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. Author manuscript; available in PMC: 2010 Jan 4.
Published in final edited form as: Dev Cell. 2004 Aug;7(2):251–262. doi: 10.1016/j.devcel.2004.07.010

A Crucial Interaction between Embryonic Red Blood Cell Progenitors and Paraxial Mesoderm Revealed in spadetail Embryos

Laurel A Rohde 1,2,*, Andrew C Oates 1,3, Robert K Ho 1
PMCID: PMC2801434  NIHMSID: NIHMS164627  PMID: 15296721

Summary

Zebrafish embryonic red blood cells (RBCs) develop in trunk intermediate mesoderm (IM), and early macrophages develop in the head, suggesting that local microenvironmental cues regulate differentiation of these two blood lineages. spadetail (spt) mutant embryos, which lack trunk paraxial mesoderm (PM) due to a cell-autonomous defect in tbx16, fail to produce embryonic RBCs but retain head macrophage development. In spt mutants, initial hematopoietic gene expression is absent in trunk IM, although endothelial and pronephric expression is retained, suggesting that early blood progenitor development is specifically disrupted. Using cell transplantation, we reveal that spt is required cell autonomously for early hematopoietic gene expression in trunk IM. Further, we uncover an interaction between embryonic trunk PM and blood progenitors that is essential for RBC development. Importantly, our data identify a hematopoietic microenvironment that allows embryonic RBC production in the zebrafish.

Introduction

The differentiation of embryonic (primitive) blood cells in zebrafish initiates within two separate intraembryonic environments. The first two blood lineages detected in zebrafish are a transient group of embryonic red blood cells (RBCs) that derive solely from blood progenitors in the trunk (Kimmel et al., 1990; Lieschke et al., 2002) and early macrophage cells that originate from progenitors located in the head (Figure 1A; Herbomel et al., 1999). This spatial restriction of embryonic RBC and early macrophage differentiation may reflect a role for the local environment in specifying these different fates. During adult (definitive) hematopoiesis in mammalian systems, signals from the microenvironment, the so-called “stem cell niche,” are thought to regulate hematopoietic stem cells (HSCs), the self-renewing progenitors of all blood lineages (Lemischka and Moore, 2003).

Figure 1. RBC Gene Expression Is Absent in spt Embryos.

Figure 1

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(A) Regions producing early macrophages (head, purple) and RBCs (trunk, red) in 8s embryos. Flat-mount in dorsal view, anterior at top. Gray shading indicates trunk and tailbud area shown in Figures 1–5.

(B) gata1 (arrowheads), jak2a, αe1globin, and βe1globin expression in 24 hpf wild-type (wt) and spt embryos.

(C–G) Trunk and tailbud regions. (C) gata1 and biklf/klf4 hematopoietic expression (arrowhead) at 7s is absent in spt. Ectodermal biklf/klf4 expression remains (arrows). (D–G) scl (D), lmo2 (E), hhex (F), and gata2 (G) expression is initially absent in spt, appears at 5s–7s (arrowheads). Nonneural ectodermal gata2 expression in spt (arrows).

In the zebrafish embryo, RBCs derive from intermediate mesoderm (IM), a bilateral tissue extending along the anterior/posterior axis between trunk paraxial mesoderm (PM) and lateral plate mesoderm (LPM). Zebrafish hematopoietic tissue has been referred to as LPM; however, morphology (Al-Adhami and Kunz, 1977) and molecular markers suggest that IM and LPM are separate tissues. In addition to RBCs, IM gives rise to endothelial and pronephric lineages (Lieschke et al., 2002; Serluca and Fishman, 2001). At mid-segmentation stages, trunk blood and endothelial cells migrate to the midline, forming a single structure, the Intermediate Cell Mass, from which RBCs enter circulation at 24 hpf (hours post fertilization; Detrich et al., 1995; Gering et al., 1998).

Zebrafish homologs of vertebrate hematopoietic regulators include those important for the development of early blood progenitors, such as scl, lmo2, hhex, and gata2 (Detrich et al., 1995; Liao et al., 1998; Liao et al., 2000; Thompson et al., 1998), as well as those involved in specification and terminal differentiation of the RBC lineage such as gata1, biklf/klf4, jak2a, and embryonic globin genes (Brownlie et al., 2003; Detrich et al., 1995; Oates et al., 1999; Quinkertz and Campos-Ortega, 1999). Sequential expression of these key genes in zebrafish hematopoietic domains is similar to that observed in tetrapods (Minko et al., 2003; Silver and Palis, 1997), and is associated with progression of developing blood cells toward lineage restriction (Amatruda and Zon, 1999).

In Xenopus, mouse, and chick, developing embryonic blood and endothelial cells require signals from adjacent tissues (Baron, 2003). Although non-cell-autonomous requirements for zebrafish hematopoiesis have been suggested (Liao et al., 2002; Parker and Stainier, 1999), a tissue supplying cues to IM has not yet been identified. One potential source is suggested by zebrafish spadetail (spt) embryos in which RBC loss is associated with an absence of trunk PM (Kimmel et al., 1989; Thompson et al., 1998). spt embryos have a lesion in the T-box transcription factor, tbx16 (Griffin et al., 1998; Ruvinsky et al., 1998), which is normally required cell autonomously during gastrulation for proper localization of trunk PM precursors (Ho and Kane, 1990). Loss of spt/tbx16 results in mislocation of these cells to the tail, leaving the trunk severely deficient in PM (Kimmel et al., 1989). The cause of RBC loss in spt embryos is unclear, although it does not appear to result from a general blood defect, as early macrophages develop normally in the spt head and later myeloid cells are detected (Lieschke et al., 2002). Previous investigation of hematopoietic and endothelial gene expression in spt embryos (Thompson et al., 1998; Oates et al., 1999) suggested that spt functions after blood progenitor specification by affecting transition to RBC fate, possibly at the level of an HSC (Amatruda and Zon, 1999).

To further define the spt blood defect, we examined early events in the hematopoietic program, finding a severe loss of both blood progenitor and RBC gene expression. Using reciprocal cell transplantation between wt and spt embryos, we discovered not only that spt function is required cell autonomously within IM during early hematopoiesis, but that an interaction with trunk PM is additionally required for RBC lineage-specific expression in IM. Our data demonstrate a novel role for trunk PM in establishing a critical environment for differentiation of the zebrafish embryonic RBC lineage.

Results

Hematopoietic Gene Expression Defects in spt Embryos

Circulating embryonic RBCs are absent in spt embryos along with expression of RBC markers, gata1 and jak2a, at 24 hpf (Figure 1B; Kimmel et al., 1989; Oates et al., 1999; Thompson et al., 1998). Consistent with these previous findings, embryonic αe1globin and βe1globin expression is also lost at 24 hpf (Figure 1B). To investigate the onset of RBC lineage differentiation in spt embryos, we assayed early gata1 expression, as it is the first RBC-specific marker and is required for blood progenitors to differentiate RBCs (Lyons et al., 2002). gata1 is detected by the 6 somite-stage (6s; 12 hpf) in wt trunk IM posterior to somite 6 (Detrich et al., 1995) but is not initiated in spt embryos (Figure 1C). Morpholino knock-down suggests a role for biklf/klf4 expression in the maintenance of gata1 expression (Kawahara and Dawid, 2001). At 7s (12.5 hpf), when biklf/klf4 hematopoietic expression is distinct from overlying ectodermal expression in wt embryos, we detect no expression in spt trunk IM (Figure 1C). Thus, the hematopoietic program in spt embryos is blocked prior to expression of the earliest RBC markers, strongly suggesting that RBC differentiation is not initiated.

Prior to RBC lineage marker expression, genes critical for early events in the vertebrate hematopoietic program are expressed in zebrafish trunk IM starting at 2s (10.5 hpf). We looked for this early gene expression in spt IM to determine if loss of RBCs might result from defects in blood progenitor formation. scl, lmo2, and hhex are expressed within developing blood and endothelial progenitors in zebrafish and are thought to function in the specification of hemangioblasts, bipotent cells hypothesized to produce both blood and endothelial cells (Gering et al., 1998, 2003; Liao et al., 1998; Liao et al., 2000; Thompson et al., 1998). Mammalian data suggest that gata2 acts by regulating the proliferation and survival of HSCs, the progeny of the hemangioblast (Tsai and Orkin, 1997). At 3s (11 hpf), there was a complete loss of scl, lmo2, and gata2 expression in spt trunk IM, and hhex likewise failed to initiate at 5s (11.5 hpf; Figures 1D–1G). However, from 5s to 7s, expression of these genes gradually appeared in a small number of spt trunk IM cells (Figures 1D–1G). By 12s, the number of expressing cells increased markedly, but had not reached wt levels (Figures 1D–1G). Therefore, gene expression associated with two potential early hematopoietic events, hemangioblast specification and HSC development, is defective in spt IM, suggesting that an early defect in blood progenitor formation underlies RBC loss in spt embryos.

The earliest described marker of the zebrafish trunk hematopoietic IM is the putative nuclear protein draculin (dra), whose function in hematopoiesis has not yet been demonstrated (Herbomel et al., 1999). In gastrulating wt embryos, dra is expressed in ventrolateral mesodermal cells of the hypoblast (Herbomel et al., 1999) in a domain that separates from PM marked by her1 (Müller et al., 1996) by Bud stage, at which time dra expression appears largely restricted to IM (10 hpf; Figure 2A). We found dra expression reduced throughout the hypoblast of the spt gastrula at 70% epiboly (8 hpf), after which expression decreased until no longer detected in the trunk at Bud or later during segmentation stages (Figure 2A). In wt, dra expression is retained in developing RBCs and early macrophages (Herbomel et al., 1999).

Figure 2. IM and LPM Gene Expression in spt Embryos.

Figure 2

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(A) dra (blue) and her1 (red) expression at 70% epiboly (lateral view, dorsal to right), tailbud (Bud), and 12s. dra expression domain in wt separates from her1 expressing PM (arrowhead, 70%). dra is reduced in spt at 70% and absent at Bud through 12s.

(B and C) fli1 and pax2.1 expression in wt and spt at 3s–12s.

(D) Endothelial flk1 expression in wt and spt at 12s. (E) LPM dhand expression in wt and spt at 7s. (F) Co-in situ hybridization in 6s wt for scl (blue), dhand (red), and myoD (blue, arrowheads). (G) Co-in situ hybridization in 6s wt and spt embryos for fli1 (blue), dhand (red), myoD (blue, arrowheads). Bracketed areas enlarged.

Consistent with previously described expression of the hematopoietic regulator, pu1/spi1 in the spt head blood domain (Lieschke et al., 2002), we found head expression of scl, lmo2, gata2, and hhex also unperturbed (data not shown), suggesting that spt embryonic blood defects are limited to the trunk. In summary, we have shown that spt trunk IM exhibits a combination of delayed, severely reduced, or lost hematopoietic gene expression, including dra, the earliest known marker of these cells. As the spt mutation specifically affects trunk blood, we next asked whether this reflects either a general loss of trunk IM or an overall delay in differentiation of its derivatives.

IM and LPM Marker Expression in the spt Trunk

Coexpression of blood, pronephros, and endothelial markers in wt zebrafish embryos (Davidson et al., 2003; Gering et al., 1998, 2003), and fate mapping studies (Lieschke et al., 2002; Serluca and Fishman, 2001), indicate that these tissues develop closely within IM, perhaps originating from common progenitors. fli1 is expressed in wt trunk IM by Bud stage and is initially coexpressed with gata2, but is later maintained only in endothelial cells (Brown et al., 2000; Thompson et al., 1998). We find fli1 expressed in spt IM with the same time course as in wt embryos; however, as detected at stages 3 s to 12 s, the fli1 expression domain in spt embryos is widened (Figure 2B). Expression of another endothelial marker flk1 is detected in spt IM at 12s (Figure 2D), in a pattern similar to scl (Figure 1D), suggesting that scl expression in spt IM at 12s is confined to a population of developing endothelial cells. pax2.1 expression in pronephric precursors (Majumdar et al., 2000) also occurs along a normal time course in spt IM and, like fli1, appears broader in comparison to wt embryos (Figure 2C). pax2.1 expression in spt embryos at 12s is disordered, consistent with previous reports of disorganized pronephric and endothelial structures in older spt embryos (Kimmel et al., 1989; Thompson et al., 1998). Additionally, the anterior limit of pax2.1 expression in spt IM is similar to wt, suggesting that significant alteration in IM anterior-posterior identity has not occurred. Disruption of the earliest hematopoietic markers in spt trunk IM, and later RBC loss, is therefore not due to a general failure to differentiate IM or to a delay in IM development, suggesting that the defect is specific to hematopoietic IM.

We find hematopoietic IM distinct from LPM marked by dhand expression (Figure 2E; Angelo et al., 2000). By 6s in wt embryos, scl, fli1, and dhand are already expressed in restricted mediolateral domains (Figures 2F and 2G). In spt embryos at 6s, LPM and IM as marked by dhand and fli1 remain distinct (Figure 2G). Thus, spt embryos appear to maintain a correct mediolateral organization of trunk mesoderm.

Finally, in the mouse, gata1−/− proerythroblasts undergo apoptosis (Weiss and Orkin, 1995), raising the possibility that spt RBC progenitors have died. Acridine orange staining revealed no difference between spt and wt IM at early segmentation stages through 24 hpf (data not shown).

spt Is Required Cell Autonomously for RBC Formation

To test if RBC loss in spt embryos is caused by a requirement for spt within developing RBCs, we transplanted combinations of differently labeled wt and spt donor cells at pregastrula stages into unlabeled wt host embryos (Figure 3A). At 27 hpf, we found that if cells from two wt donors contributed to IM, RBCs with both labels were found in circulation (n = 13/17 embryos; Figure 3B). In contrast, if wt and spt donor cells contributed to IM, only wt donor cells were detected as RBCs (n = 4/4 embryos; Figure 3C). Importantly, both wt and spt donor cells formed pronephric and endothelial structures (Figure 3D). These results demonstrate that spt cells, while able to differentiate as IM (Kimmel et al., 1989; Thompson et al., 1998), are unable to form RBCs even when placed within the appropriate wt environment. spt function is therefore required cell autonomously for the formation of RBCs.

Figure 3. spt Is Required Cell Autonomously for Production of RBCs.

Figure 3

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(A) Pregastrulation cells from labeled donors cotransplanted into unlabeled wt hosts. Boxed areas of 24 hpf wt as shown below.

(B and C) Donor RBCs circulating across yolk in live 27 hpf wt host. (B) Control, RBCs derive from both wt donors (arrowheads [B’ and B”]).

(C) RBCs derive from the wt donor cells (green [C’]), but not spt (red [C”]).

(D) Posterior of embryo in (B). spt (red [D’ and DMerge]) and wt (green [D” and DMerge]) donor cells form vasculature (arrowheads) and pronephros (arrows).

(E–G) Composite confocal sections through gata1 expression domain (red) in wt host embryos at 8s. Donor cells in green. (E) Control, wt donor-derived IM expresses gata1 (arrowheads). (F–G) spt donor-derived IM cells do not express gata1 (arrowheads, bracket). (H and I) Confocal sections through dra expression domain (red) in half of posterior wt host at Bud. (H) Control, wt donor cells (green) express dra. (I) spt donor cells (green) fail to express dra (arrowheads).

spt Is Required Cell Autonomously for Early Hematopoietic Expression

To establish if spt plays a cell-autonomous role during early RBC development, we transplanted spt cells into wt host embryos at pregastrula stages, then assayed for gata1 and dra expression at 8s and Bud stage, respectively (Table 1; Figures 3E–3I). When spt donor cells formed IM, no gata1 expression was detected in these cells, although the surrounding wt host cells expressed gata1 (n = 0/18 events, Table 1; Figures 3F and 3G). Similarly, at Bud stage when spt cells were located within the dra expression domain in a wt host, the spt cells did not express dra (n = 0/5 embryos, Table 1; Figure 3I). As expected, wt cells transplanted into wt hosts expressed gata1 and dra (gata1 n = 21/25 events, dra n = 8/8 embryos, Table 1; Figures 3E and 3H). Therefore, spt is required cell autonomously for expression of gata1, a gene critical to RBC differentiation, and dra, a marker of early blood progenitors.

Table 1.

Cell-Transplantation Experiments

Donor-Derived Trunk Tissue
Cell Transplant Situation Scored CNS Noto PM Alone IM Alone IM+PM
A. Cell Transplants Analyzed for gata1 Expression at 8s
wt → wt (nt = 140) hosts (nh [%])
events (ne)
gata1 pos. IM (npos [%])		 35 (25)
35
35 (100)		 76 (54)
78
78 (97)		 50 (36)
54
51 (94)		 22 (16)
25
21 (84)		 36 (26)
38
34 (90)
wt → spt (nt = 56) hosts (nh [%])
events (ne)
gata1 pos. IM (npos [%])		 12 (21)
12
0		 7 (13)
7
0		 33 (60)
38
0		 7 (13)
10
0		 15 (27)
19
12 (63)
spt → wt (nt = 23) hosts (nh [%])
events (ne)
gata1 pos. IM (npos [%])		 6 (26)
6
6 (100)		 10 (44)
10
10 (100)		 0
0
0		 13 (57)
18
0		 0
0
0
B. Cell Transplants Analyzed for dra Expression at 8s
wt → wt (nt = 59) hosts (nh [%])
events (ne)
dra pos. IM (npos [%])		 21 (36)
21
21 (100)		 32 (54)
32
32 (100)		 24 (41)
24
23 (96)		 14 (24)
19
18 (95)		 18 (31)
19
17 (90)
wt → spt (nt = 40) hosts (nh [%])
events (ne)
dra pos. IM (npos [%])		 3 (8)
3
0		 9 (23)
9
0		 18 (45)
22
0		 9 (23)
16
8 (50)a 		22 (55)
34
32 (94)
C. Cell Transplants Analyzed for dra Expression at Bud
Non-Axial Mesoderm
wt → wt (nt = 33) hosts (nh [%])
dra pos. IM (nd [%])		 24 (73)
8 (30)
spt → wt (nt = 19) hosts (nh [%])
dra pos. IM (nd [%])		 10 (53)b
0
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Donor-derived tissues are: Central nervous system (CNS); Notochord (Noto); PM without adjacent donor IM (PM alone); IM without adjacent donor PM (IM alone); and a combination of apposed IM and PM (IM+PM). An event is a contiguous region of donor cell contribution to a particular tissue(s).

nt = total hosts examined; nh = hosts containing given donor-derived tissue type (% = nh/nt); ne = events of donor cell contribution to a given tissue(s); npos = events of gata1 or dra expression in host IM at 8s (when donor cells contribute to CNS, Noto, or PM alone) or in donor IM (when donor cells contribute to IM alone, or IM+PM) (% = npos/ne); nd = host containing dra positive donor cells (% = nd/nh)

a

Positive expression found only in donor cells in tailbud region.

b

5 host embryos contained spt donor cells within the wt host dra domain

Interaction between IM and PM Is Required for gata1 Expression

To test if spt function in IM is sufficient to rescue RBC formation or if environmental cues normally required for RBC formation are lacking in spt mutants, we transplanted wt cells at pregastrula stages into spt embryos. This manipulation resulted in spt hosts at 8s that contained regions in which wt donor cells formed different combinations of trunk tissues, which we identified using both position and morphology. We assayed for gata1 expression within trunk regions of spt host embryos that contained either wt donor-derived PM alone, or IM alone, or a combination of apposed IM and PM. All data were collected from host trunk regions within normal anterior and posterior limits of gata1 expression at 8s. Transplanted wt cells that formed large regions of trunk PM, including somites, in the PM-deficient spt host were not sufficient to rescue expression of gata1 in adjacent spt IM (n = 0/38 events, Table 1; Figure 4B). Wt donor-derived nervous system and notochord likewise did not rescue gata1 expression in spt IM (Table 1), consistent with our finding that spt function is required within IM cells for RBC formation.

Figure 4. PM Is Required for gata1 Expression in Adjacent IM.

Figure 4

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(A–E) Composite confocal sections at level of gata1 expressing IM (red). Transplanted wt cells (green) formed the following trunk tissues in hosts: PM without adjacent donor-derived IM (PM); IM without adjacent donor-derived PM (IM); and a combination of apposed IM and PM (IM+PM). Somite boundaries marked by arrowheads. (A) Control, wt cells express gata1 in a wt host. (B–E) spt hosts containing wt donor cells. Expected anterior limit of gata1 expression marked (arrows). (B) Trunk PM fails to rescue gata1 expression in spt host. (C) IM alone does not express gata1. IM cells adjacent to PM express gata1. (D and E) IM and IM+PM in spt hosts. (D’ and E’) Illustration of wt IM cells (outline) and wt PM somitic structures (gray blocks).

In regions of spt host embryos in which wt donor cells contributed only to trunk IM (Figures 4C and 4E), wt cells, like the surrounding spt host IM, failed to express gata1 (n = 0/10 events, Table 1). This indicates that cell-autonomous spt function in IM is not sufficient to rescue RBC formation in spt embryos and suggests that an environmental hematopoietic factor(s), ordinarily present in wt embryos could be additionally absent in spt embryos. Here we show that gata1 expression depends upon an interaction occurring between the IM and trunk PM. In regions of spt host embryos in which wt donor cells formed a combination of IM and adjacent trunk PM, strong gata1 expression was now observed in wt IM cells (n = 12/19 events, Table 1; Figures 4C–4E). In all cases in which wt donor cells contributed to nonaxial trunk mesodermal tissue, gata1 expression was only detected in wt donor-derived IM that was apposed to PM formed by wt donor cells (n = 12/67 events, Table 1). This rescue is in contrast to the above results in which wt donor-derived PM alone or IM alone was not sufficient for gata1 expression. Our cell-transplantation data thus reveal a novel interaction between hematopoietic IM and trunk PM for embryonic RBC lineage formation.

Rescue of dra Expression

To determine if an interaction with PM is also required for dra expression in wt donor cell IM, we assayed rescue throughout the trunk and tailbud IM at 8s (Table 1; Figure 5). Similar to gata1, we saw no dra expression in wt donor cells that formed IM alone in the trunk of spt hosts (n = 0/8 events, Table 1; Figures 5B and 5C). Expression of dra in the trunk was only seen in cases where wt donor-derived IM was adjacent to wt PM (n = 32/34 events, Table 1; Figures 5B–5D). Therefore, in trunk IM at 8s, dra expression also requires both functional spt and an interaction with wt PM. In contrast to the results seen in the trunk, wt donor-derived IM in the tailbud of spt hosts was observed to express dra in the absence of nearby wt PM (n = 8/8 events, Table 1; Figure 5D), although potentially in contact with large numbers of spt PM precursor cells present in the host tailbud. Wt PM alone failed to rescue dra expression in trunk and tailbud spt host IM (Figures 5B and 5C).

Figure 5. PM Is Required for Anterior Trunk Expression of dra at 8s.

Figure 5

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(A–D) Composite confocal sections at the level of dra expression in 8s hosts. Transplanted wt cells (green) and dra expression (red). See Figure 4 for description of PM, IM, and IM + PM wt donor-derived tissue. (A) Control, wt cells express dra in a wt host (arrowheads). (B–D) Wt IM in spt trunks does not express dra. Wt trunk IM adjacent to wt PM (* in [D]) expresses dra. Wt PM is unable to induce dra expression in spt host. (D) Wt IM alone (arrows) in spt host tailbud region expresses dra.

Wt Trunk PM Is Not Sufficient to Rescue RBC Formation in spt Embryos

As we have shown, in spt trunk IM there is a late appearance of limited scl, lmo2, hhex, and gata2 expression (Figure 1). Although we were unable to detect circulating RBCs from spt IM cells in wt host embryos (Figure 3), the possibility remains that wt PM might induce spt IM cells to express RBC markers later than 8s. We therefore tested the ability of wt PM to rescue expression of αe1globin in spt IM at 24 hpf. Wt host embryos containing wt donor-derived PM showed normal αe1globin expression (n = 5; Figures 6A and 6B), indicating no disruption by the pregastrula transplantation. In spt host embryos, wt PM was not sufficient to rescue αe1globin expression in spt host IM (n = 12 embryos; Figures 6C and 6D), nor were wt donor cells in other nonhematopoietic regions (data not shown). Wt cells in the spt host hematopoietic region expressed αe1globin (n = 4 embryos; Figures 6C and 6D), but, consistent with our earlier results, wt PM was in the vicinity of these cells. Dependence of expression upon PM is unclear, however, as the spatial relationship between the αe1globin-positive cells and wt donor-derived PM may not reflect the arrangement at the onset of hematopoiesis, and differentiated spt trunk PM appears by 24 hpf (Kimmel et al., 1989). In conclusion, despite limited, late expression of hematopoietic genes, spt IM does not respond at stages examined here to an interaction with wt PM.

Figure 6. Wt Trunk PM Fails to Rescue RBC Production from spt Host.

Figure 6

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(A–D) Left are confocal sections of host trunk at PM level (para-sagittal sections, anterior to left). Right panels are sections through midline hematopoietic region (sagittal section) of left embryo. Transplanted wt cells in green, and αe1globin in red. (A and B) Control, wt cells incorporated into PM somites of 24 hpf wt host (asterisks in [A]). (C and D) 24 hpf spt host containing wt trunk somites (asterisks in [C]). αe1globin expression is not rescued in spt host cells, but is expressed in wt cells (arrowheads). Expression (C” and D”) restricted to wt cells (C’ and D’).

Discussion

spadetail (spt) exhibits a severe loss of both trunk PM and RBCs (Kimmel et al., 1990; Thompson et al., 1998). We demonstrate that RBC loss in spt mutants is the consequence of a novel 2-fold requirement for spt function (Figure 7): (1) spt function is required cell autonomously within RBCs and (2) spt function is also needed within PM to position it in the trunk, thereby creating the proper environment for RBC development in neighboring IM. Thus, the previously proven cell-autonomous requirement for spt in trunk PM during convergence (Ho and Kane, 1990) can now also be described as a non-cell-autonomous requirement for blood development.

Figure 7. Model of spt Cell-Autonomous Function and IM/PM Interaction during Zebrafish Embryonic RBC Production.

Figure 7

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PM (green) and IM (red) lineages develop closely throughout epiboly and are molecularly observed to be separate populations by Bud stage. spt functions cell autonomously in trunk PM for gastrulation movements. spt also functions cell autonomously in trunk hematopoietic IM for expression of dra at Bud and gata1 at 8 s, and for ultimate production of RBCs by 24 hpf. PM supplies an interaction (blue arrows) that is crucial to RBC development. PM must interact with IM at or prior to the onset of gata1 expression (solid blue arrow). This interaction may be required for expression of early hematopoietic markers, such as dra and scl, and formation of early blood progenitors (dashed blue arrows).

Interaction between IM and Trunk PM

In zebrafish, RBC-producing IM is closely associated with prospective PM from gastrulation onward. At Bud stage, a distinct division into apposed regions of IM and PM is observed using molecular markers (Figure 2). Transplanted wt cells that form IM in the normally PM-deficient trunk of a spt host do not express gata1 at 8s unless adjacent to wt trunk PM (Figure 4; Table 1), indicating that these two tissues must interact at some point prior to gata1 onset. Considering that gata1 expression is a key first step toward RBC lineage differentiation, this IM/PM interaction is essential to form embryonic RBCs (Figure 7).

Expression of the early hematopoietic marker dra is also dependent on PM in the trunk at 8s; however, we found dra expression in transplanted wt IM cells located in spt host tailbuds despite the absence of wt PM (Figure 5D). We offer two possible explanations for this occurrence. First, a requirement for PM might be supplied by spt cells that normally differentiate as PM in the tail (Kimmel et al., 1989), and/or by mislocated spt trunk PM cells. Second, posterior cells within an embryo are less developed; therefore dra-expressing cells in the tailbud may be progenitors undergoing early hematopoiesis independent of PM. Consistent with a less stringent requirement for PM, we find dra expression is easier to rescue than gata1 (63% events gata1, 94% events dra (IM+PM), Table 1).

Environmental Influences on Vertebrate Blood Development

Previous studies of zebrafish hematopoietic mutants support a role for environmental cues in embryonic blood development. bloodless (bls) embryos have a severe loss of embryonic gata1 expression; however, bls cells express gata1 in wt hosts at 24 hpf, indicating a non-cell-autonomous role for bls (Liao et al., 2002). In cloche mutants, loss of both blood and endothelial cells arises from a cell-autonomous defect; however, early RBC differentiation in cloche depends solely on an unidentified non-cell-autonomous contribution (Parker and Stainier, 1999). Parker and Stainier (1999) found that wt cells in a cloche host expressed gata1 only in association with a large group of nonexpressing donor cells, which they hypothesized to be supplying an endothelial-derived signal. In contrast, we found that rescue of gata1 and dra expression in transplanted wt cells in a spt trunk is always associated with PM, independent of IM clone size (Figures 4 and 5).

Visceral endoderm in the mouse and chick, and ectoderm in Xenopus, are sources of signals received by embryonic blood progenitors (Baron, 2003). Our results suggest that nonautonomy of embryonic blood formation may be conserved across vertebrates. However, the source and molecule fulfilling this nonautonomous requirement may vary. Unlike mouse and chick, endoderm is not required in the zebrafish, as casanova mutants, which fail to form endoderm, still make RBCs (Parker and Stainier, 1999). Ectodermal-derived signals in zebrafish hematopoiesis have yet to be determined, although PM may relay cues from ectoderm. Interestingly, IM/PM communication is thought to occur in chick and Xenopus during pronephric differentiation (Mauch et al., 2000; Seufert et al., 1999).

What might be the factor(s) from zebrafish PM? Based on the necessity for apposition of gata1-expressing IM cells and PM, such a factor is restricted to act at short range. In mouse, visceral endoderm signals appear to instruct blood and endothelial development within the underlying blood islands, and rely on activity of visceral endoderm-derived Indian hedgehog and Vascular endothelial growth factor (VEGF) signaling (Baron, 2003). Zebrafish VEGF is expressed in PM during segmentation stages (Liang et al., 1998), and putative VEGF receptors flk1 and flt1 are expressed in IM (Liao et al., 1997; Thompson et al., 1998), suggesting a potential for hematopoietic function similar to mouse. Indeed, overexpression of zebrafish VEGF mRNA isoforms induces scl and gata1 expression prematurely and ectopically (Liang et al., 2001). However, morpholino knock-down of vegfA (Nasevicius et al., 2000) or loss of the flk1 VEGF receptor (Habeck et al., 2002) does not compromise blood production. Hedgehog (Hh) signaling also seems not to be required for zebrafish hematopoiesis, since neither shh, ihh, or ehh ligands are expressed in PM (Currie and Ingham, 1996; Krauss et al., 1993), nor are patched1, patched2, or smoothened receptors expressed in IM (Chen et al., 2001; Lewis et al., 1999), and loss of Hh signaling has not been reported to cause blood defects (Chen et al., 2001). Thus, known homologs of genes involved in mouse hematopoietic induction do not seem to be obvious candidates for mediating zebrafish IM/PM communication.

During Xenopus gastrulation, BMP-4 is required for an interaction between ectoderm and underlying mesoderm to produce blood islands (Kikkawa et al., 2001). Walters et al. (2002) suggest that these ectodermal signals regulate lineage differentiation of previously specified blood progenitors; however, data from Walmsley et al. (2002) support an action earlier in hematopoiesis. Zebrafish BMP signaling mutants are severely dorsalized and fail to form RBCs along with other posterior structures such as pronephros (Schier, 2001), making an independent role for BMP signaling in RBC development unclear. Transplanted swirl/bmp2b and somitabun/smad5 mutant cells form RBCs in a wt host (Hild et al., 1999; Kishimoto et al., 1997; Nguyen et al., 1998), indicating BMP signaling through these components is not required within zebrafish RBCs. Likewise, a direct role for Wnts and FGFs, as reported during Xenopus and mammalian hematopoiesis (de Haan et al., 2003; Kumano and Smith, 2000; Reya et al., 2003), has not been described for zebrafish. Current evidence therefore does not support direct involvement of known hematopoietic inducers either during the IM/PM interaction or through spt cell-autonomous function.

spt Function in the IM

spt regulates expression of paraxial protocadherin, a cell-adhesion molecule implicated in PM convergence during early gastrulation (Yamamoto et al., 1998). Given this morphogenetic role, which might reflect a general mechanism for T-box genes in development (Ahn et al., 2002), one hypothesis is that spt is cell-autonomously involved in hematopoiesis though control of IM cell movements. Indeed, IM convergence defects during gastrulation may result in broadened fli1 and pax2.1 domains (Figures 2B and 2C). However, convergence defects in trunk mesoderm alone do not appear to be sufficient to curtail hematopoiesis (Marlow et al., 2004; A.C.O., unpublished data).

As spt is cell-autonomously required for early dra, and gata1 expression (Figure 3), it is possible that spt acts during events that lead to specification of blood precursors, such as the proposed hemangioblast and HSC (Figure 7). Prior to gastrulation, spt is expressed in presumptive mesoderm within the marginal zone (Kimmel et al., 1990); however, later expression in the trunk is found mainly in posterior PM (Griffin et al., 1998; Ruvinsky et al., 1998). When scl and gata1 are first expressed in IM, Spt protein is detected only within the most posterior trunk and tail mesoderm and does not mirror blood expression during segmentation stages (Amacher et al., 2002; L.A.R., unpublished data). Thus, timing of spt expression within IM supports the hypothesis that spt acts prior to expression of hematopoietic regulators such as scl, lmo2, hhex, gata2, and gata1. Indeed, expression of these genes may bypass a prior cell-autonomous requirement for spt, as overexpression of zebrafish hhex can ectopically induce flk1 and gata1 expression in non-mesodermal regions (Liao et al., 2000). The IM/PM interaction may act later than early blood genes given that while co-overexpression of scl and lmo2 induces ectopic fli1 in nonaxial mesoderm along the entire axis, gata1 expression can be induced only within IM (Gering et al., 2003). Gering et al. (2003) suggest that this limited gata1 induction results from a requirement for endogenous cofactors localized within IM. Our data suggest that this might additionally be due to a need for close proximity to trunk PM.

Comparison of key hematopoietic gene expression in wt and spt embryos (Figure 1) suggests that these genes normally exhibit two phases of expression. The first phase of scl, lmo2, gata2, and hhex expression may be blood specific and initiates in wt, but not spt, between stages 2s to 5s in IM posterior to somite 6. The second phase begins after 5s in anterior trunk IM at the level of somites 1 to 5; this “anterior expansion” has been previously described for scl, lmo2, and hhex (Davidson et al., 2003; Gering et al., 2003; Liao et al., 2000). In wt embryos, endothelial cells, but not blood, derive from the anterior expression domain (Lieschke et al., 2002). Strikingly, the appearance of scattered scl-, lmo2-, gata2-, and hhex-expressing IM cells in spt embryos at stages 5s to 7s, correlates with the onset of wt anterior expression. Previous reports have interpreted these scl-expressing cells in spt embryos as arrested HSCs (Oates et al., 1999), but our findings suggest that late expression of these genes in spt mutants reflects a normal progression of the endothelial program. In wt posterior IM, a late-occurring wave of endothelial-specific expression would normally be masked by gene expression associated with developing blood.

In summary, our data suggest an important role for the hematopoietic microenvironment in zebrafish embryonic RBC production. The observed spatial separation of embryonic RBC (trunk) and early macrophage blood lineages (head) in zebrafish is suggestive of a possible instructive role for trunk PM at the level of lineage choices made by HSCs (Figure 7). Future experiments apposing trunk PM and early macrophage blood progenitors will test the ability of trunk PM to instruct a RBC fate.

Experimental Procedures

Embryo Collection

Wild-type (wt) embryos from *AB and TL (Johnson and Zon, 1999) and commercial lines (Princeton, NJ; Chicago, IL). spadetail b104 (spt) homozygote embryos collected from heterozygote spawning and wt siblings used as staging references (Kimmel et al., 1995). Embryos cultured at 28.5°C in embryo medium (Westerfield, 2000), dechorionated manually, and fixed 2 days in 4% PFA in PBS at 4°C and stored in MeOH at −20°C.

Pregastrula Cell Transplantation

Donor embryos microinjected at 1–2 cell stages with 5% fixable fluorescein-conjugated dextran (40 kDa, Molecular Probes) in 0.2 M KCl. Cell transplantation previously described by Ho and Kane (1990). Donor cells (10–50) from high-sphere staged embryos were transplanted into the margin of similarly staged hosts (Kimmel et al., 1990). Hosts cultured in EM + 0.2% penicillin-streptomycin (Bio-Whittaker). spt homozygote donors identified at segmentation stages. Rates of host death and damage varied with clutch health 5%–20%.

In Situ Hybridization

FITC or DIG (Roche) labeled antisense riboprobes synthesized (Promega) for: gata1, gata2 (Detrich et al., 1995); jak2a (Oates et al., 1999); αe1globin, βe1globin (Brownlie et al., 2003); biklf/klf4 (Oates et al., 2001); scl (Liao et al., 1998); lmo2, fli1, flk1 (Thompson et al., 1998); hhex (Liao et al., 2000); dra (Herbomel et al., 1999); pax2.1 (Krauss et al., 1991); dhand (Angelo et al., 2000); myoD (Weinberg et al., 1996). Single and double in situ hybridization performed as described (Prince et al., 1998).

Cell Transplantation Analysis and Imaging

Fixed hosts screened for fluorescently labeled donor cells on a Leica MZFLIII fluorescent dissection microscope. Hosts containing donor-derived IM, or other tissues requiring close inspection, were deyolked, flat-mounted, and analyzed on a Zeiss Axiovert confocal (LSM510). Aided by knowledge of PM, IM, and LPM gene expression patterns, we determined donor-derived cell types using the following criteria: PM was identified by proximity to notochord, presence of epithelial boundaries, multi-cell-layer thickness, and compact mesenchymal cell shape; IM was identified by position relative to both PM and hematopoietic gene positive cells in the same host, and by a less compact distribution, larger cell size, more ventral position, and fewer cell layers compared to PM. A transplant “event” as recorded in Table 1 is defined as a contiguous region in which wt donor cells formed a specific tissue(s). One or two events for a particular tissue(s) usually found per host. Gene expression visualized fluorescently from Fast Red precipitate detected with rhodamine filters.

Acknowledgments

We thank M. Brand, F. Bucholz, C.P. Heisenberg, E. Lammert, I. Skromne, and D.G. Ahn for comments, and the Zon, Stainier, and Thisse labs for probes. We are appreciative of fish care provided by B. Bielang, H. Dow, and T. Roskoph. Research support provided by grants from the LICR to A.O., and NSF and NIH to R.K.H.

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