Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor

Abstract

The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell-derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.

Figure 1.

Mouse Mix is activated early during ES cell differentiation and is conditionally expressed in i-Mix ES cell lines. (A) RT-PCR analysis of mMix expression in EBs (E14 line). Controls (-RT, -DNA) were routinely performed. NT indicates minus template control. (B) Strategy for conditional expression of mMix in ES cells. The vector plox-Mix1, encoding amino-terminal FLAG-tagged mMix, was inserted into the X chromosome using Cre-mediated recombination. In the presence of DOX, rtTA protein expressed from the ROSA26 locus on chromosome 6 (chr6) can bind to a tetracycline operator (tetOP), and expression of mMix is activated. (C) Western blot analysis of 1 of the 3 i-mMix ES cell lines (clone 2) cultured at different concentrations of DOX. Actin was used as a loading control. (D) Nuclear expression of FLAG-tagged mMix protein in cells from induced i-Mix EBs. Magnification, × 40.

Figure 2.

The mesodermal developmental program is accelerated in response to mMix. (A) General experimental protocol for induction and analysis of i-Mix EBs cultured with or without DOX. DOX (0.1 μg/mL) was added on day 1 after plating under differentiation conditions. (B) Semiquantitative RT-PCR analysis of gene expression in i-Mix EBs cultured with or without DOX. EBs were harvested on days 2.0 (lanes 2, 12), 2.3 (lanes 3, 13), 2.6 (lanes 4, 14), 3.0 (lanes 5, 15), 3.3 (lanes 6, 16), 3.6 (lanes 7, 17), 4.0 (lanes 8, 18), 4.3 (lanes 9, 19), 4.6 (lanes 10, 20), and 5.0 (lanes 11, 21). NT (lane 22) indicates minus template control. Expression of T was transient, preceding activation of Flk1 and c-kit. The differentiation program in DOX-induced i-Mix cells was accelerated (see text). (C) Quantitative real-time RT-PCR analysis of primitive streak and early mesodermal gene expression in i-Mix EBs cultured with or without DOX. Expression was normalized to that of a housekeeping gene (for this figure, Gapdh) and expressed as a ratio (see “Materials and methods”).

Figure 3.

Development of hemoglobin pigment is accelerated in response to mMix. Representative photomicrographs are shown for i-Mix EBs cultured for 6, 7, or 8 days in the presence (+DOX) or absence (–DOX) of doxycycline. Scale bar, 200 μm.

Figure 4.

Activation of the hemangioblastic and hematopoietic developmental programs is accelerated and enhanced in response to mMix. Quantitative real-time RT-PCR analysis of gene expression in i-Mix EBs cultured with (▪) or without () DOX. Markers of the hemangioblast and genes involved in hematopoiesis were activated in response to induced mMix. Expression was normalized to that of a housekeeping gene (for this figure, Gapdh) and expressed as a ratio. For additional details, see Figure 2.

Figure 5.

Formation of Flk1⁺ and c-kit⁺ cell populations is accelerated and enhanced in response to mMix. FACS analysis of cell surface Flk1 and c-kit protein expression in i-Mix EBs cultured in the presence or absence of DOX for 3 or 5 days (DOX added on day 1). Single-cell suspensions were labeled with anti-Flk1 and –c-kit sera and analyzed by FACS. Parental (wild-type) ES and uninduced (–DOX) i-Mix cells produced comparable numbers of Flk⁺, c-kit^hi, and Flk⁺ckit⁺ double-positive cells (not shown). This experiment was repeated 3 times with similar results.

Figure 6.

Mesodermal and hematopoietic progenitor potentials of ES cells are increased in response to mMix. i-Mix EBs were cultured in the presence (▪) or absence () of DOX, harvested on the indicated days, and dispersed to single-cell suspensions. Cells were plated in methylcellulose progenitor assays in triplicate, and colonies were scored as indicated. Colony counts are expressed as mean ± SEM per 10 000 cells plated. (A) Mesodermal progenitors (transitional and blast colonies). EBs were harvested on days 3 and 4 and plated in methylcellulose blast colony cultures. Colonies were scored after 4 days. (B) Primitive erythroid and megakaryocyte colonies. EBs were harvested on day 4 or 5, plated in primitive hematopoietic progenitor cultures, and colonies were scored after 4 or 6 days. (C) Definitive hematopoietic colonies. EBs were harvested on day 4 or 5, plated in Methocult, and scored on day 8 or 10.

Figure 7.

Conditional activation of mMix does not alter cell fate decisions within the hematopoietic lineage. (A) Primitive hematopoietic (EryP and megakaryocyte) colonies formed in methylcellulose progenitor assays upon replating of day 4 i-Mix EBs cultured in the presence or absence of DOX. The colonies were scored after 6 days. (B) Definitive hematopoietic colonies formed upon replating in methycellulose of day 4 i-Mix EBs cultured in the presence or absence of DOX. The colonies were scored after 8 days.