1. by Jonathan M. Rowlinson, and Martin Gering
Hey2 acts upstream of Notch in hematopoietic stem cell specification in zebrafish embryos
by Jonathan M. Rowlinson, and Martin Gering
Blood
Volume 116(12):
September 23, 2010
©2010 by American Society of Hematology

2. Hey2 is essential for HSC formation.
Hey2 is essential for HSC formation. (A-B) The number of runx1-expressing HSCs (black arrow and arrowhead) was severely reduced in hey2 morphants at 28 hpf. Embryos injected with a control MO had normal runx1 expression at 28 hpf (n = 24/24; data not shown). Ikaros+ (C-D) and c-myb+ (E-F) blood progenitors were missing in the DA (black arrow and arrowhead) and in the CHT (blue arrow and arrowhead) of hey2 morphants at 48 hpf. (G-H) Homozygous hey2/grlm145 mutants possessed fewer runx1+ HSCs (black arrow and arrowhead) than wt control embryos. (I-K) In the CHT, the number of cd41-gfp+ cells was significantly lower in hey2 morphants than in uninjected embryos (Student t test, P < .001). Error bars represent the standard error of the mean for both groups of embryos. In panels A-H, numbers of embryos with normal gene expression are given as a fraction of the number of embryos analyzed. All views of embryos are lateral, with anterior at the left and dorsal at the top. The embryos in panels C-D were treated with phenylthiourea to prevent pigmentation. All morphants were injected with 5 ng of hey2 or control MO.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology

3. Hey2 expression in the lateral mesoderm is induced by cloche, scl/tal1, and etsrp and is maintained in the DA by Hedgehog and Vegf signaling.
Hey2 expression in the lateral mesoderm is induced by cloche, scl/tal1, and etsrp and is maintained in the DA by Hedgehog and Vegf signaling. (A) At the 10s stage (14 hpf), hey2 was expressed in the ALM (green arrow), the bilateral heart field (blue arrow), the otic vesicle (orange arrow), parts of the midbrain and hindbrain (purple arrow), the neural crest (turquoise arrow), the somites (yellow bracket), the PLM (black arrow), and the tail bud (red arrow). (B) In clo mutants, hey2 expression was lost in the ALM and PLM (white arrowheads). (C) In scl/tal1 morphants (5 ng of MO), PLM expression was lost (white arrowhead). (D) In etsrp morphants (10 ng of MO), hey2 expression in the ALM was lost (white arrowhead). (E) Inhibition of the Hh pathway with cyclopamine (100μM) from the 2-8 cell stage did not eliminate hey2 expression at the 10s stage in any of the tissues analyzed. (F) Hey2 expression was present in the ALM and PLM of mibta52b mutants (green and black arrows) but was absent in the otic vesicle and the tail bud (white arrowheads) and was reduced in the somites (yellow bracket). (G) At 24 hpf, hey2 was expressed in the dorsal aorta (black arrow), the ISVs (red arrow), the somites (yellow arrow), the neural crest (turquoise arrow), and the telencephalon (pink arrow), in addition to parts of the midbrain and hindbrain. In mibta52b mutants (H) and rbpja/b morphants (2 ng of MO; I), hey2 expression in the DA (black arrow) and in ISVs (red arrow) was retained, whereas telencephalon expression was lost (white arrowhead). By contrast, VegfR inhibitor–treated (J; 5μM) and cyclopamine-treated (K; 100μM) and etsrp-morphant (L; 10 ng) embryos displayed normal expression of hey2 in the telencephalon (pink arrow) but failed to express hey2 in the DA and the ISVs (white arrowheads). Embryos in panels J-K were treated from 90% epiboly. All views of embryos are lateral, with anterior at the left and dorsal up.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology

4. Hey2 is essential for expression of Notch receptors in the DA angioblast cord.
Hey2 is essential for expression of Notch receptors in the DA angioblast cord. (A) Kdrl was expressed in the DA (black arrow), PCV (blue arrow), and ISVs (red arrow) of the trunk and tail of 24-hpf control embryos. (B) Separate cords of DA and PCV kdrl-expressing angioblasts were present in hey2 morphant embryos. Expression of the arterial genes efnb2a (C-D) and notch1b (E-F) was lost in hey2 morphants. Note that spinal cord expression of notch1b was unaffected (E-F green arrow). VegfAa expression in somites was normal in hey2 morphant embryos (G-H white arrows). The Notch ligand gene dll4 was expressed in the DA of hey2 morphants (Ii-Jii black arrows). Loss of Hey2 protein caused increased endogenous hey2 mRNA expression in the DA and in somites (K-L orange arrow) of hey2 morphants, which suggests the presence of a negative autoregulatory loop. In kdrl- and dll4-stained hey2 morphant embryos, ISV staining was reduced (B,J; n(kdlr) = 41/49; n(dll4) = 51/90). (M) At 24 hpf, flt4 expression was restricted to the PCV in control embryos. (N) In the DA of hey2 morphants, it was retained in addition to normal venous expression. (O-P) Another venous marker, msr, was expressed in the vein of control and hey2 morphant embryos. All views of embryos in panels A-F and I-P are lateral, with anterior left and dorsal up. Dorsal views of embryos with anterior left are provided in panels G-H. Close-ups of the trunk of embryos are shown in C-F. The inserts (Ii and Ji) show transverse sections through the posterior trunk. Numbers of embryos with normal gene expression in the DA (A-F,I-P) or in the somites (G-H) are given as a fraction of the number of embryos analyzed. All morphants were injected with 5 ng of hey2 MO.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology

5. Hey2 is expressed downstream of Vegf signaling in the DA
Hey2 is expressed downstream of Vegf signaling in the DA. Inhibition of the Hh signaling pathway with the Smoothened inhibitor cyclopamine (100μM) or of the Vegf signaling pathway with a VegfR inhibitor (5μM) from 90% epiboly (9 hpf) caused a loss of hey2 e...
Hey2 is expressed downstream of Vegf signaling in the DA. Inhibition of the Hh signaling pathway with the Smoothened inhibitor cyclopamine (100μM) or of the Vegf signaling pathway with a VegfR inhibitor (5μM) from 90% epiboly (9 hpf) caused a loss of hey2 expression in the DA (Ai,iii,Bi,iii arrow). Although injection of 100 pg of vegfA mRNA did not change hey2 expression in control embryos, it rescued hey2 expression in cyclopamine-treated embryos (Aii,iv). In a similar rescue experiment, heat-shock–induced ubiquitous expression of NICD at the 17s (17.5-hpf) stage failed to rescue hey2 expression. All views of embryos are lateral, with anterior left and dorsal up. Numbers of embryos with hey2 gene expression in the DA are given as a fraction of the number of embryos analyzed. inh. indicates inhibitor.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology

6. Notch signaling acts downstream of Hey2 in arterial gene expression.
Notch signaling acts downstream of Hey2 in arterial gene expression. Hey2 morphants failed to express the arterial genes efnb2a and notch1b in the DA angioblast cord (A,C,E,G black arrow). Heat-shock–induced ubiquitous activation of the Notch pathway rescued efnb2a (D) and notch1b (H) expression in the DA of hey2 morphants and caused ectopic induction of arterial gene expression in all embryos analyzed (B,D,F,H blue arrow). All views of embryos are lateral, with anterior left and dorsal up. Numbers of embryos with arterial gene expression in the DA are given as a fraction of the number of embryos analyzed. All morphants were injected with 4 ng of hey2 MO.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology

7. Hey2 acts upstream of Notch in HSC specification.
Hey2 acts upstream of Notch in HSC specification. (A) Expression of runx1 in the DA was lost in cyclopamine (100μM)-treated and hey2 (4 ng of MO)–morphant embryos (i,iii,v). Although 80 ng of vegfA mRNA expression convincingly rescued runx1 expression in cyclopamine-treated embryos (iv), it failed to do so in hey2 morphants (vi). Ectopic expression of runx1 was observed in 4/14 wt (ii) and in 75 of 199 cyclopamine-treated (iv) VegfAa121 mRNA-injected embryos. (B) Expression of runx1 in the DA was lost in cyclopamine-treated (iii; 100μM, treated from 9 hpf), VegfR inhibitor–treated (v; 5μM, treated from 9 hpf), hey2-morphant (vii; 4 ng), scl/tal1-morphant (ix; 5 ng), and etsrp-morphant (xi; 10 ng) embryos. Heat-shock–induced NICD expression at the 17s (17.5 hpf) stage caused ectopic expression of runx1 in control embryos (ii). Furthermore, it rescued and caused ectopic expression of runx1 in cyclopamine-treated (iv), VegfR inhibitor–treated (vi), and hey2-morphant (viii) embryos. By contrast, it failed to induce runx1 expression in tal1/scl-morphant (x) and etsrp-morphant (xii) embryos. All views of embryos are lateral, with anterior left and dorsal up. High-magnification images of the posterior trunk region of all embryos are provided. Numbers of runx1-expressing embryos are given as a fraction of the number of embryos analyzed. inh indicates inhibitor.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology

8. The gene regulatory network controlling the differentiation of developing HSC. (A) This diagram illustrates the position of hey2 within the gene regulatory network that controls arterial gene expression and HSC development during zebrafish embryogenesis.
The gene regulatory network controlling the differentiation of developing HSC. (A) This diagram illustrates the position of hey2 within the gene regulatory network that controls arterial gene expression and HSC development during zebrafish embryogenesis. Genes shown in green are required for hey2 expression in the PLM at the 10s stage. Genes shown in blue are needed for maintenance of hey2 expression in the DA angioblast cord. Please note that our data neither clarify the position of individual Scl/Tal1 isoforms in the network relative to Hey2 nor provide insights into a specific late role of Scl/Tal1 in HSC formation. (B) This diagram illustrates whether or not runx1 expression can be rescued by activation of the Notch pathway in embryos that lack particular gene products. Loss of genes/gene products in green can be compensated by activation of the Notch pathway, whereas loss of genes/gene products in orange cannot. The diagram summarizes data from the present report and the literature.45 These data define a regulatory state, best described as early endothelial, that a cell needs to reach if it is to respond to a Notch signal by activating runx1 expression.
Jonathan M. Rowlinson, and Martin Gering Blood 2010;116:
©2010 by American Society of Hematology