Lyl-1 regulates primitive macrophages and microglia development.

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/"early EMP" and transient-definitive/"late EMP" waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis.

Live Monitoring of Blastemal Cell Contributions during Appendage Regeneration.

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.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs.

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.

Fgf signaling controls pharyngeal taste bud formation through miR-200 and Delta-Notch activity.

Taste buds, the taste sensory organs, are conserved in vertebrates and composed of distinct cell types, including taste receptor, basal/presynaptic and support cells. Here, we characterize zebrafish taste bud development and show that compromised Fgf signaling in the larva results in taste bud reduction and disorganization. We determine that Fgf activity is required within pharyngeal endoderm for formation of Calb2b(+) cells and reveal miR-200 and Delta-Notch signaling as key factors in this process. miR-200 knock down shows that miR-200 activity is required for taste bud formation and in particular for Calb2b(+) cell formation. Compromised delta activity in mib(-/-) dramatically reduces the number of Calb2b(+) cells and increases the number of 5HT(+) cells. Conversely, larvae with increased Notch activity and ascl1a(-/-) mutants are devoid of 5HT(+) cells, but have maintained and increased Calb2b(+) cells, respectively. These results show that Delta-Notch signaling is required for intact taste bud organ formation. Consistent with this, Notch activity restores Calb2b(+) cell formation in pharyngeal endoderm with compromised Fgf signaling, but fails to restore the formation of these cells after miR-200 knock down. Altogether, this study provides genetic evidence that supports a novel model where Fgf regulates Delta-Notch signaling, and subsequently miR-200 activity, in order to promote taste bud cell type differentiation.

LYL-1 deficiency induces a stress erythropoiesis.

OBJECTIVE: LYL-1 is a transcription factor containing a basic helix-loop-helix motif closely related to SCL/TAL-1, a regulator of erythroid differentiation. Because LYL-1 is expressed in erythroid cell populations, we addressed its role in erythropoiesis using knockin mice. MATERIALS AND METHODS: Erythropoiesis of LYL-1(-/-) mice was studied by progenitor assays, flow cytometry, reconstitution assays, and functional tests. Expression of LYL-1, SCL, and GATA-1 was assessed at messenger RNA level by quantitative reverse transcription polymerase chain reaction. RESULTS: LYL-1(-/-) mice displayed decreased erythropoiesis with a partial arrest in differentiation, and enhanced apoptosis associated with decreased Bcl-x(L) expression in the bone marrow (BM). In addition, LYL-1(-/-) BM cells were severely impaired in their abilities to reconstitute the erythroid lineage in competitive assays, suggesting a cell autonomous abnormality of erythropoiesis. In parallel, erythroid progenitor and precursor cells were significantly increased in the spleen of LYL-1(-/-) mice. Expression of LYL-1 was differentially regulated during maturation of erythroblasts and strikingly different between spleen- and BM-derived erythroblasts. Expression of LYL-1 decreased during erythroid differentiation in the spleen whereas it increased in the BM to reach the same level in mature erythroblasts as in the soleen. Loss of Lyl-1 expression was accompanied with an increase of SCL/TAL-1 and GATA-1 transcripts in spleen but not in BM-derived erythroblasts. Furthermore, phenylhydrazine-induced stress erythropoiesis was elevated in LYL-1(-/-) mice and mutant BM and spleen erythroid progenitors were hypersensitive to erythropoietin. CONCLUSIONS: Taken together, these results suggest that LYL-1 plays a definite role in erythropoiesis, albeit with different effects in BM specifically regulating basal erythropoiesis, and spleen, controlling stress-induced erythropoiesis.

lyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny.

The TAL-1/SCL and LYL-1 genes encode two closely related basic helix-loop-helix transcription factors involved in child T-acute lymphoblastic leukemia through chromosomal rearrangements and transcriptional deregulation. During ontogeny, Tal-1/SCL is required for hematopoietic cell generation, both in the yolk sac, where erythro-myeloid cells are first produced, then in the intra-embryonic compartment, where hematopoietic stem cells independently arise. We describe here the expression pattern of lyl-1 in mouse embryos from 7 to 14 days post coitus using in situ hybridization, as well as beta-Galactosidase (beta-Gal) expression in lyl-1-lacZ knock-in embryos, which express a C-terminally truncated Lyl-1 protein fused to the beta-Galactosidase (Lyl-1Delta/beta-Gal). In addition, we compare lyl-1 expression pattern with that of tal-1/scl. Similar to Tal-1/SCL, Lyl-1 mRNA expression occurs in the developing cardiovascular and hematopoietic systems. However, contrary to tal-1/scl, lyl-1 is not expressed in the developing nervous system. In lyl-1-lacZ knock-in heterozygous and homozygous embryos, beta-Gal expression completely correlates with Lyl-1 mRNA expression in the intra-embryonic compartment and is present: (1) in the developing hematopoietic system, precisely where hematopoietic stem cells emerge, and thereafter in the fetal liver; (2) in the developing vascular system; and (3) in the endocardium. In contrast, whereas Lyl-1 mRNA is expressed in yolk sac-derived endothelial and hematopoietic cells, Lyl-1Delta/beta-Gal is either absent or poorly expressed in these cell types, thus differing from Tal-1/SCL, which is highly expressed there at both mRNA and protein levels.

The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell function and B-cell differentiation.

Hematopoietic stem cells (HSCs) arise, self-renew, or give rise to all hematopoietic lineages through the effects of transcription factors activated by signaling cascades. Lyl-1 encodes a transcription factor containing a basic helix-hoop-helix (bHLH) motif closely related to scl/tal, which controls numerous decisions in embryonic and adult hematopoiesis. We report here that Lyl-1 null mice are viable and display normal blood cell counts, except for a reduced number of B cells resulting from a partial block after the pro-B stage. Nevertheless, the deletion of Lyl-1 results in a diminution in the frequency of immature progenitors (Lin(-), CD34(-), sca-1(+), c-kit(+) [LSK], and LSK-side population [LSK-SP]) and in S(12) colony-forming unit (CFU-S(12)) and long-term culture-initiating cell (LTC-IC) content in embryonic day 14 fetal liver (E14 FL) and adult bone marrow (BM). More important, Lyl-1(-/-) E14 FL cells and BM are severely impaired in their competitive reconstituting abilities, especially with respect to B and T lineage reconstitution. Thus, ablation of Lyl-1 quantitatively and functionally affects HSCs, a cell population that transcribes Lyl-1 more actively than their differentiated progenies. Our results demonstrate for the first time that Lyl-1 functions are important for HSC properties and B-cell differentiation and that they are largely distinct from scl functions.