Robust hematopoietic progenitor cell commitment in the presence of a conflicting cue

Abstract

Hematopoietic lineage commitment is regulated by cytokines and master transcription factors, but it remains unclear how a progenitor cell chooses a lineage in the face of conflicting cues. Through transcript counting in megakaryocyte-erythroid progenitors undergoing erythropoiesis, we show that the expression levels of the pro-erythropoiesis transcription factor EKLF (also known as KLF1) and receptor EpoR are inversely correlated with their pro-megakaryopoiesis counterparts, FLI-1 and TpoR (also known as MPL). Notably, as progenitors commit to the erythrocyte lineage, EpoR is upregulated and TpoR is strongly downregulated, thus boosting the potency of the pro-erythropoiesis cue erythropoietin and effectively eliminating the activity of the pro-megakaryopoiesis cue thrombopoietin. Based on these findings, we propose a new model for exclusive decision making that explicitly incorporates signals from extrinsic cues, and we experimentally confirm a model prediction of temporal changes in transcript noise levels in committing progenitors. Our study suggests that lineage-specific receptor levels can modulate potencies of cues to achieve robust commitment decisions.

Keywords: Conflicting cue; Hematopoiesis; Lineage commitment.

Fig. 1.

Effect of Epo and Tpo treatment on erythrocyte differentiation. UT-7/GM cells were passaged into medium containing only Epo, only Tpo, or both. On days 3 and 6, part of the Epo culture was passaged separately into medium containing both cytokines. All cultures were assessed for erythrocyte differentiation by o-dianisidine staining on day 14. Results are mean±s.e.m. from duplicate experiments.

Fig. 2.

The observed proportion of erythrocytes is inconsistent with purely stochastic theory of lineage commitment. According to the stochastic theory of lineage commitment, Tpo affects proportions of committing erythrocytes only through modulation of growth and survival of committing megakaryocytes. Simulation of this model reveals that when Tpo is added to Epo-only medium on day 3, the observed proportion of o-dianisidine-stained cells on day 4 is substantially higher than expected under the stochastic theory.

Fig. 3.

Effect of Epo and Tpo treatment on EKLF and FLI-1 levels in individual cells. UT-7/GM cells were passaged into medium containing only Epo, only Tpo, or both. On days 3 and 6, part of the Epo culture was passaged separately into medium containing both cytokines. (A) Merged microscopy image of UT-7/GM cells treated with Epo and Tpo on day 12, created by flattening 3D stacks. EKLF (red) and FLI-1 (green) transcripts are shown in cell nuclei (blue). The image was enhanced to show contrast. (B) Median EKLF and FLI-1 transcripts per cell under Epo or Epo+Tpo treatment. Day 0 refers to the day when UT-7/GM cells were passaged into medium containing only Epo, or both Epo and Tpo. Error bars denote the boot-strapped 95% confidence interval for the median. (C) Correlations between EKLF and FLI-1 levels during treatment with Epo or Epo+Tpo.

Fig. 4.

Impact on EKLF and FLI-1 levels after introduction of Tpo to an Epo-induced culture. UT-7/GM cells were starved of growth factor and subsequently passaged into medium with only Epo or Epo+Tpo. On day 3, part of the culture in Epo-only medium was passaged into Epo+Tpo medium. On day 6, cells that were switched from Epo-only to Epo+Tpo medium exhibited significantly higher EKLF transcript expression [*median 33% higher, 95% confidence interval (11%, 59%)], but similar FLI-1 transcript expression [**median 3% higher, 95% confidence interval (−10%, 20%)] when compared to cells maintained in Epo+Tpo medium throughout. The box represents the 25th–75th percentiles, and the median is indicated. For each box, the interquartile range (IQR) is defined as the difference between the 75th and 25th percentile values; the top whisker is at 1.5× IQR above the 75th percentile, and the bottom whisker is at 1.5× IQR below the 25th percentile or zero, whichever is greater.

Fig. 5.

Expression of lineage-specific receptors during erythrocyte commitment. (A) Median transcripts per cell in UT-7/GM cells treated with Epo. Error bars denote the boot-strapped 95% confidence interval for the median. (B) Phase plots show correlation between EKLF and EpoR, and EKLF and TpoR transcripts during erythrocyte differentiation.

Fig. 6.

The ECAA model predicts noise profiles for transcription factor and receptor expression. (A) The CAA topology (in black) consists of two auto-regulating and mutually-inhibiting transcription factors (T_A and T_B). Extending this network to allow extrinsic cues (L_A and L_B) to indirectly promote cognate transcription factor synthesis and to allow transcription factors to inhibit extrinsic cue signals (in green) yields the full ECAA topology. (B) Proposed model for erythrocyte commitment includes mutual antagonism between EKLF and FLI-1, and TpoR downregulation in the erythrocyte lineage. In addition, and in accordance with our experimental findings, EpoR is explicitly upregulated by EKLF. (C,D) Simulated noise profiles for T_A and T_B during treatment with L_A. (E,F) Noise in EKLF and EpoR, and FLI-1 levels in UT-7/GM cells during treatment with Epo. Transcript counts for EKLF were obtained and used to compute the noise [s.d./arithmetic mean] for transcription factor and receptor mRNA levels.