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Disruption of AP1S1, causing a novel neurocutaneous syndrome, perturbs development of the skin and spinal cord.

Montpetit A, Côté S, Brustein E, Drouin CA, Lapointe L, Boudreau M, Meloche C, Drouin R, Hudson TJ, Drapeau P, Cossette P - PLoS Genet. (2008)

Bottom Line: The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development.Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation.Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.

View Article: PubMed Central - PubMed

Affiliation: McGill University, Montréal, Québec, Canada.

ABSTRACT
Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit sigma1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.

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Ap1s1 knockdown is associated with abnormal distribution of laminin and cadherin.Despite changes in the size and shape of the tail, p63-labelled nuclei (orange) of basal keratinocytes were present in KD larvae. (B). The insets show enlarged views of small groups of keratinocytes. Immunolabelling for laminin (green) in WT (C) showed normal distribution of the basement membrane along the fin fold margin, whereas in the KD larvae the residual laminin labeling was diffuse and disorganized (D). Compared to WT keratinocytes (E) labeled with p63 (orange), cadherin (green) is decreased at the cell membrane of the KD larvae (F). However, the localization of cytokeratin in WT (G) and in KD larvae (H) appeared similar. Scale bars: 10 µm, Insets in A, B, = 20 µm.
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pgen-1000296-g003: Ap1s1 knockdown is associated with abnormal distribution of laminin and cadherin.Despite changes in the size and shape of the tail, p63-labelled nuclei (orange) of basal keratinocytes were present in KD larvae. (B). The insets show enlarged views of small groups of keratinocytes. Immunolabelling for laminin (green) in WT (C) showed normal distribution of the basement membrane along the fin fold margin, whereas in the KD larvae the residual laminin labeling was diffuse and disorganized (D). Compared to WT keratinocytes (E) labeled with p63 (orange), cadherin (green) is decreased at the cell membrane of the KD larvae (F). However, the localization of cytokeratin in WT (G) and in KD larvae (H) appeared similar. Scale bars: 10 µm, Insets in A, B, = 20 µm.

Mentions: To determine if an increase in cell death underlies the skin phenotype in the KD embryo, we stained these larvae with the vital dye acridine orange [15]. We did not observe a difference compared with control (not shown), suggesting that the skin and fin disorganization was not due to an initial outgrowth followed by tissue degradation. We further tested whether the skin malformation was due to a problem in early epidermal patterning by using immunolabeling for p63, a marker of basal keratinocyte nuclei [16]. Despite the prominent changes in the size and the shape of the tail, p63-positive keratinocytes were present both in WT (Figure 3A) and KD larvae (Figure 3B). To look for a change in the population of proliferating cells, we performed immunolabelling with the phosphorylated-histone-H3 (PH3) antibody to visualize cells undergoing histone modification during mitosis, which did not reveal any obvious difference between the KD and control larvae (not illustrated). Similar results were obtained with co-immunostaining against p63 and PH3, suggesting unaffected proliferation level of basal keratinocytes population in the KD larvae (not illustrated). To further investigate whether the keratinocytes in the KD larvae exhibit specific abnormalities, we immunolabeled WT and KD larvae for laminin (Figures 3C and 3D) and for cadherin (Figures 3E and 3F). Laminins, in particular laminin 5, are synthesized by keratinocytes and are their main anchor to the basement membrane [17], while cadherins are localized to the keratinocyte cell membrane and are essential in maintaining cell-cell adhesion [18]. In the WT, laminin was detected at the outer edges of the fin (Figure 3C) while in the KD larvae (Figure 3D) the detected laminin appeared diffuse, with an abnormal localization. Furthermore, in the KD larva, cadherin immunolabeling was less obvious at the cell membrane of keratinocytes doubly-labeled with cadherin (green) and p63 (orange) (Figure 3F) In contrast, the localization of cytokeratin, a major cytoskeletal protein expressed exclusively in epithelial cells [19],[20] seemed to be preserved in KD larvae (Figures 3G and 3H).


Disruption of AP1S1, causing a novel neurocutaneous syndrome, perturbs development of the skin and spinal cord.

Montpetit A, Côté S, Brustein E, Drouin CA, Lapointe L, Boudreau M, Meloche C, Drouin R, Hudson TJ, Drapeau P, Cossette P - PLoS Genet. (2008)

Ap1s1 knockdown is associated with abnormal distribution of laminin and cadherin.Despite changes in the size and shape of the tail, p63-labelled nuclei (orange) of basal keratinocytes were present in KD larvae. (B). The insets show enlarged views of small groups of keratinocytes. Immunolabelling for laminin (green) in WT (C) showed normal distribution of the basement membrane along the fin fold margin, whereas in the KD larvae the residual laminin labeling was diffuse and disorganized (D). Compared to WT keratinocytes (E) labeled with p63 (orange), cadherin (green) is decreased at the cell membrane of the KD larvae (F). However, the localization of cytokeratin in WT (G) and in KD larvae (H) appeared similar. Scale bars: 10 µm, Insets in A, B, = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2585812&req=5

pgen-1000296-g003: Ap1s1 knockdown is associated with abnormal distribution of laminin and cadherin.Despite changes in the size and shape of the tail, p63-labelled nuclei (orange) of basal keratinocytes were present in KD larvae. (B). The insets show enlarged views of small groups of keratinocytes. Immunolabelling for laminin (green) in WT (C) showed normal distribution of the basement membrane along the fin fold margin, whereas in the KD larvae the residual laminin labeling was diffuse and disorganized (D). Compared to WT keratinocytes (E) labeled with p63 (orange), cadherin (green) is decreased at the cell membrane of the KD larvae (F). However, the localization of cytokeratin in WT (G) and in KD larvae (H) appeared similar. Scale bars: 10 µm, Insets in A, B, = 20 µm.
Mentions: To determine if an increase in cell death underlies the skin phenotype in the KD embryo, we stained these larvae with the vital dye acridine orange [15]. We did not observe a difference compared with control (not shown), suggesting that the skin and fin disorganization was not due to an initial outgrowth followed by tissue degradation. We further tested whether the skin malformation was due to a problem in early epidermal patterning by using immunolabeling for p63, a marker of basal keratinocyte nuclei [16]. Despite the prominent changes in the size and the shape of the tail, p63-positive keratinocytes were present both in WT (Figure 3A) and KD larvae (Figure 3B). To look for a change in the population of proliferating cells, we performed immunolabelling with the phosphorylated-histone-H3 (PH3) antibody to visualize cells undergoing histone modification during mitosis, which did not reveal any obvious difference between the KD and control larvae (not illustrated). Similar results were obtained with co-immunostaining against p63 and PH3, suggesting unaffected proliferation level of basal keratinocytes population in the KD larvae (not illustrated). To further investigate whether the keratinocytes in the KD larvae exhibit specific abnormalities, we immunolabeled WT and KD larvae for laminin (Figures 3C and 3D) and for cadherin (Figures 3E and 3F). Laminins, in particular laminin 5, are synthesized by keratinocytes and are their main anchor to the basement membrane [17], while cadherins are localized to the keratinocyte cell membrane and are essential in maintaining cell-cell adhesion [18]. In the WT, laminin was detected at the outer edges of the fin (Figure 3C) while in the KD larvae (Figure 3D) the detected laminin appeared diffuse, with an abnormal localization. Furthermore, in the KD larva, cadherin immunolabeling was less obvious at the cell membrane of keratinocytes doubly-labeled with cadherin (green) and p63 (orange) (Figure 3F) In contrast, the localization of cytokeratin, a major cytoskeletal protein expressed exclusively in epithelial cells [19],[20] seemed to be preserved in KD larvae (Figures 3G and 3H).

Bottom Line: The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development.Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation.Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.

View Article: PubMed Central - PubMed

Affiliation: McGill University, Montréal, Québec, Canada.

ABSTRACT
Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit sigma1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.

Show MeSH
Related in: MedlinePlus