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S. Goossens et al.
ng/mL IL7 (or 1-0.2 ng/mL IL7 where indicated) (R&D Systems or Peprotech).
OP9-DL1 bone marrow stromal cells30 were maintained in MEMalpha medium (Gibco), supplemented with 20% fetal bovine serum (FCS; Hyclone), penicillin (100 U/mL)-streptomycin (100 μg/mL), 2 mM L-glutamine (Gibco) and incubated at 37°C with 7% CO2 and 95% humidity. E13.5 fetal liver cells were stained with biotin-conjugated lineage cocktail antibodies [Gr-1 (Ly-6G & 6C), Ter119 (Ly-76), CD3ε, and B220 (CD45R); eBioscience], PE-conjugated streptavidin (BD Biosciences, 1:200) and CD117-APC (cKit, Immunosource, 1:200). Lin-cKit+ cells were sorted using a FACSAria II machine (BD Biosciences) and FACSDiva software. Dead cells were discarded via DAPI stain.
Real-time quantitative polymerase chain reactions
Total RNA was isolated using a RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized using the First Strand cDNA Synthesis Kit (Roche) with oligo(dT) primers, starting from equal amounts of RNA. Real-time quantitative polymerase chain reactions (qRT- PCR) were performed using the LightCycler 480 SYBR Green I Master (Roche), monitored on a LichtCycler 480 system (Roche) and analyzed using qBase software from Biogazelle. Gene expres- sion was standardized against the housekeeping genes β-actin, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), RPL13 and TBP. All primers used are listed in Online Supplementary Table S2.
Statistical analysis
Data are presented as the mean ± standard deviation. Comparisons between two data groups were performed using a two-sided Student t-test. *P<0.05, **P<0.01, ***P<0.001 (vs. con- trol).
Results
Overexpression of Zeb2 but not Zeb1 induces spontaneous T-cell acute lymphoblastic leukemia in the mouse
We previously demonstrated that hematopoietic-specif- ic overexpression of Zeb2 from the mouse ROSA26 locus resulted in spontaneous T-ALL development.21 To docu- ment the functional similarities and/or differences between the two ZEB family members, we recently gen- erated a similar Zeb1 conditional overexpression mouse model using the same targeting strategies as previously described for Zeb231 (Online Supplementary Figure S1A). In these R26-Zeb1tg mice, an aminoterminal HA-tag Zeb1 cDNA, preceded by a floxed transcriptional stop cassette and followed by an IRES-eGFP, was targeted to the ROSA26 locus. The details on how these mice were gen- erated have been submitted for publication elsewhere.
To compare the oncogenic potential of Zeb1 and Zeb2, we crossed these newly generated R26-Zeb1tg mice to the same Cre line as we used for the R26-Zeb2tg, the Tie2-cre, which targets hematopoietic stem cells and their proge- ny.32 This resulted in a moderate 2- to 3-fold overexpres- sion of the total Zeb1 mRNA levels in the thymus, similar to that in the R26-Zeb2tg mice (Online Supplementary Figure S1B). These Tie2cre, R26-Zeb1tg/tg mice (henceforth referred to as R26-Zeb1tg/tg) were born in normal Mendelian ratios and no obvious phenotypes were observed at a young age. While we previously reported that mice over- expressing Tie2-cre, R26-Zeb2tg/tg (henceforth referred to as R26-Zeb2tg/tg) spontaneously develop T-ALL starting from the age of 5 months with a penetrance of 53% at 15
months of age, none of the R26-Zeb1tg/tg-overexpressing mice (n=15) showed any signs of leukemia development (Online Supplementary Figure S1C). These data indicate that, in contrast to Zeb2, Zeb1 is not an oncogenic driver of murine T-ALL.
Overexpression of Zeb2 results in thymic hypoplasia and delayed T-cell development
Upon dissection, we noted that juvenile Zeb2-overex- pressing mice, and not Zeb1-overexpressing mice, consis- tently had smaller thymi compared to their littermate con- trols. Except for a reduction of the medullary areas, no sig- nificant changes were observed in thymus architecture (Figure 1A). Next, detailed flow cytometric analysis was performed on thymi of 8-week old R26-Zeb2tg versus con- trol Cre-negative littermates. Upon Zeb2 overexpression, a significant and dose-dependent decrease in total thymo- cyte numbers was observed, associated with a significant decrease in percentages and absolute numbers of mature CD3+ and CD4+CD8+ double-positive Thy1+ cell popula- tions, combined with increased percentages of the imma- ture CD4-CD8- double negative (DN) Thy1+ population (Figure 1B). These findings indicate that the thymic hypoplasia in R26-Zeb2tg animals results from a partial block or delay at an early stage during T-cell development. Within the R26-Zeb2tg DN-gated cells, an abnormal popu- lation of CD44- cells with intermediate levels of CD25 was observed, which was absent in the control thymus (Figure 1C). These cells are Thy1+, CD49b-, B220- and CD19-, confirming their T-cell lineage identity. Positive staining for CD28 and intracellular TCRβ indicated that these are early post-β-selected cells blocked or delayed at the DN3 to DN4 transition, more specifically at DN3C. In line with this notion, expression analysis of mouse and human T-cell populations (Online Supplementary Figure S2) revealed that endogenous ZEB2 levels are normally down- regulated at this DN3-4 transition point.
Notably, no T-cell differentiation block or delay was seen in the Zeb1-overexpressing mice (Online Supplementary Figure S1D), indicating that Zeb2 overex- pression has effects on early T-cell differentiation that dif- fer from those of Zeb1 overexpression.
The Zeb2-mediated differentiation delay
is a hematopoietic cell-autonomous phenotype
As the Tie2-cre line also targets endothelial cells next to hematopoietic cells, we wanted to exclude that paracrine changes in the thymic microenvironment or architecture cause the delay in T-cell differentiation in R26-Zeb2tg mice. To this end, we performed in vitro T-cell differentiation experiments with purified hematopoietic progenitor cells. Sorted fetal liver hematopoietic progenitors (Lin-, cKit+) from R26-Zeb2tg/tg or Cre-negative control E13.5 embryos were seeded on OP9-DL1 bone marrow stromal cells that express the Notch delta ligand-1,30 in the presence of 5 ng/mL Flt3 ligand and 5 ng/mL IL7. In vitro T-cell develop- ment was monitored for 4 weeks. A significant delay in T-cell development was already prominent within 1 week in the Zeb2-overexpressing co-cultures (Figure 2A), exemplified by an increased frequency of cKit+ DN cells. After 2 weeks, an increase of the DN3-like population (CD3-,CD8-,CD44-,CD25int) and a concomitant decrease in more mature cells (CD8+) were also observed, pointing to a hematopoietic cell-autonomous delay or block in early T-cell development upon Zeb2 overexpression (Figure 2A).
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haematologica | 2019; 104(8)