Discipline : Biology

Doctoral School

Gènes-Génomes-Cellules

Thesis subject title:Functions of microRNA during developmental hematopoiesis

  • Laboratory name and web site: INSERM U1009; Institut Gustave Roussy-PR1; 114 rue Edouard Vaillant; 94805 VILLEJUIF Cedex. FRANCE
  • PhD supervisor (contact person):

Name: GODIN Isabelle

Position: Research Director/CNRS; Team leader

email:

Phone number: (33) 142-11-41-43

  • Thesis proposal (max 1500 words):

The function of RNA interference is beginning to be unravelled in the ES cell model at early stages of mesoderm determination, as well as during adult hematopoiesis, in both Hematopoietic Stem Cells (HSC) and committed blood lineages (reviewed in1). In contrast, there is currently no information on the involvement of RNA interference at stages when the hematopoietic system is established.

During ontogeny, blood cells are sequentially produced in various sites, first in the yolk sac (YS), then in the fetal liver and thymus, and finally in the bone marrow. Except in the YS, where the generation and differentiation of hematopoietic progenitors occurs in situ, the other sites only provide the environment suitable for the differentiation and/or maintenance of hematopoietic progenitors, so that hematopoiesis in these sites thus depends on the initial colonization by extrinsic progenitors.The source of colonizing cells has been attributed to the YS until the discovery that its contribution to embryonic hematopoiesis is transient (Review:2). YS progenitors, which appear from embryonic day (E)7.5, display a differentiation potential restricted to the erythro-myeloid lineages, as well as a limited maintenance potential. A second, and independent, wave of mesoderm determination towards the hematopoietic fate occurs, from E8-8.5, in the caudal intra-embryonic mesoderm, in a site called Splanchnopleura (Sp)(3-4). This process leads to the generation of HSC (multipotent and capable of multilineage long term reconstitution activity in irradiated recipients) within the Sp-derived site, called Para-aortic Splanchnopleura (P-Sp: E9-E10.5), then Aorta, Gonads and Mesonephros (AGM: E10.5-11.5). It is nowadays accepted that HSC generated in the P-Sp/AGM are responsible for the colonization of hematopoietic rudiments and the development of lifelong hematopoiesis (reviewed in5).

One of the still unsolved major questions in the field concerns the regulation of the potential of YS and P-Sp/AGM progenitors: how do the differences of potential displayed by the progenitors from the two sites arise? Do they acquire their hematopoietic identity in a different way? What roles do their respective environment play in the regulationof the differentiation and maintenance potential?

The dynamic regulation of transcription factors is essential for the specification, the proliferation and the maturation of hematopoietic progenitors during ontogeny as well as in the adult. We therefore hypothesized that RNA interference may also contribute to the regulation of developmental hematopoiesis. RNA interference modulates gene activity through non-coding RNA-mediated degradation of target mRNAs or inhibition of their translation into protein, thereby achieving finely tuned control of protein levels (6). A key factor in microRNAs production is the enzyme Dicer, which is responsible for the maturation of pre-microRNAs into mature microRNAs. Dicer invalidation is embryonic lethal at E7 (7), so that the function of RNA interference during the early steps of embryogenesis is currently investigated using either the ES model or conditional deletion of Dicer under the control of tissue specific promoters, an approach that proved successful to analyze RNA interference regulation of various developing systems (skeletal muscle, nervous system, etc).

To compensate for the early lethality of dicer knock out embryos and analyze the function of interfering RNA during vascular development, Stéphane Germain's group (Collège de France; INSERM U1050) developed a Dicer deficient mouse model by crossing Dicerflox/flox mice (8) with mice expressing the CRE recombinase under the control of the tie2 promoter. The tyrosine kinase receptor Tie2 is expressed by vascular cells, but also by extra-embryonic (YS) and intra-embryonic (P-SP/AGM, fetal liver, etc) hematopoietic progenitors during the whole process of hematopoietic development. We thus established a collaboration with S. Germain's group to analyze the regulation of developmental hematopoiesis by RNA interference, using these Dicerflox/flox:tie2-cre mice. The first step of this study provides evidence that RNA interference contributes to the regulation of both developmental hematopoiesis and lymphangiogenesis. Indeed, mutant embryos, which die at E13, have a pale and hypocellumar fetal liver. They also display misconnected blood and lymphatic vessels leading to blood-filled lymphatics, a process in which hematopoietic cells are involved. Hematopoietic development in dicerflox/flox:tie2-cre embryos is collapsed, with a particularly drastic effect on the production of megakaryocyte and of immature erythro-myeloid progenitors in the yolk sac. The process of HSC generation in the P-Sp/AGM is nearly abolished, leading to highly deficient fetal liver hematopoiesis.

The thesis project aims to identify the miRNAs responsible for the defects in hematopoietic ontogeny and to precise the function of each identified microRNA in the regulation of the various steps of developmental hematopoiesis. We are currently performing a compared miRNA qPCR expression profiling, in wild type and mutant samples.

To determine the function of each deregulated miRNAs during developmental hematopoiesis, we will first validate the miRNAs identified in our screen by establishing its expression pattern using Exiqon’s miRCURY LNA™ microRNA Detection Probes for in situ hybridization (See Knowing when and in which cell typea given microRNAis expressed will provide some information on the process it might regulate, leading to the selection of best candidates for the functional assays.

We will further check which of the selected candidates mimics the defects observed in mutant embryos, using LNA antiMIR antisens technology. We will knockdown specific candidate, selected as differentially expressed in wild type and mutant YS and/or P-SP/AGM and showing a clear signal in thesesites. We will transduce wild type YS and/or P-SP/AGM with the corresponding LNA antiMIR antisens. We will subsequentlyimplement the various approaches used in the group (9) to characterize the modifications in thedevelopment of hematopoietic progenitors: -In vitro analyses (methyl-cellulose assay and limiting dilution assays and Flow cytometry) allow the quantification and characterization of the type of progenitors produced;- In vivo reconstitution experiments are used to assess the maintenance potential. In parallel, we will also perform rescue experiment involving the re-expression of candidate microRNA in mutant (and over-expression in wild type) hematopoietic YS and/or P-SP/AGM.

We expect that the information gathered in the course of this project will shed a new light on the network responsible for the fine-tuning developmental hematopoiesis.

References

  1. Garzon R, Croce CM. MicroRNAs in normal and malignant hematopoiesis. Curr Opin Hematol. 2008;15:352-358.
  2. Godin I, Cumano A. The hare and the tortoise: an embryonic haematopoietic race. Nat Rev Immunol. 2002;2:593-604.
  3. Cumano A, Dieterlen-Lievre F, Godin I. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell. 1996;86:907-916.
  4. Cumano A, Ferraz JC, Klaine M, Di Santo JP, Godin I. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution. Immunity. 2001;15:477-485.
  5. Cumano A, Godin I. Ontogeny of the Hematopoietic System. Annual Review of Immunology. 2007;25:745-785
  6. Du T, Zamore PD. microPrimer: the biogenesis and function of microRNA. Development 2005;132:4645-4652.
  7. Bernstein E, Kim SY, Carmell MA, et al. Dicer is essential for mouse development. Nat Genet. 2003;35:215-217.
  8. Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc Natl Acad Sci U S A. 2005;102:10898-10903.
  9. Bertrand JY, Giroux S, Cumano A, Godin IHematopoietic stem cell development during mouse embryogenesis. "Developmental hematopoiesis: Methods and protocols" In Methods in Molecular Medicine. Humana Press 105; M. Baron eds (2005) : 273-288
  • Publications of the laboratory in the field (max 5):
  1. Luc S, Luis TC, Boukarabila H, MacaulaIC, Buza-Vidas N, Bouriez-Jones T, Lutteropp M, Woll PS, Loughran SJ, Mead AJ, Hultquist A, Brown J, Mizukami T, Matsuoka S, Ferry H, Anderson K, Duarte S, Atkinson D, Soneji S, Domanski A, Farley A, Sanjuan-Pla A, Carella C, Patient R, de Bruijn M, Enver T, Nerlov C, Blackburn C, Godin I, Jacobsen SEWThe Earliest Thymic T Cell Progenitors Sustain B Cell and Myeloid Lineage Potentials. Nature Immunology (Sous presse)
  2. Kaushik A-L, Giroux SJD, Klaine M, JalilA, Lécluse Y, Godin I Gene transfer into mouse haemogenic sites, as a mean to functionally approach the control of mesoderm determination towards a haematopoietic fate. In " Electroporation and Sonoporation in Developmental Biology " Nakamura H ed, Springer Japan, 2009: pp201-217
  3. Cumano A, Godin IOntogeny of the hematopoietic system.Annual Review in Immunology, 2007; 25: 745-785
  4. Giroux S, Kaushik A-L, Capron C, A Jalil, Kelaidi C, Sablitzky F, Dumenil D, Albagli O, Godin Ilyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny.Mechanism of Development/ Gene Expression Pattern 2007; 7: 215–226
  5. Bertrand JY, Giroux S, Golub R, Klaine M, Jalil A, Boucontet L, Godin I, Cumano A Characterization of purified intra-embryonic HSC as a tool to define their site of origin. PNAS, 2005, 102 (1) : 134-139
  • Specific requirements to apply, if any:

Theoretical or practical experience in either developmental biology or hematology would be appreciated.