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. 2014 Aug 21;512(7514):319-23.
doi: 10.1038/nature13623. Epub 2014 Aug 13.

Jam1a-Jam2a Interactions Regulate Haematopoietic Stem Cell Fate Through Notch Signalling

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Free PMC article

Jam1a-Jam2a Interactions Regulate Haematopoietic Stem Cell Fate Through Notch Signalling

Isao Kobayashi et al. Nature. .
Free PMC article

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.

Figures

Extended Data Figure 1
Extended Data Figure 1. Alignment and phylogenetic analysis of Jam1a
a, The genomic loci of the jam1a and jam1b gene. Arrows indicate the orientation of each gene. b, Alignment of zebrafish Jam1a, Jam1b, and human JAM1. The multiple alignment was produced using ClustalW. Asterisks indicate a fully conserved residue. Colons and periods indicate strong and weak similarity, respectively. Dashes indicate gaps. Red, light blue, blue, green, yellow boxes indicate signal peptide (Sp), immunoglobulin-like domain (Ig), transmembrane domain (TM), PDZ-binding domain (PBD), and coiled-coil domain (CCD), respectively. Shaded boxes show a cisdimerization motif. c, Phylogenetic analysis of Jam proteins. Cii, Ciona intestinalis; Dre, Danio rerio; Hsa, Homo sapiens; Mmu, Mus musculus; Tni, Tetraodon nigroviridis; Gga, Gallus gallus; Ssc, Sus scrofa; Bta, Bos taurus; Fca, Felis silvestris catus. Cii Jam3 was used as an out-group. The numbers at the relevant branches refer to bootstrap values of 1000 replications. d, Schematic diagrams of human JAM1 and zebrafish Jam1a (left) and Jam1b (right).
Extended Data Figure 2
Extended Data Figure 2. The expression of jam1a and lineage tracing of jam1a- expressing cells
a-c, The expression of jam1a at 14, 18, and 24hpf. Black, blue, and red arrows indicate the posterior lateral mesoderm (PLM), pronephros, and dorsal aorta (DA), respectively. d, Single or two-color whole-mount in situ hybridization with jam1a (purple) and/or fli1 (red) probes. The expression domain of fli1 merged with that of jam1a (arrows). e, qPCR analysis of jam1a in purified fli1:GFP+ cells at 14, 17, and 20hpf. Relative expression levels in each cell population were calculated from the expression levels in the kidney tissue. Error bars, s.d. f, GFP expression of -2.2jam1a:GFP at 30hpf. GFP expression was strongly detected in posterior pronephros, lateral lines, and otic vesicles. g, The expression of CreERT2 in -5.1jam1a:CreERT2 at 24hpf. h. Three different schedules of 4-hydroxytamoxifen (4OHT) treatment (12 - 16hpf, 18 - 22hpf, and 24 - 28hpf) in -5.1jam1a:CreERT2; bactin2:loxP-BFP-loxP-DsRed (blue-to-red reporter line). The numbers indicate hour post fertilization (hpf), and red insets in the blue arrows indicate the period of the 4OHT treatment. i, Flow cytometric analysis of kidney marrow cells in 12 - 16hpf group (left, n = 5), 18 - 22hpf group (middle, n = 5), and 24 - 28hpf group (right, n = 2) at 2 months of age. One 12 - 16hpf animal showed switched DsRed+ cells in kidney marrow cells. j, DsRed+ cells from 12 - 16hpf kidneys are distributed in all blood cell populations, including neutrophils and eosinophils (N+E), precursors and monocytes (P+M), and lymphocytes and thrombocytes (L+T). k, l, Confocal imaging of the DA and caudal haematopoietic tissue (CHT) in a 12 - 16hpf embryo at 48hpf. Right panels show high magnification views of the boxed regions in left panels. Embryos are oriented with anterior to the left. There are many ‘switched’ DsRed+ cells in the ventral floor of the DA and in the CHT (arrowheads). Bars, 20μm. Data are representative of two independent experiments with two different clutches of embryos (a-e, g) or eight embryos (k, l) or pooled from two independent experiments (i, j).
Extended Data Figure 3
Extended Data Figure 3. Characterization of jam1a MOex7-injected embryos
a, RT-PCR results from jam1a MOex7-injected embryos. cDNA from embryos uninjected or injected with various doses of jam1a MOex7 (100 – 500μM) was subjected to RT-PCR analysis using specific primers, which amplify from exon 4 to 10 of jam1a. ef1a was used as a control. The expected size of PCR products in uninjected embryos is 746 base pairs (bp) (black arrow). Exon 7 (108bp)-skipped products were detected in jam1a MOex7-injected embryos (red arrow). The dose of 300μM was used in this study. b, Exon 7-skipped products were verified by sequencing. The dotted line indicates the junctions between exon 6 and exon 7 (uninjected, upper panel) or exon 8 (jam1a MOex7, lower panel). c, A schematic diagram of the mRNA splicing in jam1a MOex7-injected embryos. The red bar indicates the binding site of jam1a MOex7. d, Schematic diagrams of Jam1a protein in wild type (left) or jam1a MOex7-injected embryos (right). Since exon 7 encodes the transmembrane domain (TM), jam1a MOex7-injected embryos express a mutant protein lacking the TM. Sp, signal peptide; Ig, immunoglobulin-like domain; PBD, PDZ-binding domain. e, The relative expression of wild type jam1a mRNA in uninjected or jam1a MOex7 (300μM)-injected embryos at 24hpf. The reverse primer was designed in exon 7. Error bars, s.d. f-i, The expression of cmyb and rag1 in uninjected or jam1a MOex7-injected embryos. Arrowheads indicate the dorsal aorta (f, g) or the thymus (h, i). Data are representative of two independent experiments with two different clutches of embryos (a, e-i).
Extended Data Figure 4
Extended Data Figure 4. jam1a is required for HSC specification
a-c, Fluorescently labeled HSCs in cmyb:GFP; kdrl:mCherry. The number of cmyb:GFP;kdrl:mCherry double-positive cells in the dorsal aorta (DA) were counted in uninjected or jam1a MOatg-injected embryos at 48hpf (arrows). Embryos are oriented with anterior to the left. The average number of double-positive cells is significantly lower in jam1a MOatg-injected embryos (n = 10) compared with uninjected embryos (n =10), *p < 0.001 by Student's t-test; error bars, s. d. d-q, The expression of gata1 (an erythroid marker), lplastin (a myeloid marker) at 24hpf or 4dpf, kdrl (a pan-endothelial marker) in the trunk or caudal haematopoietic tissue (CHT), tbx20 (a marker for the roof of DA), flt4 (a vein marker), cdh17 (a pronephros marker), desma (a somite marker), nkx3.1 (a sclerotome marker), shha (a notochord marker) at 16hpf or 26hpf, and vegfa at 17hpf or 26hpf in uninjected or jam1a MOatg-injected embryos. Arrowheads indicate CHT (f), DA (i), or notochord (o). Data are representative of two independent experiments with ten embryos (a-c) or two different clutches of embryos (d-q).
Extended Data Figure 5
Extended Data Figure 5. Characterization of jam2a MOex5-injected embryos
a, RT-PCR results from jam2a MOex5-injected embryos. cDNA from embryos uninjected or injected with various doses of jam2a MOex5 (200 – 600μM) was subjected to RT-PCR analysis using specific primers, which amplify from exon 4 to 10 of jam2a. ef1a was used as a control. The expected size of PCR products in uninjected embryos is 537 base pairs (bp) (black arrow). Intron 4 (72bp)-trapped products were detected in jam2a MOex5-injected embryos (red arrow). The dose of 400μM was used in this study. b, Intron 4-trapped products were verified by sequencing. The dotted line indicates the junctions between exon 4 and exon 5 (uninjected, upper panel) or intron 4 (jam2a MOex5, lower panel). c, A schematic diagram of the mRNA splicing in jam2a MOex5-injected embryos. The red bar indicates the binding site of jam2a MOex5. d, Schematic diagrams of Jam2a protein in wild type (left) or jam2a MOex5-injected embryos (right). Since intron 4 contains an in-frame stop codon, jam2a MOex5-injected embryos express a truncated mutant protein. Sp, signal peptide; Ig, immunoglobulin-like domain; TM, transmembrane domain; PBD, PDZ-binding domain. e-l, The expression of runx1, efnb2a, cmyb, and rag1 in uninjected or jam2a MOex5-injected embryos. Arrowheads indicate the dorsal aorta (e-j) or the thymus (k, l). m, A transverse section of a fli1:GFP; phldb1:mCherry embryo injected with jam2a MOex5 at 16hpf. A high magnification view of the boxed region is shown in the right panel. A dotted line in the right panel indicates the contact surface area between fli1:GFP+ PLM cells and phldb1:mCherry+ somitic cells. Bars, 10μm. n, The average contact surface area between a PLM cell and the somite in uninjected, jam1a MOatg-, or jam2a MOex5-injected embryos. The contact surface area per cell (μm/cell) was calculated from at least one hundred fli1:GFP+ cells in an embryo, and the averages were obtained from three embryos of each. *p < 0.01, by Student's t-test. Data are representative of two independent experiments with two different clutches of embryos (a, e-l) or three embryos (m, n).
Extended Data Figure 6
Extended Data Figure 6. jam2a is required for HSC specification
a, A schematic diagram of the preparation of jam2a mutant (jam2ahu3319) embryos. Embryos from an incross of genotyped homozygous (homo) jam2ahu3319 animals were examined by whole-mount in situ hybridization (WISH). b-g, The expression of runx1 and cmyb in the dorsal aorta (DA) and rag1 in the thymus in wild type or jam2ahu3319 embryos. The numbers shown in each panel indicate the frequency of embryos showing each expression pattern. h, i, The expression of fli1 at 18hpf in wild type or jam2ahu3319 embryos. Arrows indicate a subset of fli1+ cells that did not reach the midline. j-o, The expression of endothelial marker genes (kdrl, efnb2a, and flt4) in wild type or jam2ahu3319 embryos at 28hpf. Approximately 90% of embryos showed normal vascular plexus, while the rest of embryos showed a reduction of efnb2a and kdrl. Arrowheads indicate the DA (b-e, l, m), the thymus (f, g), or posterior cardinal vein (n, o). p, Histological analysis of the adult kidney in a wild type or jam2ahu3319 animal at 2 months of age. Many blood cells are observed in the marrow area. Haematoxylin-eosin (HE) staining. q, Representative results of flow cytometric analysis of kidney marrow cells from a wild type or jam2ahu3319 animal at 3 months of age. All blood cell populations are detected in jam2ahu3319 animals. L+T, lymphocytes and thrombocytes; N+E, neutrophils and eosinophils; P+M, precursors and monocytes. Data are representative of two independent experiments with two different clutches of embryos (b-o) or three different animals (p, q).
Extended Data Figure 7
Extended Data Figure 7. Enforced expression of the Notch intracellular domain rescues HSCs in jam1a or jam2a morphants
Heat-shock (hsp70:Gal4, a, b, e) or endothelial (fli1:Gal4, c, d, f) induction of Notch intracellular domain (NICD) in uninjected, jam1a MOatg-, or jam2a MOatg-injected embryos. Left panels show whole-mount immunofluorescence visualization of Myc-tagged NICD, and right panels show the expression of runx1 at 26hpf. Arrowheads indicate the dorsal aorta. Data are representative of two independent experiments with two different clutches of embryos (a-f).
Extended Data Figure 8
Extended Data Figure 8. Somitic Dlc and Dld are involved in the activation of endothelial Notch signalling
a, 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).
Extended Data Figure 9
Extended Data Figure 9. Aortic Tp1:GFP expression is restored by overexpression of dlc or dld in jam1a morphants
a-h, The aortic expression of notch1b, notch3, dlc, and dll4 in uninjected or jam1a MOatg-injected embryos at 26hpf. Arrowheads indicate the dorsal aorta (DA). i-p, Lateral views of the DA in Tp1:GFP (i-l) and transverse sections of Tp1:GFP; fli1:DsRed (m-p) at 28hpf. Embryos were uninjected, injected with jam1a MOatg alone, or co-injected with jam1a MOatg and dlc or dld mRNA. Arrows indicate relatively low activation of Tp1:GFP in the ventral floor of the DA. The expression of Tp1:GFP was restored in the ventral floor of the DA by co-injection with dlc or dld. Bars, 10μm. Data are representative of two independent experiments with two different clutches of embryos (a-h), eight embryos (i-l), or three embryos (m-p).
Extended Data Figure 10
Extended Data Figure 10. A model of Notch signal transduction in HSC precursors
Jam1a+ PLM cells initially flank the somites then migrate to the midline along the ventral face of the somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Binding of Jam1a and Jam2a in trans is required for transmission of Notch signals into the PLM derivatives that subsequently generate aortic haemogenic endothelium (left side). In jam1a-deficient embryos, although PLM cells arise and initially migrate normally, their migration is delayed upon contact with the somite. Moreover, they show low activation of Notch signalling due to poor interaction with the somite, resulting in the failure of HSC specification in the aortic floor (right side).
Figure 1
Figure 1. Loss of jam1a results in the loss of HSCs
a, Vector constructs of transgenic animals used for lineage tracing. PA, polyA. b, Two different schedules of 4-hydroxytamoxifen (4OHT) treatment (‘early’ and ‘late’). Red insets in the blue arrows indicate the period of the 4OHT treatment. c, Flow cytometric analysis of adult kidney marrow cells. d, The percentages of DsRed+ cells in kidney marrow in the ‘early’ (n = 7) or ‘late’ group (n = 10). Red bars indicate the mean percentage. *p < 0.002, by Student's t-test. e, Flow cytometric and morphological analysis of DsRed+ cells. L, lymphocytes, N, neutrophils; E, eosinophils; M, monocytes; T, thrombocytes, P, precursors. May-Grünwald Giemsa staining. Bars, 10μm. f-k, Expression of runx1 and efnb2a in uninjected, jam1a MOatg-, or MOex7-injected embryos. l-s, Expression of cmyb, rag1, scl-α/β, and scl-α in uninjected or jam1a MOatg-injected embryos. Arrowheads indicate the dorsal aorta (f-m, p-s) or thymus (n, o). Data are pooled from two independent experiments (c-e) or representative of two independent experiments with two different clutches of embryos (f-s).
Figure 2
Figure 2. PLM cell migration is delayed in jam1a morphants
a-f, The expression of fli1 in uninjected, jam1a MOatg-, or MOex7-injected embryos. Arrowheads indicate a subset of fli1+ cells that did not reach the midline by 17hpf. g, h, Time-lapse images of fli1:GFP; phldb1:mCherry double-transgenic embryos. The regions from the tenth to twelfth somite are shown at each time point. Arrowheads indicate a subset of fli1:GFP+ cells that did not reach the midline. i, j, Transverse sections of fli1:GFP; phldb1:mCherry embryos uninjected (15.5hpf) or injected with jam1a MOatg (16hpf). High magnification views of the boxed regions are shown in the right panels. Dotted lines indicate the contact surface area between PLM cells (arrows) and somitic cells. Bars, 10μm. k, l, The number of somites were counted in jam1a MO control- or MOatg-injected embryos at 14hpf based on the expression of desma. The average numbers of somites in embryo groups are shown on each graph. There was no significant difference between jam1a MO control- (n = 28) and MOatg-injected embryos (n = 26, p = 0.61, by Student's t-test). ss, somite-stage. Data are representative of two independent experiments with two different clutches of embryos (a-f, k, l) or three embryos (i, j) or three independent experiments with nine embryos (g, h).
Figure 3
Figure 3. Loss of somitic jam2a phenocopies the Jam1a defect
a, Expression of jam2a at 16hpf. b, Relative expression levels of jam1a and jam2a in purified fli1:GFP+ and alpha-actin:GFP+ cells at 14hpf. Error bars, s.d. c, A transverse section of a fli1:GFP embryo stained with jam2a (purple, white arrowheads) and anti-GFP antibody (brown, black arrowheads) at 16hpf. The right panel shows a high magnification view of the boxed region. d, Co-immunoprecipitation (Co-IP) using anti-Flag antibody. The immunoprecipitates were examined by Western blotting using anti-Flag or anti-HA antibody. Inputs represent 10% of cell lysates used in the Co-IP experiment. Arrowheads indicate 40kDa. e, A schematic diagram of the proximity ligation assay (PLA). f, A representative result of Duolink PLA. The right panel represents a high magnification view of the boxed region. Arrowheads indicate PLA signal. g-J, The expression of runx1 and efnb2a in uninjected or jam2a MOatg-injected embryos. Arrowheads indicate the dorsal aorta. k, The expression of fli1 in jam2a MOatg-injected embryo at 17hpf. Arrowheads indicate a subset of fli1+ cells that did not reach the midline. l, The number of somites were counted in jam2a MOatg-injected embryos at 14hpf based on the expression of desma. Average somite number is shown on the graph. ss, somite-stage. Data are representative of two independent experiments with two different clutches of embryos (a-c, g-l) or three independent experiments (d, f).
Figure 4
Figure 4. Notch signalling is depleted in jam1a morphants
a, b, Transverse sections of Tp1:GFP; fli1:DsRed embryos uninjected or injected with jam1a MOatg at 18hpf. Green and red channels and merges of the boxed regions are shown in the lower panels. Arrows indicate fli1:DsRed+ cells. hc, hypochord. c-f, Flow cytometric and gene expression analysis of Tp1:GFP; fli1:DsRed embryos. Representative results of flow cytometric analysis at 22hpf (c), the mean fluorescent intensities of GFP in Tp1:GFP+; fli1:DsRed+ populations (d), relative expression levels of runx1 in the Tp1:GFPhigh and Tp1:GFPlow/mid population of fli1:DsRed+ cells in wild type embryos at 22hpf (e), and the percentages of Tp1:GFPhigh in fli1:DsRed+ populations at 22hpf (f) 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. g-j, Lateral views of the dorsal aorta (DA) in Tp1:GFP embryos and transverse sections of Tp1:GFP; fli1:DsRed embryos uninjected or injected with jam1a MOatg at 28hpf. Arrows indicate relatively low activation of Tp1:GFP in the ventral floor of the DA. Bars, 10μm. k, l, Acridine orange (AO) staining under the fli1:DsRed background in uninjected or jam1a MOatg-injected embryos at 30hpf. Arrowheads indicate AO-stained apoptotic cells. m, Relative expression levels of notch1a, notch1b, and notch3 in purified fli1:GFP+ cells obtained from uninjected or jam1a MOatg-injected embryos at 18hpf. n-q, The expression of dlc and dld in uninjected or jam1a MOatg-injected embryos at 14hpf. r-u, The expression of runx1 at 26hpf. Embryos were uninjected, injected with jam1a MOatg alone, or co-injected with jam1a MOatg and dlc or dld mRNA. Data are representative of two independent experiments with four embryos (a, b, h, j), eight embryos (g, i, k, l), four different clutches of embryos (c-f), or two different clutches of embryos (m-u).

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