Eva Welinder

A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate

It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.

Single-cell analysis of the common lymphoid progenitor compartment reveals functional and molecular heterogeneity

To investigate molecular events involved in the regulation of lymphoid lineage commitment, we crossed lambda5 reporter transgenic mice to Rag1-GFP knockin mice. This allowed us to subfractionate common lymphoid progenitors and pre-pro-B (fraction A) cells into lambda5(-)Rag1(low), lambda5(-)Rag1(high), and lambda5(+)Rag1(high) cells. Clonal in vitro differentiation analysis demonstrated that Rag1(low) cells gave rise to B/T and NK cells. Rag1(high) cells displayed reduced NK-cell potential with preserved capacity to generate B- and T-lineage cells, whereas the lambda5(+) cells were B-lineage restricted. Ebf1 and Pax5 expression was largely confined to the Rag1(high) populations. These cells also expressed a higher level of the surface protein LY6D, providing an additional tool for the analysis of early lymphoid development. These data suggest that the classic common lymphoid progenitor compartment composes a mixture of cells with relatively restricted lineage potentials, thus opening new possibilities to investigate early hematopoiesis.

IL-7 mediates Ebf-1-dependent lineage restriction in early lymphoid progenitors

Deficiencies in the IL-7 signaling pathway result in severe disruptions of lymphoid development in adult mice. To understand more about how IL-7 deficiency impacts early lymphoid development, we have investigated lineage restriction events within the common lymphoid progenitor (CLP) compartment in IL-7 knockout mice. This revealed that although IL-7 deficiency had a minor impact on the development of LY6D(-) multipotent CLPs, the formation of the lineage restricted LY6D(+) CLP population was dramatically reduced. This was reflected in a low-level transcription of B-lineage genes as well as in a loss of functional B-cell commitment. The few Ly6D(+) CLPs developed in the absence of IL-7 displayed increased lineage plasticity and low expression of Ebf-1. Absence of Ebf-1 could be linked to increased plasticity because even though Ly6D(+) cells develop in Ebf-1-deficient mice, these cells retain both natural killer and dendritic cell potential. This reveals that IL-7 is essential for normal development of Ly6D(+) CLPs and that Ebf-1 is crucial for lineage restriction in early lymphoid progenitors.

Positive intergenic feedback circuitry, involving EBF1 and FOXO1, orchestrates B-cell fate

Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D+ cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D+ progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D+ CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D+ CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.

The transcription factors E2A and HEB act in concert to induce the expression of FOXO1 in the common lymphoid progenitor

Recent studies have identified a number of transcriptional regulators, including E proteins, EBF1, FOXO1, and PAX5, that act together to orchestrate the B-cell fate. However, it still remains unclear as to how they are linked at the earliest stages of B-cell development. Here, we show that lymphocyte development in HEB-ablated mice exhibits a partial developmental arrest, whereas B-cell development in E2A+/−HEB−/− mice is completely blocked at the LY6D− common lymphoid progenitor stage. We show that the transcription signatures of E2A- and HEB-ablated common lymphoid progenitors significantly overlap. Notably, we found that Foxo1 expression was substantially reduced in the LY6D− HEB- and E2A-deficient cells. Finally, we show that E2A binds to enhancer elements across the FOXO1 locus to activate Foxo1 expression, linking E2A and FOXO1 directly in a common pathway. In summary, the data indicate that the earliest event in B-cell specification involves the induction of FOXO1 expression and requires the combined activities of E2A and HEB.

B-lymphocyte commitment: Identifying the point of no return.

Even though B-lymphocyte development is one of the best understood models for cell differentiation in the hematopoetic system, recent advances in cell sorting and functional genomics has increased this understanding further. This has suggested that already early lymphoid primed multipotent progenitor cells (LMPPs) express low levels of lymphoid restricted transcripts. The expression of these genes becomes more pronounced when cells enter the FLT-3/IL-7 receptor positive common lymphoid progenitor (CLP) stage. However, the expression of B-lineage specific genes is limited to a B-cell restricted Ly6D surface positive subpopulation of the CLP compartment. The gene expression patterns also reflect differences in lineage potential and while Ly6D negative FLT-3/IL-7 receptor positive cells represents true CLPs with an ability to generate B/T and NK cells, the Ly6D positive cells lack NK cell potential and display a reduced T-cell potential in vivo. These recent findings suggest that the CLP compartment is highly heterogenous and that the point of no return in B-cell development may occur already in B220(-)CD19(-) cells. These findings have allowed for a better understanding of the interplay between transcription factors like EBF-1, PAX-5 and E47, all known as crucial for normal B-cell development. In this review, we aim to provide a comprehensive overview of B-cell fate specification and commitment based on the recent advances in the understanding of molecular networks as well as functional properties of early progenitor populations.

Off-responses in the human D.C. registered electroretinogram.

Abstract The eyes of human volunteers were adapted to a certain light intensity (varied in different recordings) and then exposed to total darkness. At “off” a very fast d-wave appeared, followed by a slower positive wave with a maximum at 0.9–1.5 sec and then a slow negative change with a minimum at 4–6 sec. The amplitude of these changes were roughly proportional to the intensity of the adapting light. They were followed by a positive maximum at about 45 sec, corresponding to the fast EOG peak (and the SP deflexion previously shown by us in direct recordings).

Combined effects of DL-α-aminoadipic acid with sodium iodate, ethyl alcohol, or light stimulation on the ERG c-wave and on the standing potential of albino rabbit eyes

Albino rabbits were treated with intravitreal injections of DL-α-aminoadipic acid (α-AAA) into one eye (0.1 ml of a 0.15 M solution) and 0.1 ml of saline into the contralateral eye. Thirteen to fourteen hours later the DC electroretinogram (ERG) and/or the standing potential (SP) were recorded. (1) In eight of nine animals the c-wave amplitude of α-AAA injected eyes was increased compared with that of control eyes. Following intravenous injection of Sodium Iodate (40 mg/kg in 2% solution) the c-waves of both eyes were rapidly replaced by negative potentials. In 8 of 9 animals the amplitude reduction was more marked in α-AAA-treated eyes than in control eyes, but the final amplitude was higher in the former than in the latter. The SP was reduced with difference in curve form but not significantly in amplitude between the eyes. (2) In nine other rabbits iv.-injected ethyl alcohol (0.4 g/kg in 20% solution) provoked a transient increase of SP level and c-wave amplitude in control eyes and smaller but similar changes in α-AAA injected eyes. (3) In another five animals the SP was recorded following a step from darkness to continous light stimulation. The light peak was less pronounced in α-AAA treated eyes than in control eyes.

EARLY EFFECTS OF SODIUM IODATE ON THE DIRECTLY RECORDED STANDING POTENTIAL OF THE EYE AND ON THE c‐WAVE OF THE DC REGISTERED ELECTRORETINOGRAM IN ALBINO RABBITS

The early effects of intravenously administered sodium iodate (NaIO3) on the directly recorded standing potential (SP) of the eye and on the c‐ and b‐waves of the DC registered ERG were studied in 8 anaesthetized albino rabbits. In 5 of 6 animals obtaining 40 mg NaIO3/kg bwt. the SP decreased immediately following the injection, and had attained a level 3.5–4 mV below the original one after 1 h. The c‐wave declined rapidly and 6 min after the injection it was replaced by a large cornea‐negative potential. The b‐wave was relatively unchanged except in one animal. In 2 rabbits treated with 30 mg NaIO3/kg bwt. and in the 6th animal obtaining 40 mg NaIO3/kg bwt. an SP increase instead of a decrease was seen, and the c‐wave was more slowly (about 22 min after the injection) replaced by the cornea‐negative potential. The b‐wave was somewhat increased. These results demonstrate the dose‐related and interindividual variability in the SP reaction to NaIO3 and are in good agreement with the well‐known ultrastructural pigment epithelial injury and c‐wave changes produced by this substance.

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

Background: Transcription factor doses play important roles in normal and malignant B-lymphocyte development. Results: We show dose-dependent regulation of B-cell specification and expansion of committed progenitors. Conclusion: Transcription factor dose impacts several aspects of B-cell development. Significance: Knowing the effects of reduced transcription factor dose aids our understanding of the molecular events underlying leukemia and B-cell development. Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1+/− pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.