CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix

Bonet-Fernández, Juan-Manuel; Aroca-Aguilar, José-Daniel; Cortón, Marta; Ramírez, Ana-Isabel; Alexandre-Moreno, Susana; García-Antón, María-Teresa; Salazar, J. Gomez Ma de; Ferre-Fernández, Jesús-José; Atienzar-Aroca, Raquel; Villaverde, Cristina; Iancu, Ionut-Florin; Tamayo, Alejandra; Méndez-Hernández, Carmen-Dora; Morales-Fernández, Laura; Rojas, Blanca; Ayuso, Carmen; Coca‐Prados, Miguel; Martínez-de-la-Casa, José-María; García‐Feijoó, Julián; Escribano, Julio

doi:10.1007/s00439-020-02164-0

Cited by 29 publications

(28 citation statements)

References 50 publications

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“…Differences in IOP between subgroups also reached statistical significance (p=0.002). Those with JOAG had a lower median maximum recorded IOP (29 [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] mmHg) compared to those with an associated acquired condition (36 [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] mmHg,…”

Section: Early-onset Glaucomamentioning

confidence: 99%

Childhood and Early Onset Glaucoma Classification and Genetic Profile in a Large Australasian Disease Registry

et al. 2021

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Section: Early-onset Glaucomamentioning

confidence: 99%

Childhood and Early Onset Glaucoma Classification and Genetic Profile in a Large Australasian Disease Registry

et al. 2021

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“…Fluorescence immunohistochemistry was performed as previously described [43,44]. Briefly, gelatin embedded histological sections of juvenile (28 dpf) or adult zebrafish (7 months) were treated with immunoblocking solution [10% fetal bovine serum (FBS), 1% DMSO and 1% Triton X-100 in DPBS] at room temperature for 1 h. Gonadal tissue and myocilin were identified using an antibody against the germ cell marker vasa (1:200 [45], GTX128306, GeneTex, Hsinchu City, Taiwan) or the anti-myocilin (TNT, 1:150) antibody, followed, respectively, by incubation with a Cy2-conjugated anti-chicken IgY (1:1000) or anti-rabbit IgG secondary antibody (1:1000).…”

Section: Fluorescence Immunohistochemistrymentioning

confidence: 99%

Knockout of myoc Provides Evidence for the Role of Myocilin in Zebrafish Sex Determination Associated with Wnt Signalling Downregulation

Atienzar-Aroca

Aroca-Aguilar

Alexandre-Moreno

et al. 2021

Biology

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Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known as the first glaucoma gene. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 h post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but, remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provides in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.

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“…Fluorescence immunohistochemistry was performed as previously described 75,76 . Brefly, gelatin embedded histological sections of larvae (eight dpf), juvenile (28 dpf) or adult zebrafish (7 months) were treated with immunoblocking solution [10% fetal bovine serum (FBS), 1% DMSO and 1% Triton X-100 in DPBS] at room temperature for 1 h. Gonadal tissue and myocilin were identified using an antibody against the germ cell marker vasa (1:200, GeneTex, GTX128306) or the anti-myocilin (TNT, 1:150) antibody, followed respectively, by incubation with a Cy2-conjugated anti-chicken IgY (1:1000) or anti-rabbit IgG secondary antibody (1:1000).…”

Section: Methodsmentioning

confidence: 99%

“…Phenylthiourea-treated and fixed embryos (96 hpf) were incubated with a chicken primary antibody (1:50) raised against a N-terminal peptide of the human myocilin protein (anti-TNT) 14 followed by incubation with a Cy2 goat anti-chicken IgY Fluorescence immunohistochemistry. Fluorescence immunohistochemistry was performed as previously described 75,76 . Brefly, gelatin embedded histological sections of larvae (eight dpf), juvenile (28 dpf) or adult zebrafish (7 months) were treated with immunoblocking solution [10% fetal bovine serum (FBS), 1% DMSO and 1% Triton X-100…”

Section: Fwihcmentioning

confidence: 99%

Knockout ofmyocreveals the role of myocilin in zebrafish sex determination associated with Wnt signalling downregulation

Atienzar-Aroca

Aroca-Aguilar

Alexandre-Moreno

et al. 2020

Preprint

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Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known for its association with glaucoma. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in embryonic (96 hours post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but remarkably, we observed absence of females among the KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provide in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.

show abstract

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix

Cited by 29 publications

References 50 publications

Childhood and Early Onset Glaucoma Classification and Genetic Profile in a Large Australasian Disease Registry

Childhood and Early Onset Glaucoma Classification and Genetic Profile in a Large Australasian Disease Registry

Knockout of myoc Provides Evidence for the Role of Myocilin in Zebrafish Sex Determination Associated with Wnt Signalling Downregulation

Knockout ofmyocreveals the role of myocilin in zebrafish sex determination associated with Wnt signalling downregulation

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