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Temporal and spatial emergence of GATA1 complex
D9 as shown by the lack of red coloration. This correlates with absence of interaction detection by PLA constituting an important control for PLA specificity, in addition to the single probes and secondary antibodies alone used a neg- ative control.
We next characterized the dynamic expression of hematopoietic TF proteins during the time course of ES cell differentiation at D4-5 using Ldb1-KO cells as the con- trol (Online Supplementary Figure S1A). This showed that the different factors already form a complex as deter- mined by immunoprecipitation at D4 and D5 (Online Supplementary Figure S1C-D) using Ldb1-KO cells as the control (Online Supplementary Figure S1E). Online Supplementary Figure S1C shows that LDB1 and FOG1 fail to pull down GATA1 (and vice versa) in D4 differentiated cells, which is likely due to very low amounts of the bridg- ing factor LMO2. Day 5 (Online Supplementary Figure S1D) shows more GATA1, but LMO2 is still undetectable and LDB1 and GATA1 appear to fail pulling down each other. The same is seen for GATA1 and FOG1. Interestingly FOG1 appears to be regulated by (the) LDB1 (complex) as it is present in D5 WT cells, but not in Ldb1-KO cells. This agrees with our observation that LDB1 binds to the Fog1 gene in Flk1 positive cells sorted four days after ES cell dif- ferentiation and the reduction of Fog1 expression in Ldb1-KO cells analysed by RNA-Seq.10 The amounts of the proteins involved (directly or indirectly) were too low to allow their detection by immunoprecipitation or mass spectrometry (data not shown), because D4 and D5 differ- entiated ES cells are a mixture of few hematopoietic cells in the presence of many non-hematopoietic cells. For example, many cells express Oct4 (Online Supplementary Figure S1A) or cardiac genes in cardiac progenitors.20 The complexes are barely detectable by size-exclusion chro- matography (Online Supplementary Figure S1B). By con- trast, the clear PLA signals (Figure 1B) show the power of PLA to analyze complexes in individual cells. The few PLA signals in undifferentiated ES cells are background since such signals are also detected with single antibody con- trols.
In summary, PLA monitors the dynamic changes of dif- ferent protein complexes even of low amounts present in a subset of cells. The PLA signals in D5 EB appear to dis- tinguish a subpopulation of cells, suggesting that some specification is already in progress towards hematopoietic cells in the mixed three-dimensional cell aggregates.
The GATA1 complexes (GATA1/LDB1 and GATA1/FOG1) are observed in mouse E12.5 FL cells
(Pre-)HSC move to the FL at embryonic day E10.5 to 11.5. We applied PLA on FL sections at E12.5 to under- stand the temporal appearance of the two GATA1 com- plexes in definitive blood cells. Figure 2 shows the com- parison of the GATA1/LDB1 and GATA1/FOG1 complex- es, together with negative controls of GATA1, FOG1 or LDB1 single-primary antibody. Although FL tissue is com- pact and single cells can be difficult to distinguish, clearly some cells contained very dense GATA1/LDB1 PLA sig- nals when compared to surrounding cells (Figure 2A). Close up images show that the signal co-localizes with the DAPI staining (of note, those cells have little cytoplasm relative to the size of the nucleus) and that cells with no signal (next to strong positive ones) include FL endothelial cells which are expected to be negative for GATA1. A sim- ilar result was found in fetal aorta (not shown). Specific PLA
signals were also detected for LDB1/LMO2, which is part of the same GATA1/LDB1 complex (Figure 2B) suggesting GATA1/LDB1 and LDB1/LMO2 and by inference the GATA1/LDB1/LMO2 complex are present at a high level in a subpopulation of cells. This is in accordance with our previous co-immunoprecipitation and ChIP-sequencing co-localisation data of these factors in MEL and in Flk1+ cells sorted four days after ES cell differentiation.5,8,10 In contrast, GATA1/FOG1 signals appear in similar numbers of cells but are less abundant/weaker and more evenly dis- tributed (Figure 2, upper panel A). In agreement with the PLA results, immunofluorescent staining for individual LDB1, GATA1 and FOG1 proteins in FL sections showed higher co-expression of GATA1 and LDB1 in a subpopula- tion of cells than GATA1 and FOG1 (Online Supplementary Figure S2), although it should be noted these are signals from different antibodies (see Methods).
FL contains erythroid cells at different stages of differen- tiation and the PLA results suggest that the LDB1 com- plex, is more important at particular stages of erythroid cells in agreement with data showing that GATA1 increas- es before the end stage of erythroid differentiation8 prima- rily in the LDB1 complex.
GATA1/LDB1 complex is highly localized in early erythroid differentiating cells in sorted FL
In order to identify the cells containing high PLA signals, E12.5 or E13.5 FL cells were sorted using glycophorin (TER119) and transferrin receptor (CD71) antibodies into four populations: P1 (CD71–/TER119–), P2 (CD71+/TER119–), P3 (CD71+/TER119+) and P4 (CD71–/TER119+) in order to separate different stages from proerythroblasts to orthochromatic erythroblasts21,22 (Figure 3A).
First, gene expression was measured in the four popu- lations (Figure 3B). At early stages (P1 and P2), expression of Ldb1, Gata1 and Fog1 starts increasing followed by a decrease at P3 for Ldb1 and at P4 for Gata1 and Fog1, which follow each other. Gata2, c-Kit and c-Myb genes are expressed highly in P1 which contains precursor cells (and other cell types), their expression decreases during differ- entiation, whereas the β-globin gene increased dramatical- ly from P3 to P4. The CD71/TER119 sorted cells were used for RNA-seq analysis in two independent biological replicates for each P1 to P4 population of E12.5 FL cells. Principal component analysis (Figure 3C) shows that bio- logical replicates of each population cluster and can be separated from each other. As expected, the RNA-seq result in those populations (Online Supplementary Figure S3) is very similar to the genes analysed by qPCR (Figure 3B and Online Supplementary Figure S3). There is an increase of expression of the transcription factors Ldb1, Gata1 and Fog1 from P1 to P2 (together with E2A) and continuing to P3 for Gata1 and Fog1 (together with Klf1). Gata2, c-myb and c-kit expression is inversely correlated and follows the same trend as Eto2 and Irf2bp2, with a decrease peaking at P3 followed by erythroid specific markers such as β-globin, Alas2 and Gypa and the transcription factor Lmo2. The result shows that the sorting method clearly separates the different stages of the erythroid cells. The significantly dif- ferentially expressed genes (±0.6 log log two-fold change and P-value ≤0.05) between the populations is shown in the Online Supplementary Table S3. Of note Ldb1 expres- sion presents a two-fold increase between P1 and P2, but is not included in the Online Supplementary Table S3 due to
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