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Comparative Study
. 2005 Apr 15;19(8):965-78.
doi: 10.1101/gad.1298305.

Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling

Tom N Taghon  1 , Elizabeth-Sharon David, Juan Carlos Zúñiga-Pflücker, Ellen V Rothenberg
Affiliations
Comparative Study

Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling

Tom N Taghon et al. Genes Dev. .

Abstract

Using the OP9-DL1 system to deliver temporally controlled Notch/Delta signaling, we show that pluripotent hematolymphoid progenitors undergo T-lineage specification and B-lineage inhibition in response to Notch signaling in a delayed and asynchronous way. Highly enriched progenitors from fetal liver require > or =3 d to begin B- or T-lineage differentiation. Clonal switch-culture analysis shows that progeny of some single cells can still generate both B- and T-lineage cells, after 1 wk of continuous delivery or deprivation of Notch/Delta signaling. Notch signaling induces T-cell genes and represses B-cell genes, but kinetics of activation of lineage-specific transcription factors are significantly delayed after induction of Notch target genes and can be temporally uncoupled from the Notch response. In the cells that initiate T-cell differentiation and gene expression most slowly in response to Notch/Delta signaling, Notch target genes are induced to the same level as in the cells that respond most rapidly. Early lineage-specific gene expression is also rapidly reversible in switch cultures. Thus, while necessary to induce and sustain T-cell development, Notch/Delta signaling is not sufficient for T-lineage specification and commitment, but instead can be permissive for the maintenance and proliferation of uncommitted progenitors that are omitted in binary-choice models.

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Figures

Figure 1.
Figure 1.
Kinetics of early T- and B-lineage differentiation from multipotent precursors. (A) c-Kit+Lin- fetal liver cells were cocultured on OP9-control and OP9-DL1 stromal cells and analyzed by FACS at various time points as indicated. All dot plots shown are gated on CD45+ hematopoietic cells and representative for three independent experiments. Numbers in quadrants indicate the percentage of each corresponding population. (B) Schematic overview of the initial different phenotypical changes in developing B and T cells, derived from A.
Figure 2.
Figure 2.
Absolute cell numbers of early B- and T-lineage developmental intermediates from multipotent precursors. The absolute number of cells for each indicated cell population at each time point were calculated based on the total number of cells in the culture and their percentage in the total cell population. Data shown is derived from cultures shown in Figure 1 and are representative of three independent experiments.
Figure 3.
Figure 3.
Time course of gene-expression changes during initial B- and T-lineage specification. Quantitative real-time RT-PCR gene expression analysis of c-Kit+Lin- cells, cocultured for 1-6 d on OP9-control and OP9-DL1 stromal cells as indicated. Samples shown are derived from cultures shown in Figure 1 and are representative of three independent experiments. Units of expression are given relative to levels in the purified starting population.
Figure 4.
Figure 4.
Delayed establishment of T- and B-cell commitment relative to differentiation. (A) Experimental design; c-Kit+Lin- cells were cultured in parallel on OP9-control or OP9-DL1 cells for 1-6 d. At these two time points, both OP9-control and OP9-DL1 cultured were disrupted and replated, half of their progeny onto fresh OP9-control and half onto fresh OP9-DL1 stromal cells. (B) FACS analysis of cultures disrupted and replated after 1 d of preculture, analyzed after an additional 6 d of culture. (C) FACS analysis of cultures disrupted and replated after 6 d of preculture, analyzed after an additional 6 d of culture.
Figure 5.
Figure 5.
Asynchronous lineage commitment in progeny of single, differentiating precursor cells. FACS analysis of single c-Kit+CD27+Lin- FL cells that were initially precultured onto OP9-control or OP9-DL1 stromal cells for 7 d. After 7 d, the progeny of each of the outgrowing cells was replated, half onto OP-control and half onto OP9-DL1 stromal cells to determine the frequency of lineage-committed clones. One representative example is shown for lineage commitment (A) and no lineage commitment (B) after OP9-DL1 preculture and for lineage commitment (C) and no lineage commitment (D) after OP9-control preculture.
Figure 6.
Figure 6.
Reversibility of initial lineage-specific gene expression changes in c-Kit+ CD27+ progenitors. (A) Experimental design; c-Kit+Lin- cells were cultured in parallel on OP9-control or OP9-DL1 cells for 4 d. At day 4, c-Kit+CD27+ cells were sorted from both cultures and both OP9-control and OP9-DL1 derived c-Kit+CD27+ cells were replated onto both OP9-control and OP9-DL1 stromal cells as indicated. (B) Quantitative real-time gene expression analysis of sorted c-Kit+CD27+ cells after 4 d of culture on OP9-control or OP9-DL1. (C) Quantitative real-time gene expression analysis of sorted 4-d precultured c-Kit+CD27+ cells that were replated onto fresh OP9-control and OP9-DL1 stromal cells with samples taken after 0, 2, 4, and 6 d of additional culture. Units of expression for each gene are relative to their levels in the c-Kit+CD27+ cells from the appropriate preculture, as shown in B.
Figure 7.
Figure 7.
Different T-cell developmental kinetics of hematolymphoid precursors are not a result of differences in Notch signaling. (A) Rationale of the experiment: Are the varied and asynchronous responses of individual precursor cells occurring in the presence of equal or differential Notch signaling? (B) Quantitative real-time gene expression analysis of CD27+ c-kit+ CD25- and CD27+ CD25+ sorted cells after 4 d of OP9-DL1 culture (left two graphs) and of CD27+ c-kit+ CD19- and CD19+ cells after 4 d of OP9-control culture (right two graphs). Units of expression are given relative to levels in the purified starting population. Note the change of scale for pTα and CD3ε. (C) T-cell developmental capacities of cells used for gene expression analysis in B. Cells were analyzed by FACS after OP9-DL1 coculture at various time points as indicated. All dot plots shown are gated on CD45+ hematopoietic cells and representative of two independent experiments.
Figure 8.
Figure 8.
Models of the responses of individual hematolymphoid precursors to Notch/Delta signaling. Effect of culture on OP9-DL1 or OP9-control for precursors with ability to delay T/B-lineage commitment. (Left) Fast-responding cells behave as bipotent precursors described by the standard instructive model for Notch/Delta signaling: deterministic model of Notch actions in T-cell development. In this model, the activation of the T-cell alternative automatically excludes the B-cell alternative. (Right) This model does not explain the persistence under polarizing conditions of cells with the capacity for both, as seen in the slow responding cells. In this model, different precursors can respond asynchronously to Notch/Delta signaling or its absence. Cells remaining “multipotent” in this model, as T- or B-cell development is first being induced in other cells, are capable of making the transit to T- or B-cell differentiation at a later time, as permitted by culture conditions, as shown in Figure 5B and D.
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