Martin Gering

Research

In my lab, we are interested in how blood cells form during vertebrate embryogenesis. Our main focus is on haematopoietic stem cells (HSCs), the cells that maintain our blood system throughout life. In mammals, they reside in the bone marrow (BM). HSCs are immature cells that are multipotent (i.e. can give rise to cells of all blood lineages) and that have retained the ability to self-renew (i.e. divide and give rise to daughter cells that are themselves HSCs again). These cells are of enormous clinical importance as they constitute the active component of BM transplants that are used to re-establish blood formation in patients who have lost their blood system due to bone marrow failure or therapeutic treatment for leukaemia or solid cancers. Because of their longevity, HSCs are the ideal target for gene therapy approaches to curing inherited blood disorders. Autologous (using the patient’s own BM) and allogeneic (using BM donated by an antigen-matched donor, often a close relative) BM transplants are used dependent on whether functional, disease-free HSCs can be obtained from the patient. Autologous BM transplants are preferred as allogeneic transplants bear the increased risks of graft rejection or graft-versus-host disease. In the future, we will hopefully be able to offer to patients currently receiving allogeneic transplants patient-specific HSCs that are derived from disease-free adult tissue. These adult cells would need to be reprogrammed into a naïve embryonic state and subsequently turned into HSCs that can be maintained in vitro. Although we know how to grow mature blood cells from embryonic stem cells, generation of transplantable HSCs from ES cells has proven difficult (without expressing transgenes from retroviral vectors in them), as has maintenance and expansion of isolated BM HSCs in vitro (Bordignon, 2006; Keller, 2005).

The vertebrate embryo forms HSCs and maintains them into adulthood. Using the zebrafish as a model system, we are examining the cellular origin and the molecular programming of forming HSCs in the embryo. As in all vertebrate embryos, haematopoieisis in zebrafish occurs in two waves, a transient primitive wave that gives rise to primitive red blood cells and some myeloid cells, and a second, definitive wave that forms all blood cell lineages, including the lymphoid lineages (T and B cells). HSCs form in this second wave of haematopoiesis. While in mammalian, amphibian and avian embryos, primitive and definitive haematopoiesis are spatially separated in the extraembryonic yolk sac (ventral blood island in frogs) and in the intraembryonic aorta-gonads-mesonephros (AGM) region, respectively (Durand and Dzierzak, 2005; Godin and Cumano, 2002), both waves occur in close proximity in the intermediate cell mass (ICM) of the zebrafish embryo (Fig. 1A). The ICM is a chord of cells located in the trunk midline between the notochord, the endoderm and the somites. that forms primitive red blood cells and the two major trunk vessels, the dorsal aorta (DA) and the posterior cardinal vein. The putative HSCs first appear just before the primitive red blood cells enter circulation at 24 hours post fertilisation (hpf). They are identified by the expression of the transcription factors Runx1 and c-Myb, and by their close association with the ventral wall of the DA (Fig. 1B, C and D (Gering and Patient, 2005)). We could demonstrate that runx1 is essential for definitive haematopoiesis as loss of Runx1 in runx1 morphants (embryos injected with a runx1 antisense morpholino that interferes with the normal splicing of the runx1 primary transcript (Fig. 1E)) causes loss of c-myb-expressing definitive progenitors (Gering and Patient, 2005; Kalev-Zylinska et al., 2002) and loss of T cells in the thymus (Fig. 1F;(Gering and Patient, 2005)). Since T cells are the first easily identifiable progeny of the HSC in vertebrate embryos, the loss of T cells in runx1 morphants suggests that the runx1-expressing cells in the ICM are HSCs or their precursors. Recent short-term labelling experiments performed by others (Jin et al., 2007; Murayama et al., 2006) show that cells ventral to the DA do indeed via the caudal tail mesenchyme (Fig. 1G) eventually seed the thymus as well as the kidney (Fig. 1H), the place of adult haematopoiesis in the fish. Whether these cells include cells that maintain haematopoiesis in the long term (LT-HSCs) is yet to be determined.

Fig. 1: Definitive haematopoiesis in the zebrafish embryo. (A) Before the red blood cells (stained in red for β-embryonic globin expression) enter circulation, definitive haematopoietic cells (stained in purple for runx1 expression) can be identified just dorsal to the primitive erythrocytes (arrows) in the intermediate cell mass. (B) As erythrocytes enter blood circulation runx1 expression marks the location of the definitive blood cells between the major blood vessels of the trunk, the dorsal aorta (DA) and the posterior cardinal vein (PCV). White arrows indicate the direction of the blood flow in the blood vessels. (C and D) Cross sections through the trunk of zebrafish embryos confirm expression of runx1 and c-myb in the cells between the two major trunk vessels. (E) RT-PCR confirms that a runx1 splice morpholino interferes with the splicing of the runx1 primary transcript. RT-PCR for ef1a is used as a loading control. (F) Loss of runx1 in the morphant causes a severe reduction in the number of rag1-positive thymocytes. (G) c-myb expressing cells in the ventral tail mesenchyme (arrow) mark a transient site of haematopoiesis. (H) By day 5, c-myb positive cells seed the thymus and the kidney. NT- neural tube, NC- notochord; All views, except (F) are lateral. The view in (F) is dorsal. In (A), (B), (E) and (F) anterior is to the left. Dorsal is top in (A), (B), (C), (D) and (E).

The cells of the ICM that participate in primitive and definitive haematopoiesis originate from the lateral trunk mesoderm (Gering et al., 1998; Gering et al., 2003; Zhong et al., 2001). Despite their similar origin in the post-gastrula embryo, primitive and definitive blood cells have very different signalling requirements. Studies on mutant zebrafish embryos and on zebrafish embryos treated with small molecule inhibitors that block defined signalling pathways have shown that definitive haematopoiesis requires active Hedgehog, vascular endothelial growth factor (Vegf) and Delta-Notch signalling, while primitive erythropoiesis is completely unaffected in embryos mutated or inhibited in any of these signalling pathways (Gering and Patient, 2005). Data by Len Zon’s lab confirm the Notch signalling requirement in definitive haematopoiesis and suggest an instructive role for Notch (Burns et al., 2005). Interestingly, the same three signalling pathways are also needed for arterial gene expression in the DA (Lawson et al., 2001; Lawson et al., 2002). Together with the finding that runx1-expressing cells are a ventral subpopulation of the DA angioblast chord these data suggest a common cellular origin of the DA endothelium and the HSCs. The molecular mechanism that governs HSC divergence from the DA angioblasts is unknown.

Fig. 2: Definitive, but not primitive, haematopoiesis requires active Hedgehog, Vegf and Notch signalling. Embryos that carry a mutation in smoothened (smo, B) or mindbomb (mib, F), and embryos that have been treated with a Vegf receptor inhibitor (D) lack runx1-expressing definitive blood cells. None of these mutant or inhibitor-treated embryos displays a defect in primitive erythropoiesis (A, C and E).

We are currently addressing the following issues:

• We are only beginning to understand the molecular mechanisms that govern the divergence of primitive and definitive haematopoiesis and the segregation of HSCs and DA endothelial cells. Using antisense morpholino oligos and small molecule inhibitors we are examining the roles of a number of genes and signalling pathways in these processes. Cell transplantations at blastula stage are used to reveal cell-autonomous and non-cell-autonomous requirements.

• Putative haematopoietic stem/precursor cells have been identified based on gene expression and location. Cells in this location have also been shown to seed the kidney, the adult site of haematopoiesis, but it has not been demonstrated that they are able to participate in long-term haematopoiesis. Using fluorescent reporter transgene expression in these cells, we want to isolate these cells by fluorescence-activated cell sorting, transplant them into wild-type embryos and follow them in the host to show that they are able to contribute to long-term haematopoiesis.

• In zebrafish, HSCs are maintained in the embryonic caudal tail mesenchyme and in the larval and adult kidney. Little is known about the interactions of the HSCs with their niches. Using differential gene expression detected either by fluorescent in situ hybridisation or revealed in transgenic zebrafish lines that express fluorescent protein reporter genes, we are trying to identify the cells that provide the stem cell niche in the embryo as a first step towards investigating the molecular mechanisms that control stem cell maintenance in the different niches.

• Genes are re-employed in different tissues and at different time points during embryonic development and adult life. We need to find ways to interfere with a gene’s role in zebrafish in a conditional manner. In collaboration with Dr William Brown’s lab, we are, therefore, developing new approaches to manipulate the zebrafish genome using bacteriophage-derived, site-specific recombinases.

Our work is supported by project grants from the MRC and the BBSRC.

References

Bordignon, C. (2006). Stem-cell therapies for blood diseases. Nature 441, 1100-1102.

Burns, C.E., Traver, D., Mayhall, E., Shepard, J.L., and Zon, L.I. (2005). Hematopoietic stem cell fate is established by the Notch-Runx pathway. Genes Dev 19, 2331-2342.

Durand, C., and Dzierzak, E. (2005). Embryonic beginnings of adult hematopoietic stem cells. Haematologica 90, 100-108.

Gering, M., and Patient, R. (2005). Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos. . Dev Cell 8, 389-400.

Gering, M., Rodaway, A.R.F., Gottgens, B., Patient, R.K., and Green, A.R. (1998). The SCL gene specifies haemangioblast development from early mesoderm. EMBO J 17, 4029-4045.

Gering, M., Yamada, Y., Rabbitts, T.H., and Patient, R.K. (2003). Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1. Development 130, 6187-6199.

Godin, I., and Cumano, A. (2002). The hare and the tortoise: an embryonic haematopoietic race. Nature Reviews 2, 593-604.

Jin, H., Xu, J., and Wen, Z.L. (2007). Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development. Blood 109, 5208-5214.

Kalev-Zylinska, M.L., Horsfield, J.A., Flores, M.V., Postlethwait, J.H., Vitas, M.R., Baas, A.M., Crosier, P.S., and Crosier, K.E. (2002). Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis. Development 129, 2015-2030.

Keller, G. (2005). Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19, 1129-1155.

Lawson, N.D., Scheer, N., Pham, V.N., Kim, C.H., Chitnis, A.B., Campos-Ortega, J.A., and Weinstein, B.M. (2001). Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development 128, 3675-3683.

Lawson, N.D., Vogel, A.M., and Weinstein, B.M. (2002). Sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 3, 127-136.

Murayama, E., Kissa, A., Zapata, A.G., Mordelet, E., Briolat, V., Lin, H.F., Handin, R.I., and Herbomel, P. (2006). Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development Immunity 25, 963-975.

Zhong, T.P., Childs, S., Leu, J.P., and Fishman, M.C. (2001). Gridlock signalling pathway fashions the first embryonic artery. Nature 414, 216-220.