IntroductionHematopoiesis is maintained by a pool of hematopoietic stem cells (HSCs) defined by their capacity to self-renew and, hence, to maintain the pool and to differentiate into all mature progenies. These properties underlie their ability to reconstitute the hematopoietic system. 1 HSCs are identified by molecular markers 2 and are divided into long-term HSCs (LT-HSCs), short-term HSCs (STHSCs), and multilineage progenitors (MPPs). 3,4 As they differentiate, HSCs give rise to committed progenitors, such as common lymphoid progenitors (CLPs) or common myeloid progenitors (CMPs), which generate mature cells within the different hematopoietic lineages. 5 The action of HSCs to self-renew or to differentiate is a tightly controlled process linked to the induction or repression of some crucial genes. 6 Gene-targeting experiments have provided support to this hypothesis by identifying transcription factors of various families or their coregulators that are implicated in HSC biology. 7,8 The basic helix-loop-helix (bHLH) family of transcription factors regulates a wide range of developmental events. 1,9 Some bHLHs, such as MyoD and NeuroD, display restricted tissue expression and control the differentiation of particular cellular lineages. 10 They form transactivating dimers by heteromerization with ubiquitous bHLH proteins, termed E proteins, such as E2A, E12, and E47 genes. 11,12 Conversely, the latter proteins are repressed by other HLH proteins, which often precludes differentiation. 10,13,14 The tal/scl gene (hereafter referred to as scl) encodes a bHLH protein. 15,16 It was identified through its implication in T-cell acute lymphocytic leukemia (T-ALL). Knock-out (KO) experiments 17,18 show that scl is autonomously required for primitive and definitive hematopoiesis. 19,20 Moreover, its enforced expression enhances megakaryocytopoiesis and erythropoiesis. 21,22 The Lyl-1 gene encodes a bHLH protein closely related to scl. 23 Its transcriptional activation upon translocation is also associated with T-ALL. 24 SCL and LYL-1 bHLH regions show 82% of amino acid identity, 24 suggesting that these 2 proteins share at least some target genes and biologic functions. 25,26 However, LYL-1 and SCL diverge largely outside the bHLH region and display a distinct expression pattern in hematopoietic cells. 23,27 From the Insitut National de la The biologic functions of SCL prompted us to investigate those of LYL-1 in hematopoiesis. In contrast to scl Ϫ/Ϫ mice, Lyl-1 Ϫ/Ϫ mice do not exhibit embryonic lethality but have a reduced number of B cells. Although the CLP compartment is normal, the immature B-cell compartments are reduced in adult mice. In addition, we show that Lyl-1 is highly expressed in stem/progenitor cells, correlating with an important role in the control of the size and function of this cell compartment. Similarly, the number of multipotent progenitor S 12 colony-forming units (CFU-S 12 s) is reduced in Lyl-1 Ϫ/Ϫ animals. Overall, these defects are distinct from those revealed upon scl KO, suggesting that...

SUMMARY The blastema is a mass of progenitor cells that enables regeneration of amputated salamander limbs or fish fins. Methodology to label and track blastemal cell progeny has been deficient, restricting our understanding of appendage regeneration. Here, we created a system for clonal analysis and quantitative imaging of hundreds of blastemal cells and their respective progeny in living adult zebrafish undergoing fin regeneration. Amputation stimulates resident cells within a limited recruitment zone to reset proximodistal (PD) positional information and assemble the blastema. Within the newly formed blastema, the spatial coordinates of connective tissue progenitors are predictive of their ultimate contributions to regenerated skeletal structures, indicating early development of an approximate PD pre-pattern. Calcineurin regulates size recovery by controlling the average number of progeny divisions without disrupting this pre-pattern. Our longitudinal clonal analyses of regenerating zebrafish fins provide evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures.

LYL-1 is a basic Helix-Loop-Helix (bHLH) transcription factor closely related to TAL. Both LYL-1 and TAL were originally identified through their implication in T acute lymphocytic leukemia. Their bHLH domain seems functionally equivalent suggesting that these two proteins share some biological function. However LYL-1 and TAL diverge largely outside the bHLH region and display a distinct, yet overlapping, expression pattern in hematopoietic cells. The role of TAL on erythropoiesis remains controversial: it is required for proper erythroid and megakaryocytic differentiation, plays an important role in the proliferation of early erythroid progenitors (BFU-E) but appears dispensable for baseline and stress erythropoiesis. This minor erythroid defect in TAL-null mice suggests that another transcription factor may replace TAL during erythroid differentiation. Since LYL-1 is also expressed in erythroid cells, we assessed its role in erythropoiesis using knock-in mice. We show that mice deficient for LYL-1 have impaired erythropoiesis. Erythroid progenitor and erythroblast numbers were significantly increased in the spleen of LYL-1−/− mice while in bone marrow (BM) erythroblasts we observed a partial differentiation blockade and enhanced apoptosis associated with decreased Bcl-xL expression. More importantly, LYL-1−/− BM cells are severely impaired in their erythroid lineage competitive reconstituting abilities. Indeed, the reconstitution capacity of erythroid lineage with LYL-1−/− cells was drastically reduced of about 10-fold. Despite this reduced BM erythropoiesis, LYL-1−/− mice had stimulated erythropoiesis. Indeed, we found a significant rise in both BFU-E and CFU-E and erythroblasts cloning efficiencies in the spleen of LYL-1−/− mice. Thus, we wondered if a compensatory mechanism by TAL and GATA-1 was operating in LYL-1−/− mice. TAL and GATA-1 transcripts were more expressed in the mature erythroblast populations from the spleen of LYL-1−/− mice compared to control. As GATA-1 is necessary to activate stress erythropoiesis we investigated the role of LYL-1 in stress erythropoiesis by treating mice with phenylhydrazine (PHZ). LYL-1−/− mice were extremely sensitive to PHZ treatment with a rapid and profound drop in hematocrit followed by rapid recovery and associated with a significant rise in circulating reticulocytes and an increase of spleen CFU-E and BFU-E. Moreover, LYL-1−/− erythroid progenitors in BM and spleen displayed EPO hyper-responsiveness. In conclusion, our results definitely show modified erythropoiesis in LYL-1−/− mice that parallels the defects described in TAL−/− mice. Our results suggest that both transcription factors may have partially redundant functions on erythropoiesis, in contrast to their distinct function in HSCs that we previously described. Finally, double TAL−/− and LYL-1−/− KO mice may help to precisely understand the transcriptional regulation of erythropoiesis.