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Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.

Kobayashi I, Kobayashi-Sun J, Kim AD, Pouget C, Fujita N, Suda T, Traver D - Nature (2014)

Bottom Line: Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs.Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos.Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0380, USA.

ABSTRACT
Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development and requires intimate contact between signal-emitting and signal-receiving cells, although little is known regarding when, where and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a (also known as f11r) and migrate axially across the ventral somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

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Somitic Dlc and Dld are involved in the activation of endothelial Notch signallinga, b, Transverse sections of fli1:GFP embryos stained with dlc or dld (purple) and anti-GFP antibody (brown) at 15hpf. Right panels show high magnification views of the boxed regions. Migrating fli1:GFP+ cells (black arrowheads) are in contact with dlc+ or dld+ somitic cells (white arrowheads). c-e, Flow cytometric analysis of Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 22hpf. Representative results of flow cytometric analysis (c), the mean fluorescent intensities of GFP in Tp1:GFP+; fli1:DsRed+ populations (d), and the percentages of Tp1:GFPhigh in fli1:DsRed+ populations (e) are shown. Blue gates and red circles indicate the Tp1:GFP+; fli1:DsRed+ and Tp1:GFPhigh; fli1:DsRed+ population, respectively. *p < 0.01, by Student's t-test. Error bars, s.d. f, g, Lateral views of the dorsal aorta (DA) in Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 28hpf. Arrows indicate the low activation of Tp1:GFP in the ventral floor of the DA. Data are representative of two independent experiments with four embryos (a, b), eight embryos (f, g), or four different clutches of embryos (c-e).
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Figure 12: Somitic Dlc and Dld are involved in the activation of endothelial Notch signallinga, b, Transverse sections of fli1:GFP embryos stained with dlc or dld (purple) and anti-GFP antibody (brown) at 15hpf. Right panels show high magnification views of the boxed regions. Migrating fli1:GFP+ cells (black arrowheads) are in contact with dlc+ or dld+ somitic cells (white arrowheads). c-e, Flow cytometric analysis of Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 22hpf. Representative results of flow cytometric analysis (c), the mean fluorescent intensities of GFP in Tp1:GFP+; fli1:DsRed+ populations (d), and the percentages of Tp1:GFPhigh in fli1:DsRed+ populations (e) are shown. Blue gates and red circles indicate the Tp1:GFP+; fli1:DsRed+ and Tp1:GFPhigh; fli1:DsRed+ population, respectively. *p < 0.01, by Student's t-test. Error bars, s.d. f, g, Lateral views of the dorsal aorta (DA) in Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 28hpf. Arrows indicate the low activation of Tp1:GFP in the ventral floor of the DA. Data are representative of two independent experiments with four embryos (a, b), eight embryos (f, g), or four different clutches of embryos (c-e).

Mentions: At 15hpf, migrating fli1:GFP+ cells were observed to make direct contact with dlc+ or dld+ somitic cells (Extended Data Fig. 8a, b), indicating that PLM cells may receive Notch signaling via presentation of somitic Notch ligands. We observed low activation of Tp1:GFP in endothelial cells in wnt16 morphants (Extended Data Fig. 8c-g), which show a reduction in somitic dlc and dld4. This suggests that Notch signalling in endothelial cells is activated at least in part by somitic Dlc and/or Dld. We investigated the expression of somitic Notch ligand genes (dlc and dld) as well as aortic Notch receptor and ligand genes (notch1a, notch1b, notch3, dlc, and delta-like 4 (dll4)) in jam1a morphants. Importantly, each was expressed normally in jam1a morphants (Fig. 4m-q, Extended Data Fig. 9a-h), suggesting that the defect in Notch signalling in jam1a morphants is due to low Notch signal transmission rather than misregulation of Notch signalling components. Consistent with this postulate, we observed less contact surface area between migrating PLM cells and the somite in both jam1a and jam2a morphants (Fig. 2i, j, Extended Data Fig. 5m, n), which correlates with low activation of Notch signalling. Our hypothesis is further supported by an additional rescue experiment in which dlc or dld is globally overexpressed in jam1a morphants in order to present more Notch ligand to HSC precursors. As presented in Fig. 4u, the expression of runx1 in the DA was almost fully rescued by co-injection of dld mRNA along with the jam1a MOatg, whereas runx1 expression was only partially rescued following co-injection with dlc mRNA (Fig. 4r-u). Furthermore, the expression of Tp1:GFP was also restored in the ventral floor of the DA by co-injection with dlc or dld mRNA (Extended Data Fig. 9i-p). These data confirm that the impairment of HSC specification in jam1a morphants is caused by inadequate activation of Notch signalling in HSC precursors and suggest that Jam1a and Jam2a normally mediate the physical interaction between these precursors and the somite, which is required for efficient Notch signal transmission (Extended Data Fig 10).


Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.

Kobayashi I, Kobayashi-Sun J, Kim AD, Pouget C, Fujita N, Suda T, Traver D - Nature (2014)

Somitic Dlc and Dld are involved in the activation of endothelial Notch signallinga, b, Transverse sections of fli1:GFP embryos stained with dlc or dld (purple) and anti-GFP antibody (brown) at 15hpf. Right panels show high magnification views of the boxed regions. Migrating fli1:GFP+ cells (black arrowheads) are in contact with dlc+ or dld+ somitic cells (white arrowheads). c-e, Flow cytometric analysis of Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 22hpf. Representative results of flow cytometric analysis (c), the mean fluorescent intensities of GFP in Tp1:GFP+; fli1:DsRed+ populations (d), and the percentages of Tp1:GFPhigh in fli1:DsRed+ populations (e) are shown. Blue gates and red circles indicate the Tp1:GFP+; fli1:DsRed+ and Tp1:GFPhigh; fli1:DsRed+ population, respectively. *p < 0.01, by Student's t-test. Error bars, s.d. f, g, Lateral views of the dorsal aorta (DA) in Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 28hpf. Arrows indicate the low activation of Tp1:GFP in the ventral floor of the DA. Data are representative of two independent experiments with four embryos (a, b), eight embryos (f, g), or four different clutches of embryos (c-e).
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Related In: Results  -  Collection

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Figure 12: Somitic Dlc and Dld are involved in the activation of endothelial Notch signallinga, b, Transverse sections of fli1:GFP embryos stained with dlc or dld (purple) and anti-GFP antibody (brown) at 15hpf. Right panels show high magnification views of the boxed regions. Migrating fli1:GFP+ cells (black arrowheads) are in contact with dlc+ or dld+ somitic cells (white arrowheads). c-e, Flow cytometric analysis of Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 22hpf. Representative results of flow cytometric analysis (c), the mean fluorescent intensities of GFP in Tp1:GFP+; fli1:DsRed+ populations (d), and the percentages of Tp1:GFPhigh in fli1:DsRed+ populations (e) are shown. Blue gates and red circles indicate the Tp1:GFP+; fli1:DsRed+ and Tp1:GFPhigh; fli1:DsRed+ population, respectively. *p < 0.01, by Student's t-test. Error bars, s.d. f, g, Lateral views of the dorsal aorta (DA) in Tp1:GFP; fli1:DsRed embryos uninjected or injected with wnt16 MO at 28hpf. Arrows indicate the low activation of Tp1:GFP in the ventral floor of the DA. Data are representative of two independent experiments with four embryos (a, b), eight embryos (f, g), or four different clutches of embryos (c-e).
Mentions: At 15hpf, migrating fli1:GFP+ cells were observed to make direct contact with dlc+ or dld+ somitic cells (Extended Data Fig. 8a, b), indicating that PLM cells may receive Notch signaling via presentation of somitic Notch ligands. We observed low activation of Tp1:GFP in endothelial cells in wnt16 morphants (Extended Data Fig. 8c-g), which show a reduction in somitic dlc and dld4. This suggests that Notch signalling in endothelial cells is activated at least in part by somitic Dlc and/or Dld. We investigated the expression of somitic Notch ligand genes (dlc and dld) as well as aortic Notch receptor and ligand genes (notch1a, notch1b, notch3, dlc, and delta-like 4 (dll4)) in jam1a morphants. Importantly, each was expressed normally in jam1a morphants (Fig. 4m-q, Extended Data Fig. 9a-h), suggesting that the defect in Notch signalling in jam1a morphants is due to low Notch signal transmission rather than misregulation of Notch signalling components. Consistent with this postulate, we observed less contact surface area between migrating PLM cells and the somite in both jam1a and jam2a morphants (Fig. 2i, j, Extended Data Fig. 5m, n), which correlates with low activation of Notch signalling. Our hypothesis is further supported by an additional rescue experiment in which dlc or dld is globally overexpressed in jam1a morphants in order to present more Notch ligand to HSC precursors. As presented in Fig. 4u, the expression of runx1 in the DA was almost fully rescued by co-injection of dld mRNA along with the jam1a MOatg, whereas runx1 expression was only partially rescued following co-injection with dlc mRNA (Fig. 4r-u). Furthermore, the expression of Tp1:GFP was also restored in the ventral floor of the DA by co-injection with dlc or dld mRNA (Extended Data Fig. 9i-p). These data confirm that the impairment of HSC specification in jam1a morphants is caused by inadequate activation of Notch signalling in HSC precursors and suggest that Jam1a and Jam2a normally mediate the physical interaction between these precursors and the somite, which is required for efficient Notch signal transmission (Extended Data Fig 10).

Bottom Line: Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs.Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos.Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0380, USA.

ABSTRACT
Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development and requires intimate contact between signal-emitting and signal-receiving cells, although little is known regarding when, where and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a (also known as f11r) and migrate axially across the ventral somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

Show MeSH
Related in: MedlinePlus