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Arginylation-dependent neural crest cell migration is essential for mouse development.

Kurosaka S, Leu NA, Zhang F, Bunte R, Saha S, Wang J, Guo C, He W, Kashina A - PLoS Genet. (2010)

Bottom Line: Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture.Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death.Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

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Wnt1-Ate1 mice have defects in neural crest cell migration.(A–H) X-gal staining of E9.5 control (A,B) and Wnt1-Ate1 (C–E) embryos derived from Wnt1-Ate1-R26R mouse line grouped by somite count (indicated on the top left for each embryo). A, lower magnification image of a control embryo at 24 somite stage. (B–E), back areas of different wild-type and Wnt1-Ate1 embryos with higher somite count, corresponding to the region boxed in (A), showing different neural crest migration patterns as described in the text. For Wnt1-Ate1, three littermates are shown, illustrating different pattern and severity of defects as described in the text; asterisk indicates the embryo, for which somite count was not performed and the staging relied on the comparison with its littermates shown on both sides. (F–H), back areas of wild-type and Wnt1-Ate1 embryos with lower somite count. Bar, 1 mm, for the images shown in (B–H). 10 wild-type and 10 mutant embryos were analyzed. See Figure S14 for whole embryo views. (I,J) In situ hybridization of E9.5 (I) and E10.5 (J) control and Wnt1-Ate1 embryos using Sox10 neural crest marker. 2 wild-type and 2 Wnt1-Ate1 embryos were analyzed for each developmental stage. See Figure S15 for whole embryo views.
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pgen-1000878-g004: Wnt1-Ate1 mice have defects in neural crest cell migration.(A–H) X-gal staining of E9.5 control (A,B) and Wnt1-Ate1 (C–E) embryos derived from Wnt1-Ate1-R26R mouse line grouped by somite count (indicated on the top left for each embryo). A, lower magnification image of a control embryo at 24 somite stage. (B–E), back areas of different wild-type and Wnt1-Ate1 embryos with higher somite count, corresponding to the region boxed in (A), showing different neural crest migration patterns as described in the text. For Wnt1-Ate1, three littermates are shown, illustrating different pattern and severity of defects as described in the text; asterisk indicates the embryo, for which somite count was not performed and the staging relied on the comparison with its littermates shown on both sides. (F–H), back areas of wild-type and Wnt1-Ate1 embryos with lower somite count. Bar, 1 mm, for the images shown in (B–H). 10 wild-type and 10 mutant embryos were analyzed. See Figure S14 for whole embryo views. (I,J) In situ hybridization of E9.5 (I) and E10.5 (J) control and Wnt1-Ate1 embryos using Sox10 neural crest marker. 2 wild-type and 2 Wnt1-Ate1 embryos were analyzed for each developmental stage. See Figure S15 for whole embryo views.

Mentions: To address the question whether Wnt1-Ate1 mice exhibit any defects in neural crest cell migration, we used the Wnt1-Ate1-R26R reporter conditional knockout line and analyzed the distribution of LacZ -expressing cells in wild-type and Wnt1-Ate1-R26R embryos at E9.5. Several litters, whose embryonic stage was determined by counting the somites, were analyzed, and the comparisons were made between embryos with comparable somite numbers. In the analyzed embryos, the somite numbers ranged between 22 and 26 in wild-type and between 21 to 28 in Wnt1-Ate1, consistent with the expected somite numbers at this stage. While the X-gal staining of the migrating neural crest cells in these embryos, especially with the larger somite numbers, was heavily masked by staining of other organs originating from Wnt1-expressing cells, such as midbrain (Figure S14), cell migration patterns could be clearly observed in the somite regions and near the pharyngeal arches, where the migrating cells appeared as streams of LacZ-expressing ‘dots’ arranged in different patterns from the back to the front of the embryo (Figure 4). In wild-type (Figure 4A and 4B), prominent populations of cells migrated from back to front as continuous lines (from the third pharyngeal arch down to the somites) and as triangular ‘streams’ directed toward the third and fourth pharyngeal arches and along each of the somites. In Wnt1-Ate1, several of these migration patterns were affected with different degrees of severity. Embryos with 24+ somites had altered cell distribution in the streams migrating toward the third and fourth pharyngeal arches and in the line migrating over the upper area of the trunk, which appeared diffuse, with fewer cells present in those areas (Figure 4C and 4D). In some embryos (Figure 4E), migration toward the pharyngeal arches appeared normal, but X-gal staining in the upper trunk area appeared weaker than control, indicating a reduced number of migrating cells in that area. In the embryos at a slightly earlier developmental stage (21–23 somites, Figure 4F–4H) these differences were more obvious, resulting in much lower overall levels of the X-gal-stained cells visible in these areas. Such extremely affected embryos also appeared smaller than wild-type embryos or knockout embryos with less severe phenotypes (see Figure S14).


Arginylation-dependent neural crest cell migration is essential for mouse development.

Kurosaka S, Leu NA, Zhang F, Bunte R, Saha S, Wang J, Guo C, He W, Kashina A - PLoS Genet. (2010)

Wnt1-Ate1 mice have defects in neural crest cell migration.(A–H) X-gal staining of E9.5 control (A,B) and Wnt1-Ate1 (C–E) embryos derived from Wnt1-Ate1-R26R mouse line grouped by somite count (indicated on the top left for each embryo). A, lower magnification image of a control embryo at 24 somite stage. (B–E), back areas of different wild-type and Wnt1-Ate1 embryos with higher somite count, corresponding to the region boxed in (A), showing different neural crest migration patterns as described in the text. For Wnt1-Ate1, three littermates are shown, illustrating different pattern and severity of defects as described in the text; asterisk indicates the embryo, for which somite count was not performed and the staging relied on the comparison with its littermates shown on both sides. (F–H), back areas of wild-type and Wnt1-Ate1 embryos with lower somite count. Bar, 1 mm, for the images shown in (B–H). 10 wild-type and 10 mutant embryos were analyzed. See Figure S14 for whole embryo views. (I,J) In situ hybridization of E9.5 (I) and E10.5 (J) control and Wnt1-Ate1 embryos using Sox10 neural crest marker. 2 wild-type and 2 Wnt1-Ate1 embryos were analyzed for each developmental stage. See Figure S15 for whole embryo views.
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Related In: Results  -  Collection

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pgen-1000878-g004: Wnt1-Ate1 mice have defects in neural crest cell migration.(A–H) X-gal staining of E9.5 control (A,B) and Wnt1-Ate1 (C–E) embryos derived from Wnt1-Ate1-R26R mouse line grouped by somite count (indicated on the top left for each embryo). A, lower magnification image of a control embryo at 24 somite stage. (B–E), back areas of different wild-type and Wnt1-Ate1 embryos with higher somite count, corresponding to the region boxed in (A), showing different neural crest migration patterns as described in the text. For Wnt1-Ate1, three littermates are shown, illustrating different pattern and severity of defects as described in the text; asterisk indicates the embryo, for which somite count was not performed and the staging relied on the comparison with its littermates shown on both sides. (F–H), back areas of wild-type and Wnt1-Ate1 embryos with lower somite count. Bar, 1 mm, for the images shown in (B–H). 10 wild-type and 10 mutant embryos were analyzed. See Figure S14 for whole embryo views. (I,J) In situ hybridization of E9.5 (I) and E10.5 (J) control and Wnt1-Ate1 embryos using Sox10 neural crest marker. 2 wild-type and 2 Wnt1-Ate1 embryos were analyzed for each developmental stage. See Figure S15 for whole embryo views.
Mentions: To address the question whether Wnt1-Ate1 mice exhibit any defects in neural crest cell migration, we used the Wnt1-Ate1-R26R reporter conditional knockout line and analyzed the distribution of LacZ -expressing cells in wild-type and Wnt1-Ate1-R26R embryos at E9.5. Several litters, whose embryonic stage was determined by counting the somites, were analyzed, and the comparisons were made between embryos with comparable somite numbers. In the analyzed embryos, the somite numbers ranged between 22 and 26 in wild-type and between 21 to 28 in Wnt1-Ate1, consistent with the expected somite numbers at this stage. While the X-gal staining of the migrating neural crest cells in these embryos, especially with the larger somite numbers, was heavily masked by staining of other organs originating from Wnt1-expressing cells, such as midbrain (Figure S14), cell migration patterns could be clearly observed in the somite regions and near the pharyngeal arches, where the migrating cells appeared as streams of LacZ-expressing ‘dots’ arranged in different patterns from the back to the front of the embryo (Figure 4). In wild-type (Figure 4A and 4B), prominent populations of cells migrated from back to front as continuous lines (from the third pharyngeal arch down to the somites) and as triangular ‘streams’ directed toward the third and fourth pharyngeal arches and along each of the somites. In Wnt1-Ate1, several of these migration patterns were affected with different degrees of severity. Embryos with 24+ somites had altered cell distribution in the streams migrating toward the third and fourth pharyngeal arches and in the line migrating over the upper area of the trunk, which appeared diffuse, with fewer cells present in those areas (Figure 4C and 4D). In some embryos (Figure 4E), migration toward the pharyngeal arches appeared normal, but X-gal staining in the upper trunk area appeared weaker than control, indicating a reduced number of migrating cells in that area. In the embryos at a slightly earlier developmental stage (21–23 somites, Figure 4F–4H) these differences were more obvious, resulting in much lower overall levels of the X-gal-stained cells visible in these areas. Such extremely affected embryos also appeared smaller than wild-type embryos or knockout embryos with less severe phenotypes (see Figure S14).

Bottom Line: Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture.Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death.Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

Show MeSH
Related in: MedlinePlus