Chromatin programming by developmentally regulated transcription factors: lessons from the study of haematopoietic stem cell specification and differentiation

Abstract Haematopoiesis has long served as a paradigm for adult stem cell systems and studies over the last 20 years have established that transcriptional control is central to the specification and subsequent differentiation of haematopoietic stem cells (HSCs). With many of the key transcription factors known, haematopoiesis provides a powerful cellular system for the analysis of mammalian gene regulatory networks. The key missing ingredient, particularly for the stem and progenitor cell stages, is a set of experimentally validated gene regulatory regions together with a molecular understanding of their biological activity. Despite progress in lower organisms, genome-wide computational identification of mammalian cis-regulatory sequences has been hindered by increased genomic complexity and cumbersome transgenic assays. In order to identify transcriptional control regions active during early embryonic haematopoietic development, we have developed a strategy that is based upon a haematopoietic stem cell enhancer (+19 enhancer) identified downstream of the SCL transcription factor, a key regulator of haematopoietic stem cell formation. Starting with this well-characterised blood stem cell enhancer, we have developed computational tools which allow genome-wide identification of functionally related enhancers. This approach has been used to identify novel enhancers involved in the regulation of early blood and endothelial development, and which exhibit predicted biological activity in vitro and in transgenic mice. Transcription factors binding to these enhancers have been identified by chromatin immunoprecipitation. Our data allow the construction of an experimentally verified nascent transcriptional network, which controls the development of blood and endothelium, and which so far contains the SCL, GATA-2, Fli-1, Elf-1 and Hex transcription factors. Additional candidate members of the network have been identified and are being subjected to functional validation. This approach represents a widely applicable strategy for characterising the transcriptional networks controlling a broad range of mammalian developmental programmes.

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VEGFA Controls Haematopoietic Stem Cell Specification In a Dose-Dependent, Isoform-Specific Manner

Blood ◽

10.1182/blood.v116.21.406.406 ◽

2010 ◽

Vol 116 (21) ◽

pp. 406-406

Author(s):

Amy Leung ◽

Aldo Ciau-Uitz ◽

Paresh Vyas ◽

Roger Patient ◽

Catherine Porcher

Keyword(s):

Stem Cell ◽

Conflicts Of Interest ◽

Biological Properties ◽

Haematopoietic Stem Cell ◽

Lateral Plate ◽

Cell Specification ◽

Knock Down ◽

Haemopoietic Stem Cell ◽

Exact Function ◽

Dose Dependent

Abstract Abstract 406 VEGFA signalling is critical for endothelial and haematopoietic development during embryogenesis. Perturbation of the VEGFA pathway results in defective haemopoietic stem cell (HSC) specification and vascular/arterial formation. Because of the intimate relationship between arterial and blood development, the contribution of VEGFA to HSC specification is unclear. Characterising the exact function of VEGFA is of primary interest for understanding mechanisms regulating haematopoietic specification. Here, we show that morpholino-induced knock-down of the co-repressor and oncoprotein ETO2 in Xenopus embryos leads to a failure of HSC specification via a VEGFA–dependent mechanism, independently from dorsal aorta (DA) formation and arterial specification. Knock-down of ETO2 represses Vegfa levels in somitic tissues along the migratory pathway from the dorsal lateral plate to the midline. This, in turn, leads to absence of Notch1 expression in the hemogenic endothelim of the DA and failure to activate the HSC transcriptional programme. DA formation and arterial specification occur normally, thus uncoupling VEGFA requirements for DA versus HSC formation. Then, we examined expression of the 3 VEGFA isoforms that differ in their physical and biological properties. In Eto2 morphant embryos, there was reduction (∼50%) in mRNA expression of the medium and long Vegfa variants in the somites. Importantly, Vegfa hypomorph embryos phenocopy the Eto2 morphant phenotype; this further demonstrates that reduction in VEGFA levels is sufficient for failure of HSC specification. Furthermore, restoration of medium Vegfa isoform (Vegfa170) levels alone in either Eto2 morphants or Vegfa hypomorph embryos was sufficient to re-establish the HSC programme. In summary, this is the first report of a dose-dependent, isoform-specific function of VEGFA at the onset of the HSC programme, independent from its functions in vessel formation and specification of the DA. We propose that signalling from VEGFA170 in somites instructs the HSC programme of the DA/HSC progenitors as they migrate towards the midline, in a paracrine manner. This VEGFA170 signal is critically required later in the haemogenic endothelium of the newly formed DA to initiate Notch1 expression and trigger the haematopoietic transcriptional programme. This work also reveals for the first time that ETO2 controls the onset of the HSC programme in a non cell-autonomous manner. This work furthers our understanding of the complex interplay between the cell populations controlling key signaling events leading to HSC specification. Manipulation of these signaling cascades may allow us to better develop strategies to produce and expand HSCs for therapeutic and regenerative purposes. Disclosures: No relevant conflicts of interest to declare.

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