the first 18 h of embryogenesis at the same rate as controls, as assessed by gross morphology, arguing against a general developmental delay in the mutants (Supplementary Material, Fig. S6 and Movie S1).Figure 6.


Impaired neural development in a zebrafish model for Lowe syndrome.

Ramirez IB, Pietka G, Jones DR, Divecha N, Alia A, Baraban SC, Hurlstone AF, Lowe M - Hum. Mol. Genet. (2011)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3313792&req=5

DDR608F6: 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.
Mentions: The presence of neurological abnormalities, namely increased seizure susceptibility, in OCRL1-deficient zebrafish embryos suggests that OCRL1 may play a role in neural development during embryogenesis. We therefore analysed the OCRL1-deficient mutant embryos in more detail. The morphology of mutant embryos was assessed at different stages of development. The most striking phenotype is the reduction in the size of the brain and eyes of OCRL1 mutant embryos, which is most obvious at 1 dpf (Fig. 6A). The reduced size of these tissues does not simply reflect smaller overall size of the mutant embryos, as this was the same as WT embryos at 1 dpf (data not shown). The midbrain–hindbrain boundary is less defined when compared with controls at this stage, and the eyes are underdeveloped. There are however no obvious cataracts or opacities in the lens of mutant embryos. The mutant embryos also have a reduction in pigmentation that is also seen at later stages (Supplementary Material, Fig. S4A and data not shown). To better assess the reduction in brain size in the mutants, we performed confocal microscopy of the head region of whole-mount embryos, and histology on transverse sections taken through different regions of the head. Both approaches revealed a significant reduction in neural tissue in the fore-, mid- and hindbrain regions of mutants at 1 dpf (Fig. 6B and C). Importantly, these differences are not due to a general developmental delay. Embryos were confirmed to be at the same stage of development according to somite number (Supplementary Material, Fig. S7). Moreover, time-lapse microscopy showed that the OCRL1-deficient embryos progress through the first 18 h of embryogenesis at the same rate as controls, as assessed by gross morphology, arguing against a general developmental delay in the mutants (Supplementary Material, Fig. S6 and Movie S1).Figure 6.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Manchester, The Michael Smith Building, Manchester M13 9PT, UK.

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.

Show MeSH
Related in: MedlinePlus