Charles Durand

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta–gonad–mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, β-NGF (a neurotrophic factor), MIP-1γ (a C–C chemokine family member) and Bmp4 (a TGF-β family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

The roles of BMP and IL-3 signaling pathways in the control of hematopoietic stem cells in the mouse embryo

During mouse ontogeny, the first adult-type hematopoietic stem cells (HSC) are autonomously generated at mid-gestation in the AGM (Aorta-Gonad-Mesonephros) region. Successively present in different anatomical sites where they will expand, HSCs will finally colonize the bone marrow (BM) where they will reside during the entire adult life. In the bone marrow, both HSC self-renewal and differentiation are controlled at cellular and molecular levels by interactions with the stromal microenvironment. So far, very little is known about the extracellular factors involved in the regulation of embryonic HSC emergence, survival and expansion. In the present review, we outline the BMP and IL-3 signaling pathways that are critical for the growth and potential of embryonic HSCs. We will also discuss how these pathways might be integrated with the ones of Notch and Mpl/thrombopoietin, also identified as important key regulators of AGM HSC activity.

Complexity of the T cell receptor Cβ isotypes in the Mexican axolotl: structure and diversity of the VDJCβ3 and VDJCβ4 chains

We have reported previously the presence of two T cell receptor β‐chain constant region (Cβ) isotypes in the Mexican axolotl. Specific Dβ and Jβ segments were present at the Vβ‐Cβ1 and Vβ‐Cβ2 junctions and nine Vβ families which associate with both isotypes were characterized. This report describes two new Cβ isotypes, Cβ3 and Cβ4. About 70 % of the amino acids in Cβ3 are identical to Cβ1 and Cβ2. A Dβ3 and a single Jβ3 were found at the Vβ‐Cβ3 junctions. The Dβ3 consensus core sequence (TACGTGGCTACGTGGG) differs to all the presently known Dβ and the CDR3β loops of the Vβ‐Cβ3 junctions (mean: 11.1 amino acids) contain a majority of aromatic, small hydrophobic and basic residues. The CDR3β loops of the other isotypes are shorter (mean: 8.5 amino acids), contain a majority of acidic residues and very few aromatic residues. The axolotl Cβ4 sequence has about 46 % similarity to Cβ1, Cβ2 and Cβ3. Dβ4 is identical to Dβ2 and six new Jβ segments are used at the Vβ‐Cβ4 junctions. Four new families of Vβ segments (Vβ10‐Vβ13) are preferentially associated to Cβ4. A strong selective pressure must operate in most vertebrates to preserve the structural stability of the extracellular part of the Cβ chain. The four axolotl Cβ seem to have evolved more freely, perhaps to favor the early emergence of a large diversity of T cell receptors in an amphibian species which is not fully immunocompetent before the 5th month of development.

RAG expression is restricted to the first year of life in the Mexican axolotl.

Abstract. The developmental expression of the RAG1 gene in the Mexican axolotl hematopoietic organs was studied. RAG1 mRNAs were first detected in trunk extracts from 6-week-old larvae, and in head and trunk extracts of 8- and 9-week-old larvae. RAG1 is expressed in the thymus at all stages of development, until its natural involution after 12 months of age. In contrast, although RAG1 transcripts were present in the spleen and liver of the young larvae, they were not detected in the liver after 4.5 months and in the spleen after 8 months. No RAG1 mRNA expression was observed in the spleens or livers of 24-month-old hyperimmunized axolotls. The developmental expression of the RAG2 protein was also analyzed in axolotl thymus, spleen, and liver extracts using specific anti-RAG2 antibodies. RAG2 was readily detected at 7 months, but not in hematopoietic organs of 12- and 24-month-old axolotls. The presence of RAG1 transcripts was limited to the sub-capsular area of the thymus lobes, as detected by in situ hybridization. Discrete clusters of labeled cells were observed in the spleen sections, and a relatively large number of labeled cells were located in the hepatic peripheral hematopoietic layer of 3-month-old axolotls. The first appearance of RAG1 gene products in the axolotl hematopoietic organs is thus well correlated with the first production of rearranged T-cell and B-cell receptor mRNAs, 40–60 days after fertilization.

STRUCTURE AND DEVELOPMENTAL EXPRESSION OF IKAROS IN THE MEXICAN AXOLOTL

Abstract TheIkaros family of transcription factors plays an essential role in hematopoiesis. We report here the structure of cDNA clones encoding two Ikaros isoforms, Ik1 and Ik2, in the Mexican axolotl. The Ik1 cDNA sequence is very similar to that of the rainbow trout, chicken, and mammalian Ik1 sequences. However, a 96 base pair region which encodes the first N-terminal zing finger (F1) is lacking from axolotl Ik1, both in clones from a cDNA library and clones isolated from direct polymerase chain reaction products. A region corresponding to exon 3 is completely absent from the axolotl Ik2 sequence and thus the Ik1 and Ik2 isoforms possess the same number of zinc finger motifs. The structure of these five CC-HH motifs is very well conserved in the axolotl, including the structural deviations from its amino acid consensus composition which are identical in all species analyzed to date. The axolotl Ik1 3′ untranslated region sequence is very long (2538 bp) and contains two UA-rich motifs known as instability determinants and which could play a role in mRNA translational efficiency. Ikaros transcripts are first detected in the ventral blood island of stage 36 embryos, about 24 h before the first heartbeats (late tailbud stage), and then in the major lymphopoietic organs of the developing larvae. In situ hybridization demonstrates that Ikaros transcripts are abundant at the periphery of the thymus lobes, in the presumptive site of early thymocyte differentiation.

Endoglin expression level discriminates long-term hematopoietic from short-term clonogenic progenitor cells in the aorta

CD105 is an auxiliary receptor for the transforming growth factor beta superfamily, highly expressed on proliferating endothelial cells and adult hematopoietic stem cells. Because CD105 mRNA expression was reported in the developing aortic region, we further characterized its expression profile in the aorta and examined the hematopoietic potential of CD105+ cells. Aortic endothelial cells, intra-aortic hematopoietic cell clusters and the purified cell fraction enriched in progenitor/hematopoietic stem cell activity expressed CD105. Aortic hematopoietic short-term clonogenic progenitors were highly enriched in the CD105intermediate population whereas more immature long-term progenitors/hematopoietic stem cells are contained within the CD105high population. This places CD105 on the short list of molecules discriminating short-term versus long-term progenitors in the aorta. Furthermore, decreasing transforming growth factor beta signaling increases the number of clonogenic progenitors. This suggests that CD105 expression level defines a hierarchy among aortic hematopoietic cells allowing purification of clonogenic versus more immature hematopoietic progenitors, and that the transforming growth factor beta pathway plays a critical role in this process.

Structure, diversity and expression of the TCRδ chains in the Mexican axolotl

Mammals and birds have two major populations of T cells, based on the molecular composition and biological properties of their antigen receptors (TCR). α β T cells recognize antigenic peptides linked to major histocompatibility complex (MHC) molecules, and γ δ T cells recognize native peptide or non‐peptide antigens independently of MHC. Very little is known about γ δ T cells in ectothermic vertebrates. We have cloned and characterized the TCRδ chains of an urodele amphibian, the Mexican axolotl (Ambystoma mexicanum). The Cδ domain is structurally similar to its mammalian homologues and the transmembrane domain is very well conserved. Four of the six Vα regions that can associate with Cα (Vα2, Vα3, Vα5 and Vα6) can also associate with Cδ, but no specific Vδ regions were found. This suggests that the axolotl TRD locus is nested within the TRA locus, as in mammals, and that this organization has beenpresent in all tetrapod vertebrates and in the common ancestor of Lissamphibians and mammals, for over 400 million years. Two Jδ regions were identified, but no Dδ segments were clearly recognized at the Vδ‐Jδ junctions. This results in shorter and less variable CDR3 loops than in other vertebrates and the size range of the Vδ‐Jδ junctions is similar to that of mammalian immunoglobulin light chains. Equivalent quantities of TRD mRNA were found in the lymphoid organs, and in the skin and the intestines of normal and thymectomized axolotls. The analysis of several Vα/δ6‐Cδ and Vβ7‐Cβ junctions showed that both the TCRδ and the TCRβ chains were limited in diversity in thymectomized axolotls.

Identification and expression of Helios, a member of the Ikaros family, in the Mexican axolotl: implications for the embryonic origin of lymphocyte progenitors

Transcription factors of the Ikaros gene family are critical for the differentiation of T and B lymphocytes from pluripotent hematopoietic stem cells. To study the first steps of lymphopoiesis in the Mexican axolotl, we have cloned the Helios ortholog in this urodele amphibian species. We demonstrated that the axolotl Helios contains a 144‐bp deletion at the 5′ end of the activation domain. Helios is expressed in both the thymus and spleen but not in the liver of the pre‐adult axolotl. During ontogeny, Helios transcripts are detected from neurula stage, before the apparition of the first Ikaros transcripts and the colonization of lymphoid tissues. Interestingly, Helios and Ikaros mRNA are found predominantly in the ventral blood islands of late tail‐bud embryos. These results suggest that in contrast to the Xenopus and amniote embryos where two sites of hematopoiesis have been characterized, the ventral blood islands could be the major site of hematopoiesis in the axolotl.

Developmental hematopoiesis - preface

Stem cell biology is one of the most rapidly developing and exciting fields in the Life Sciences. The biology of embryonic and adult stem cells is critically important for understanding development, tissue homeostasis, reprogramming and cancer. Stem cells are characterised by their capacity to self-renew and differentiate into one or several cell types and hold great promise for regenerative medicine. The purification and tissue localization of stem cells, as well as identification of intrinsic and extrinsic factors regulating their functions, are central topics in stem cell biology. Hematopoietic stem cells (HSCs) are one of the best studied models and represent a key paradigm for analysis of various stem cell types. The availability of antibodies recognizing surface markers has been instrumental for enrichment and localization of HSCs. In vivo and in vitro assays offer researchers the possibility to study hematopoietic stem and progenitor cells at the functional level. For several decades bone marrow and more recently cord blood HSCs have been used for treatment of patients with hematopoietic disorders such as leukemia.

The quest for hematopoietic stem cells in the embryo. An interview with Françoise Dieterlen-Lièvre

Françoise Dieterlen-Lièvre is probably the scientist who has most contributed to our basic knowledge of developmental hematopoiesis. She has dedicated her career to answering cutting edge questions on the origin of hematopoietic stem cells in the embryo. Her seminal contributions, widely recognized by the scientific community, have paved the way for generations of developmental hematologists questioning the origins of hematopoietic stem cells. After having demonstrated the intra-embryonic origin of hematopoietic stem cells, established the dual origin of the endothelial network in the embryo and revealed the hematopoietic function of the allantois in birds, she has switched to mammals and contributed to demonstrating that the aorta and allantois/placenta are new sites of hematopoietic production in the mouse embryo. The manifold insights generated by the pivotal work of Françoise Dieterlen-Lièvre have created multiple paradigm shifts which continue to challenge the field of developmental hematopoiesis.