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Pdgf signalling guides neural crest contribution to the haematopoietic stem cell specification niche

Nature Cell Biology volume 19, pages 457–467 (2017)Cite this article

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Abstract

Haematopoietic stem cells (HSCs) support maintenance of the haematopoietic and immune systems throughout the life of vertebrates, and are the therapeutic component of bone marrow transplants. Understanding native specification of HSCs, to uncover key signals that might help improve in vitro directed differentiation protocols, has been a long-standing biomedical goal. The current impossibility of specifying true HSCs in vitro suggests that key signals remain unknown. We speculated that such signals might be presented by surrounding ‘niche’ cells, but no such cells have been defined. Here we demonstrate in zebrafish, that trunk neural crest (NC) physically associate with HSC precursors in the dorsal aorta (DA) just prior to initiation of the definitive haematopoietic program. Preventing association of the NC with the DA leads to loss of HSCs. Our results define NC as key cellular components of the HSC specification niche that can be profiled to identify unknown HSC specification signals.

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Figure 1: NC contact the DA prior to and during HSC specification.
Figure 2: Pdgfra-mediated signalling is required for NC migration to the DA.
Figure 3: Pdgfra is required for HSC specification.
Figure 4: Arterial HE and primitive blood are normal in Pdgfra- and Pdgfrb-impaired animals.
Figure 5: NC are required for HSCs.
Figure 6: Arterial HE and primitive blood are normal in NC-impaired animals.
Figure 7: Catecholamine biosynthesis enzymes are not required for HSC specification.

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Acknowledgements

The authors thank B. Appel (University of Colorado Denver, USA), C. Burns (Harvard University, USA), M. Lardelli (University of Adelaide, Australia), D. Ransom (National Cancer Institute, USA), D. Traver (University of California at San Diego, USA), N. Trede (Juno Therapeutics, USA) and L. Zon (Harvard University, USA) for probe constructs; and J. Eberhart (University of Texas at Austin, USA), M. Granato (University of Pennsylvania, USA), C. Lien (Children’s Hospital of Los Angeles, USA), R. Stewart (University of Utah, USA) and D. Traver (University of California San Diego, USA) for providing mutant and transgenic zebrafish lines. We would also like to thank J. Peters and V. Frohlich of the St. Jude Cell and Tissue Imaging Center for assistance with microscopy and M. Weiss for providing critical feedback on the manuscript. This work was funded by NIH R00HL097150 and March of Dimes Basil O’Connor Starter Scholar Research Award no. 5-FY14-42 to W.K.C.

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Authors and Affiliations

  1. Department of Hematology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, USA

    Erich W. Damm & Wilson K. Clements

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  1. Erich W. Damm
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Contributions

E.W.D. and W.K.C. designed all experiments. E.W.D. performed all experiments. E.W.D. and W.K.C. analysed all results and wrote the manuscript.

Corresponding author

Correspondence to Wilson K. Clements.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 NC contact the dorsal aorta prior to and during HSC specification.

Trunk lateral views (a–f; dorsal up, anterior left) of NC migration progress using the NC reporter sox10:mRFP relative to endothelial cells labeled with EGFP in fli1a:EGFP animals. Images are still frames from a z-stack volume projection in single and merged channels (a–f) from time lapse confocal microscopy from 20 h.p.f. to 27 h.p.f. as indicated. Arrowheads indicate NC streams that are positioned ventral to (yellow arrowheads) or dorsal to (blue arrowheads) the notochord. Scale bar = 100 μm, mag. = 200X. isv – intersegmental vessel, da – dorsal aorta, pcv – posterior cardinal vein.

Supplementary Figure 2 Pdgfr expression before and after HSC specification.

Expression of pdgfra (a–c”) and pdgfrb(d–f”) in wild-type embryo trunk in either lateral view (a–f; dorsal up, anterior left), transverse section (a’–f’; dorsal up, level indicated by red line in a–f), and dorsal trunk view (a”–f”; anterior up) prior to (18 h.p.f., 21 h.p.f.) (a,b”,d–e”) and during (24 h.p.f.) (c–c”, f–f”) HSC specification. Developmental stages are indicated. Numbers in the bottom right corner of panels indicate numbers of zebrafish embryos analysed for the indicated gene, versus the total number of zebrafish analysed. Zebrafish randomly selected from one clutch are represented by the image (a–f”). Scale bars = 100 μm. Lateral views (dorsal up, anterior left) of whole uninjected, Pdgfra, Pdgfrb and Pdgfra/b double morphant, DMSO and inhV treated embryos at the developmental stages indicated, demonstrating overall normal morphology (g–l). Scale bars = 200 μm.

Supplementary Figure 3 Pdgfr-mediated signalling is required for NC migration to the DA.

NC migration visualized by crestin expression in lateral (dorsal up, anterior left) and transverse (dorsal up) views of DMSO (a–a’) or inhV (b–b’) treated embryos, uninjected embryos (c-c’), a subset of embryos injected with 10 ng of PdgfrbMO displaying normal NC migration (d–d’) a subset of embryos injected with 10 ng of PdgfrbMO displaying defective NC migration (e–e’) and embryos injected with both PdgfraMO and PdgfrbMO (f–f’). Numbers in the bottom right corner of panels indicate numbers of zebrafish embryos analysed for the indicated gene, versus the total number of zebrafish analysed. Zebrafish randomly selected from three clutches are represented by the image (a–f,h,i). Positions of transverse sections are indicated by the red bars (a–f). Scale bars = 100 μm (a–f, h-i). Average number of NC streams ventral to the notochord at 24 h.p.f. for the indicated conditions, error bars indicate standard error of the mean, n = 20 is the number of randomly selected zebrafish embryos from three biologically independent clutches each, the triple asterisk indicates a statistically significant result between bars, from left to right, p-value was < 0.0001 by Student’s t-test in all cases (g), see Supplementary Table 3. Lateral views (dorsal up, anterior left) of crestin expression in uninjected (h) and Pdgfra morphant (i) embryos at 36 h.p.f. NC streams that have migrated ventral to the notochord are indicated by yellow arrowheads (a–f,h–i), red brackets enclose the AGM region (a–f), green arrowheads indicate the presence of melanophores (h–i). Lateral z-stack volume projections of the trunk (dorsal up, anterior left) with vasculature in green (fli1a:EGFP) and NC in red (sox10:mRFP) in uninjected (j) and Pdgfra morphant (k) embryos, blue (k) and yellow (j) arrowheads indicate NC streams which are dorsal to and ventral to the notochord respectively, scale bar = 100 μm, mag. = 200X. nt – neural tube, no – notochord, DA – dorsal aorta, PCV – posterior cardinal vein.

Supplementary Figure 4 Pdgfra-mediated signalling is required for NC migration to the DA.

Lateral z-stack volume projections of the trunk (dorsal up, anterior left) with vasculature in green (fli1a:EGFP) and NC in red (sox10:mRFP) in embryos injected with Pdgfra MO (a–f). Still frames from time lapse confocal microscopy from 20 h.p.f. to 27 h.p.f. as indicated. Blue arrowheads indicate NC streams that are dorsal to the notochord. Scale bars = 100 μm, mag. = 200X. isv – intersegmental vessel, da – dorsal aorta, pcv – posterior cardinal vein.

Supplementary Figure 5 Pdgfr signalling is required for HSC specification.

Lateral trunk views (dorsal up, anterior left) of expression of the HSC marker runx1 in DMSO (a) and inhV treated embryos (b) or dnpdgfrb:YFP; hsp70:GAL4 embryos that were either heat shocked (c, d) or not heat shocked (e), wild-type uninjected embryos (f), subset of Pdgfrb morphants displaying normal HSC specification (g) a subset of Pdgfrb morphants displaying defective HSC specification (h), Pdgfra morphants (i) and low-dose Pdgfra and Pdgfrb combined morphants (j) at 24 h.p.f. Green arrowheads identify runx1 expressing HSC precursors. Average number of runx1+ cells in the DA for the indicated conditions (k), n is the number of randomly selected zebrafish embryos from three biologically independent clutches, error bars indicate standard error of the mean, triple asterisk indicate p-value < 0.0001, single asterisk p = 0.0411 by Student’s T-test. See Supplementary Table 3. T-lymphocyte expression of rag1 in 4 d.p.f. embryos (ventral head view, anterior up) treated with DMSO at 18, 24 and 48 h.p.f. (l,n,p) or inhV at 18, 24 and 48 h.p.f. (m,o,q), blue arrowheads indicate rag1+ T-lymphocytes, when present. Numbers in the bottom right corner of panels indicate numbers of zebrafish embryos analysed for the indicated gene, versus the total number of zebrafish analysed. Zebrafish randomly selected from three clutches are represented by the image (a–j,l–q). Scale bars = 100 μm (a–j,l-q).

Supplementary Figure 6 NC are required for HSCs.

Lateral trunk views (dorsal up anterior left) of in situs for the HSC marker runx1(blue) and the NC marker crestin (red) (a,d,g,j,m), the HSC marker cmyb (b,e,h,k,n), and the T lymphocyte marker rag1 (c,f,i,l,o, ventral head views) in uninjected or Tfap2a morphant (a–f), wild-type and sox10m241heterozygous (g–i), sox10m241 homozygous mutant (j–l) and Sdc4 morphant (m–o) embryos at the developmental stages indicated. Green and red arrowheads (a,b,d,e,g,h,j,k,m,n) indicate cells expressing runx1 and cmyb respectively, blue arrowheads (c,f,i,l,o) identify rag1 expressing T-lymphocytes when present, red brackets (a,d,g,j,m) enclose the AGM region. Numbers in bottom right corner indicate number of individuals in the clutches represented by the image (a–o). Scale bars = 100 μm (a–o). Average number of runx1+ (p) or cmyb+ (q) DA cells, or average number of NC streams ventral to the notochord at 24 h.p.f (r) in the indicated conditions. Numbers in the bottom right corner of panels indicate numbers of zebrafish embryos analysed for the indicated gene, versus the total number of zebrafish analysed. Zebrafish randomly selected from three clutches are represented by the image (a–o). Fluorescently labelled HSCs in uninjected (s) and Sdc4 morphant (t) cmyb:GFP;kdrl:mCherry transgenic embryos at 36 h.p.f., HSCs are yellow cells indicated by yellow arrowheads, scale bar = 50 μm, mag. = 200X. Average number of mCherry+GFP+ cells in the DA for the indicated conditions (u), n = 8 zebrafish each. Error bars indicate the standard error of the mean (p–r,u). Triple asterisk indicates p-values < 0.0001 in (p–r) and p = 0.0007 in (u) by Student’s t-test. See Supplementary Table 3.

Supplementary Figure 7 NC are required for HSCs; NC mutant and morphant validation.

Lateral trunk views (dorsal up, anterior left) of in situs for the NC marker crestin (a–h) and the HSC marker runx1 (i–p) at 24 h.p.f. in uninjected (a, i), Tfap2a morphant (b,j), Sdc4 morphant (c,k), Sox10 morphant (d,l), wild-type and sox10m241 heterozygous (e,m), sox10m241 homozygous mutant (f,n), wild-type and tfap2ats213 heterozygous (g,o) and sox10ts213 homozygous mutant (h,p) embryos. Red brackets enclose the AGM region (a–h), green arrowheads indicate runx1 expressing cells (i–p). Numbers in the bottom right corner of panels indicate numbers of zebrafish embryos analysed for the indicated gene, versus the total number of zebrafish analysed. Zebrafish randomly selected from three clutches are represented by the image (a–p). Scale bars = 100 μm (a–p). Lateral images (dorsal up, anterior left) of whole uninjected (q), Tfap2a morphant (r), Sdc4 morphant (s) and Sox10 morphant (t) embryos at 24 h.p.f. showing embryo morphology. RT-PCR showing aberrant mRNA splicing in Tfap2a morphant (arrows) compared with uninjected embryos (u). Lateral views (dorsal up, anterior left) of uninjected controls (v), wild-type and sox10m241 heterozygous (w), wild-type and tfap2ats213 heterozygous (x) embryos have increased numbers of melanophores compared with Sox10 morphant (v’), sox10m241 homozygous mutant (w’) and tfap2ats213 homozygous mutant (x’) embryos (red arrowheads indicate presence of increased numbers of melanophores) at the developmental stages indicated (v–x’). Scale bars = 200 μm (q–t, v–y’).

Supplementary Figure 8 Unprocessed Blots.

Unprocessed agarose gel electrophoresis image for RT-PCR experiments examining the effects of splice-blocking morpholinos targeting th and tfap2a on mRNA levels and splicing (a). Expected sizes for normal transcripts and approximate sizes for aberrantly spliced transcripts are indicated. Lanes are labelled 1 through 7 and the identity of each band and the associated embryo injection conditions are indicated in the table.

Supplementary information

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Supplementary Table 1

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Supplementary Table 2

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Supplementary Table 3

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NC contact the dorsal aorta prior to and during HSC specification (transverse projection).

Virtual transverse section (dorsal up) through the mid-trunk using confocal z-stack volume projection time lapse depicting NC migration (red, sox10:RFP) relative to axial vessel endothelial cells (green, fli1a:EGFP) over a 7 h period from 20 to 27 h.p.f. in wild-type animals. (MOV 17874 kb)

NC contact the dorsal aorta prior to and during HSC specification (lateral projection).

Lateral (dorsal up, anterior left) z-stack volume projection time lapse depicting NC migration (red, sox10:RFP) relative to axial vessel endothelial cells (green, fli1a:EGFP) over a 7 h period from 20 to 27 h.p.f. in wild-type animals. (MOV 45694 kb)

Pdgfra-mediated signalling is required for NC migration to the DA (transverse projection).

Virtual transverse section (dorsal up) through the mid-trunk using confocal z-stack volume projection time lapse depicting NC migration (red, sox10:RFP) relative to axial vessel endothelial cells (green, fli1a:EGFP) over a 7 h period from 20 to 27 h.p.f. in embryos injected with 7 ng PdgfraMO. (MOV 5990 kb)

Pdgfra-mediated signalling is required for NC migration to the DA (lateral projection).

Lateral (dorsal up, anterior left) z-stack volume projection time lapse depicting NC migration (red, sox10:RFP) relative to axial vessel endothelial cells (green, fli1a:EGFP) over a 7 h period from 20 to 27 h.p.f. in embryos injected with 7 ng of PdgfraMO. (MOV 63755 kb)

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Damm, E., Clements, W. Pdgf signalling guides neural crest contribution to the haematopoietic stem cell specification niche. Nat Cell Biol 19, 457–467 (2017). https://doi.org/10.1038/ncb3508

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