Human yolk sac-like haematopoiesis generates RUNX1- and GFI1/1B-dependent blood and SOX17-positive endothelium.

The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1/1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data defines the hierarchical requirements for both RUNX1 and GFI1/1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.


INTRODUCTION
Blood cells develop from an endothelial intermediate at multiple stages during embryonic development in vertebrates (Dzierzak and Bigas, 2018;Ivanovs et al., 2017). Studies in the mouse have revealed that this is true for erythro-myeloid progenitors (EMPs) arising from yolk sac endothelium, and for blood cells emerging from aortic endothelium in the aorta-gonadmesonephros (AGM) region (de Bruijn and Dzierzak, 2017;Frame et al., 2016;Okuda et al., 1996;Swiers et al., 2013;Wang et al., 1996). Similarities between mouse and human haematopoietic development (Dzierzak and Bigas, 2018;Ivanovs et al., 2017) suggest that the same regulatory genes critical for mouse haematopoietic development will play essential roles in blood formation from the human embryo. Since studies of genetically modified human blood cells in the context of a developing human embryo are not possible, haematopoietic differentiation of human pluripotent stem cells has emerged as the most tractable surrogate experimental system. RUNX1 dependent, because deletion of RUNX1 resulted in the failure of normal blast colony development, with replacement of mixed haematopoietic and vascular colonies by reduced numbers of core structures containing SOX17 + endothelia but no blood cells. A single wave of extra embryonic erythropoiesis, without macrophage formation, was possible in the absence of RUNX1, but was only revealed under specific culture conditions on an air-liquid interface.
Furthermore, this wave of RUNX1-independent erythropoiesis was blocked by a small molecule inhibitor of GFI1/1B signalling. As expected, RUNX1-deficient hPSCs were unable to form blood cells upon differentiation towards HOXA + intra embryonic haematopoiesis. In summary, we show that human blood cell development from differentiating hPSCs under extra embryonic, yolk sac-like conditions arises from a haemangioblast-like SOX17endothelial cell, and is sequentially dependent upon GFI1/1B and RUNX1.

Modelling extra embryonic, yolk sac-like haematopoiesis
SOX-RUNX cells were differentiated to haematopoietic mesoderm, dissociated and transferred into methylcellulose cultures for blast colony (BL-CFC) assays ( Fig. 1A and Materials and Methods).
As expected, most RUNX1C + cells (76±13.3%, n=5) expressed CD43 (Fig. 1G). A small percentage of SOX17 + cells at d3 also expressed CD43 (10.2±3.8%, n=5) implying a modest haemogenic capacity for these cells (Fig. 1G). In summary, these data demonstrated expression of SOX17 in extra embryonic, yolk sac-like endothelium, which appeared at the same time as CD43 + haematopoietic cells, and prior to RUNX1C expression in a subset of the nascent blood.
Both d3 SOX17 + ENDO and d3 SOX17 -ENDO cells formed endothelial-like networks in Matrigel TM within 24 hours (Fig. 1J,K). The SOX17 + cultures retained expression of the mCHERRY reporter, whilst most of the SOX17cells remained mCHERRY negative, suggesting that the allocation of cells to a SOX17 + fate was largely complete by d3 in methylcellulose (Fig. 1J,K). Continuing the cultures for a further 24 hours in the presence of stem cell factor generated many haematopoietic cells from SOX17 -ENDO cells but only infrequent foci of blood cells in SOX17 + ENDO cultures ( Fig   1L,M), consistent with the results of re-culture experiments (Fig. 1I).
The frequency of haematopoietic progeny from SOX17 -ENDO cells, sorted after 2 days of methylcellulose culture, was determined by limit dilution analysis and compared to the haemogenic frequency of SOX17 + ENDO and SOX17 -ENDO cells sorted from d3 of methylcellulose differentiation (Fig. 1H,N and Supplementary  In order to explore the developmental relationship between SOX17and SOX17 + endothelium, we performed live cell imaging over 65 hours of d2 SOX17 -ENDO cells sorted from methylcellulose cultures and re-plated into a Matrigel TM endothelial network assay (Supplementary Movie 1, Fig. 2 and Fig. S2). It can be seen from time lapse images taken at 10 minute intervals (Supplementary Movie 1 and Fig. 2C) that the endothelia were initially SOX17 -, and that individual, SOX17cells began to acquire expression of the SOX17 reporter after 6 hours of observation. Importantly, mCHERRY expression was acquired by 24-28 hours, and during this period there was little increase in cell numbers, precluding division of any rare contaminating SOX17 + cells present at the onset of the culture as the reason for the increase in SOX17 + cell numbers (Fig. 2D). After this period the number of blood cells rapidly increased and the number of SOX17 + cells decreased a little and stabilised. Similar kinetics of mCHERRY reporter expression were observed in a second experiment that was not subjected to time lapse imaging (Fig. S2). These data strongly support the premise that d3 SOX17 + ENDO derives from the same d2 SOX17 -ENDO precursor population that also exhibits high haemogenic activity (Fig. 1N).
Since we had observed that a small percentage of SOX17 + ENDO cells developed into CD43 + haematopoietic cells (Fig. 1G), we compared them to SOX17 -ENDO derived CD43 + cells. We sorted haematopoietic and endothelial cells at d3 of methylcellulose culture that were either SOX17 + or SOX17 -, confirming that the SOX17 + cells expressed higher levels of mCHERRY and of SOX17 and providing reassurance that the appearance of SOX17 + CD43 + cells was not the consequence of imperfect sorting (Fig. S3A,B). The expression of haematopoietic genes (RUNX1, GFI1, SPI1[PU.1], KLF1 and GATA1) was higher in the d3 SOX17 -ENDO endothelial cells, probably reflecting their greater haemogenic capacity (Fig. S3B). The CD34 + CD43 + derivatives of both SOX17 + and SOX17endothelium expressed similar levels of haematopoietic transcription factors and globin genes, and displayed similar morphology, frequency and distribution of colony forming cells (Fig. S3B-G). These data suggest that, in human extra embryonic, yolk sac-like cultures, blood cells predominantly derive from SOX17endothelium, with a very small proportion of phenotypically similar cells arising from precursors that are SOX17 + .
Mesodermal cells, and endothelial/haematopoietic samples clustered into separate cell populations ( Fig. 3D). Differential gene expression analysis revealed that up regulated genes in mesoderm were enriched for Gene Ontology terms associated with embryo development (GO:0009790) and gastrulation (GO:0007369) whilst the endothelial/haematopoietic populations were enriched for leukocyte (GO:0050900) or vascular related genes (GO:0001944) (Supplementary Table 3). While very few genes were differentially expressed between d2 and d3 SOX17 -ENDO cells, several thousand genes were differentially expressed between CD34 + CD43 + and CD34 -CD43 + haematopoietic cells and d2 SOX17 -ENDO cells (Fig. S4A,B and Supplementary Table 4).
Patterns of differentially expressed genes between haematopoietic and endothelial cells were also consistent with cells segregating into distinct SOX17-expressing endothelium or CD34 + CD43 + haematopoietic fates during blast colony differentiation (Fig. S4C). Specifically, 914/1062 (86.1%) genes up regulated in d3 SOX17 + ENDO were down regulated in CD34 + CD43 + blood cells, and 711/954 (74.5%) of genes down regulated in d3 SOX17 + ENDO were up regulated in CD34 + CD43 + blood cells ( Fig. S4D and Supplementary Table 4). Similarly, 851/1028 (82.8%) genes up regulated in CD34 + CD43 + were down regulated in d3 SOX17 + ENDO cells and 1647/1898 (86.8%) of genes down regulated in CD34 + CD43 + were up regulated in d3 SOX17 + ENDO cells ( Fig. S4E and Supplementary Table 4). The data may be summarised to state that the same genes up regulated during the transition from d2 SOX17 -ENDO to d3 SOX17 + ENDO are down regulated in the transition to CD34 + CD43 + , and vice-versa. These analyses argue for the presence of a binary 'switch' active in the d2 SOX17 -ENDO cells that will lead to either a haematopoietic or endothelial fate, and are consistent with a haemangioblast-like function of these cells. Notably, there was variation in expression between RUNX1 and GFI1B in the endothelial populations ( Fig. 3F). Higher levels of RUNX1 and GFI1B expression in the d2 and d3 SOX17 -ENDO cells correlated with a high capacity to form haematopoietic cells, whilst low levels of RUNX1 and GFI1B in d3 SOX17 + ENDO marked a largely non-haemogenic endothelium. In order to explore the role of these factors in dictating haemogenic capacity, we characterised differentiation in cell lines in which they were deleted or inhibited.

RUNX1 is required for blast colony development
To examine whether RUNX1 is a key driver of the EHT in human extra embryonic, yolk sac-like haematopoiesis, we generated RUNX1-null hPSCs (denoted

Primitive erythroid cell generation in RUNX1-KO cells is GFI1-dependent
The presence of abundant nucleated erythroid cells in Runx1-knock out mouse embryos at E12.5 (Okuda et al., 1996;Wang et al., 1996) argues that the initial wave of yolk sac erythroid differentiation remains intact, although we were initially unable to detect expansion of CD43 + blood cells in human RUNX1-KO cells after d7 (Fig. 4I,J), or colony-forming cells in the d7 RUNX1-KO differentiation cultures (Fig. 4K).
We then added FGF2 and a low concentration of CHIR from the onset of differentiation ( After d7, an increasing proportion of SOX-RUNX cells expressed CD43, often associated with RUNX1C, and down regulated GYPA. In contrast, no new CD43 + cells appeared in the RUNX1-KO cultures. These cells down regulated CD43, but retained high levels of GYPA expression, consistent with adoption of an erythroid fate (Fig. 5C,E). These data indicate that RUNX1 is not required for the generation of the first CD43-expressing cells that subsequently differentiate only to erythroid cells. This is consistent with observations in Runx1-null embryos and differentiated Runx1-null mouse ES cells, in which all myeloid cells are absent (Lacaud et al., 2002;Okuda et al., 1996;Wang et al., 1996). We compared the frequency of colony forming cells in differentiating SOX-RUNX and RUNX1-KO cultures. In SOX-RUNX cells, BL-CFC peaked at d2 of differentiation, as noted previously (Fig. 1D), followed by a wave of primarily erythroid colonies at d6-d7 (Fig. 5F).
However, in RUNX1-KO cultures, the only clonogenic cells detected were at d2, when a small number of erythroid colonies were observed arising from vascular cores when cells were cultured at high density in methylcellulose ( In order to determine the optimum differentiation day at which to transfer embryoid bodies to airliquid interface cultures for erythroid development, transfers at d2, d3 and d4 were compared (  in the SOX-RUNX cultures (<1%) and these were >95% SOX17 + (Fig. S6A,B). In the RUNX1-KO and the LSDi treated SOX-RUNX cultures, approximately 30-40% of the viable cells were SOX17 + endothelial cells (Fig. S6A,B).
However, LSDi-treated cultures, in contrast to RUNX1-KO lines, failed to generate or maintain CD43 + or GYPA + cells (Fig. 6J). PCR analysis indicated that levels of RUNX1 transcripts were lower in cultures treated with LSDi (compare SOX-RUNX with and without LSDi in Fig. 6K and Fig.   S5G), suggesting that GFI1/1B may be a regulator, as well as a target, of RUNX1. Other haematopoietic transcription factors (GATA1, KLF1, SPI1 [PU.1]) were also significantly down regulated by LSD1 inhibition (Fig. 6L), consistent with the endothelial to haematopoietic transition block. These differences in transcription factor expression in LSDi-treated cultures were seen from d4 of differentiation, antedating the emergence of CD43 + cells, and therefore excluding major differences in the cellular composition of the cultures as an explanation for this observation.

The human blast colony assay detects predominantly RUNX1-dependent haematopoiesis
RUNX1 is required for the formation of virtually all haematopoietic cells detected in the blast colony assay (Fig. 4), suggesting that this primarily reads out progenitor cells similar to mouse yolk sac EMPs, which are also Runx1 dependent (Tober et al., 2013). One prediction of this hypothesis is that blast colonies should generate granulocytes, a lineage not observed during the first wave of extra embryonic yolk sac blood formation (Palis et al., 1999). To test this, we differentiated blast colony forming cells in the presence of growth factors that preferentially support erythroid, macrophage or granulocytic cells (Fig. S7A). May-Grünwald-Giemsa stained cytospin preparations documented that erythroid cells were restricted to cultures supplemented with EPO, macrophages were dependent upon M-CSF, and maturing neutrophils and eosinophils dominated cultures supplemented with G-CSF and GM-CSF (Fig. S7B). These lineage assignments were supported by flow cytometry, showing expression of GYPA on erythroid cells, CD14 expression on macrophages and RUNX1C in granulocytes (Fig. S7C,D). Finally, PCR analysis confirmed globin, KLF1 and GATA1 expression in erythroid cells, CSF1R and SPI1 in macrophages, and EPX, SPI1 and GATA1 in granulocytes (Fig. S7E). Taken together, these data support our hypothesis that the human blast colony assay reads out RUNX1-dependent extra embryonic, yolk sac-like cells with a broad myeloid potential similar to mouse yolk sac EMPs.

DISCUSSION
We have modelled extra embryonic human haematopoiesis and dissected the role of the transcription factor RUNX1 in analyses facilitated by the use of a reporter line in which GFP reported cells expressing the haematopoietic specific, C isoform, of RUNX1 and mCHERRY, expressed from the SOX17 locus, marked endothelium. We showed that d2 SOX17 -ENDO cells Indeed, the d2 and d3 SOX17 -ENDO cells expressed similar key haematopoietic genes to haemogenic endothelia in the mouse and human intra embryonic AGM (Baron et al., 2018;Ng et al., 2016;Solaimani Kartalaei et al., 2015;Swiers et al., 2013), although HOXA expression was absent, as expected, from these extra embryonic, yolk sac-like cells. Our study indicated that the d2 SOX17 -ENDO population was the precursor of both CD34 + CD43 + haematopoietic cells and a distinct SOX17 + expressing endothelium, although we have not shown that one cell could give rise to both progeny (Fig. 7A). Our data are consistent with prior in vitro mouse ESC differentiation studies that found a transient population of Tie2 hi c-Kit + CD41endothelial cells at d2 of blast colony differentiation that gave rise to CD41 + haematopoietic progeny (Lancrin et al., 2009). Indeed, RNA seq analysis shows that the d2 SOX17 -ENDO may be analogous to these mouse cells, in that they also expressed TEK (TIE2) and KIT and were negative for ITGA2B (CD41) (Fig. 3E). Our work extends the mouse findings by demonstrating that this d2 SOX17 -ENDO not only gave rise to blood cells, but also gave rise to a largely non haemogenic endothelium, now marked by the acquisition of SOX17 and the loss of RUNX1 expression.
Confirming the requirement for RUNX1 in the haemogenic SOX17endothelium, we showed that deletion of RUNX1 abrogated all haematopoiesis save for a single wave of extra embryonic erythropoiesis (Fig. 7B). The results of these experiments can be taken to indicate that the blast colony forming assay is not dominated by precursors of the first wave of extra embryonic haematopoiesis, but predominantly reads out progenitors of a second, RUNX1-dependent wave of extra embryonic haematopoiesis, perhaps analogous to a human EMP. This interpretation is supported by our ability to differentiate blast colonies to granulocytic cells, a lineage similarly generated from mouse yolk sac EMPs (Frame et al., 2013). Furthermore, our experiments also confirmed that all human extra embryonic, yolk sac-like macrophages were absolutely RUNX1 dependent, consistent with reports in the mouse (Lacaud et al., 2002;Okuda et al., 1996;Wang et al., 1996).
We observed that loss of RUNX1 lead to reduced levels of GFI1/1B as reported in the mouse (Lancrin et al., 2012;Thambyrajah et al., 2016b), but that expression was not completely lost ( Fig.   4H and Fig. S5F). Exploring the role that residual GFI1/ Our data argue for a window of haematopoietic competence during blast colony differentiation in which expression of GFI1/1B reinforced by RUNX1 drives the generation of blood and suppresses an endothelial program (Fig. 7). Such a model fits well with the narrow developmental window during which enforced Runx1 expression in mouse embryonic endothelium is able to drive haematopoiesis (Yzaguirre et al., 2018). The factors that initiate RUNX1 expression during this permissive stage are not known.
In summary, we identified and characterised a population of SOX17haemogenic endothelial cells that is the dominant source of blood and of SOX17 + endothelium in human extra embryonic, yolk sac-like cultures. We correlated RUNX1 and GFI1/1B expression with increased haemogenic capacity, further identifying that RUNX1 is required for human blast colony development. Finally, our studies also revealed the critical role played by GFI1/1B in the emergence of the first erythroid cells and the absolute dependence of all other blood lineages on RUNX1.

Ethics
Human pluripotent stem cell studies were approved by the Monash University (reference
To generate the SOX17 mCHERRY/w RUNX1 -/-(RUNX1-KO) hPSC line, the CRISPR Design Tool (http://tools.genome-engineering.org) was utilised to design 18 nucleotide (nt) single-guide RNAs (sgRNAs) to target two sites within exon 4 of the RUNX1 gene (5' TGTCGCCGTCTGGTAGGA 3' (CRISPR SITE 1) and 5' GGTCGGTCTTCCTAGCTT 3' (CRISPR SITE 2)) ( Clones were screened for the acquisition of a 457bp deletion region of exon 4 of the RUNX1 locus containing part of the DNA binding domain using primers a and c (Fig. S5A,C). Positive clones were further selected for deletion verification by PCR using primers a and b and Sanger sequenced using primers a and c. Three positive clones (11, 14, 30) contained a complete deletion between CRISPR site 1 and CRISPR site 2.
For all lines, surface markers of undifferentiated hPSCs were expressed and genomic integrity was confirmed by Illumina HumanCytoSNP-12 v2.1 array.

Culture and differentiation of hPSCs
H9 hPSCs used in these studies were provided by the WiCell Research Institute. Cell lines were regularly tested to exclude mycoplasma contamination and confirm genomic integrity. Culture and enzymatic passaging of hPSCs lines was performed as previously reported (Ng et al., 2008b).
For analysis, EBs, liquid, air-liquid interphase and methylcellulose cultures were harvested and dissociated into single cell suspension using TrypLE Select (Invitrogen) or Collagenases Type I and IV (Worthington) and passed through a 21-23-gauge needle and 40m cell strainer.

Limit dilution estimation of frequency of haematopoietic precursor frequency
To determine the clonal frequency of haematopoietic precursors, day 2+2 and day 2+3 blast colonies were flow sorted on the basis of CD34, CD43 and SOX17 expression and cells deposited by flow cytometer at 1, 3, 10, 30, 100 and 300 cells per well into GFR-matrigel coated 96-well plates. After 5-10 days of culture in APEL medium supplemented with 100ng/ml rh SCF, 50ng/ml rh VEGF, 50ng/ml rh IL-3, 50ng/ml rh IL-6, 50ng/ml rh TPO, 20ng/ml rh BMP4, 10ng/ml rh FGF2 and 2u/ml rh EPO, wells were scored by microscopy for the presence of hematopoietic clusters of greater than 30 cells. The frequency of colony forming cells was estimated using Poisson statistics.

Endothelial network assay and time lapse imaging
GFR-Matrigel TM was solidified at 37°C for 30 minutes in wells of a 48-well plate. For the experiments shown in Fig.1, 5 x 10 4 , day 2+3 flow sorted SOX17 + ENDO or SOX17 -ENDO, cells were seeded onto polymerised GFR-Matrigel TM in APEL medium supplemented with 50ng/ml rh VEGF, 10ng/ml rh FGF2, 5ng/ml rh epidermal growth factor (EGF, PeproTech) and 10 -3 Hydrocortisone (StemCell Technologies) and incubated for 24 hours. After 48 hours, APEL medium was supplemented with 100ng/ml rh SCF. For the experiments shown in Fig. 2, Fig. S2 and Supplementary Movie 1, 1.2 x 10 5 , day 2+1.75 flow sorted SOX17 -ENDO (SOX17 -CD34 + CD43 -CD73 -) cells were seeded onto polymerised GFR-Matrigel TM in APEL medium supplemented with 50ng/ml rh VEGF, 100ng/ml rh SCF, 10ng/ml rh FGF2, 5ng/ml rh EGF and 10 -3 Hydrocortisone. For the experiment in Fig. S2, cultures were incubated in a 5%CO 2 incubator at 37°C and wells were imaged at 24 hours and 48 hours after the endothelial network assay was setup. For the experiment in Fig. 2, cultures were incubated in a 5%CO 2 incubator at 37°C for 6 hours and then placed in an environmentally controlled (37°C, 5% CO 2 in humidified air) chamber fitted to a Zeiss LSM 780 laser scanning confocal microscope for time lapse imaging.

Flow cytometry and cell sorting
Antibodies directed against the following cell surface antigens (fluorochrome, manufacturer, Samples were gated using FSC-A and FSC-H to exclude doublets. In some cases, FSC-W, SSC-A and SSC-H were also used. FSC and Propidium Iodide exclusion were used to select live cells.
Positive gates for markers of interest were determined by comparing stained samples with those in which the antibodies were not added. Frequently this could also be corroborated by gating on samples in which the marker under evaluation was not expressed. There are numerous flow cytometry plots in this manuscript. The example chosen to illustrate gating strategy is one of the experiments from which samples were sorted for RNA sequencing analysis. This is shown in Fig.   S1A.

Gene expression analysis
Total RNA was isolated from hPSCs using the RNA Isolate II Mini or Micro Kits (Bioline) or RNeasy Kit (Qiagen) as specified by the manufacturer. cDNA was reverse transcribed via random hexamer priming and Tetro cDNA synthesis (Bioline) or Superscript III (Invitrogen) kits in accordance with the manufacturers' instructions. TaqMan TM gene expression probes (Applied Biosystems) and Bioline reagents were used for quantitative real-time PCR using GAPDH as the reference gene to normalise the data.
TaqMan TM assays directed towards the following target sequences were used to detect gene  comprehensive annotation (Liao et al., 2014). Genes lowly expressed were excluded (less than 10 counts per million in fewer than two samples in series 1, and less than 1 count per million in fewer than three samples in series 2). The data was TMM normalised, voom transformed and differential gene expression was assessed using moderated t-tests from the R Bioconductor limma package (Ritchie et al., 2015;Robinson and Oshlack, 2010). Genes that had a false-discovery rate of less than 5% were called as significantly differentially expressed for the various comparisons of interest. Gene ontology analysis was performed using the ToppGene Suite (Chen et al., 2009a).

Confocal microscopy and image processing
Confocal images (Fig. 1E Table S1. Haematopoietic precursor frequency in blast colony sorted populations. Table S2. RNA-Sequencing analysis of sorted d2 mesoderm and haematopoietic blast colony populations from d2 cultures following d2 (d2+2) or d3 (d2+3) in methylcellulose. Table S3. Gene ontology terms for mesoderm and haematopoietic blast colony populations.    Click here to Download Table S1 Click here to Download Table S2 Click here to Download Table S3 Click here to Download Table S4 Click here to Download Table S5 Click here to Download Table S6 Click here to Download Table S7 Development : doi:10.1242/dev.193037: Supplementary information Movie 1. Time lapse series of endothelial network assay. Series of images taken at 10 min intervals from 0 to 3920 min (65 hours) as indicated on each image. Images run at 10fps. The first faintly red fluorescent SOX17 + cells are seen from 360 min (6 hours) and are obvious from 720 min (12 hours) onwards. Blood cells appear from 2160 min (36 hours). Scale bar, 100µm. Related to at this time point. The right-hand panels show the sequential gating strategy used to sort the SOX17 -ENDO (SOX17 -CD34 + CD43 -CD73 -) endothelial cells that were seeded into the endothelial network assay. This is an independent experiment to that shown in Fig. 2. (B, C) Images taken from the endothelial network assay at (B) 24 hr and (C) 48 hr time points. SOX17 + and SOX17adherent cells are seen as well as developing clusters of haematopoietic cells. This experiment shows more rapid differentiation than the time lapse experiment in Fig. 2, suggesting that the environmental control achieved in a sealed incubator (this experiment) is superior to differentiation in the controlled climate (37°C, 5% CO2 in humidified air) chamber used for the time lapse series. The number of SOX17 + endothelial cells in each image is indicated. The number of tightly packed haematopoietic cells precluded accurate assessment of the total cell number. Scale bar, 100µm. Development: doi:10.1242/dev.193037: Supplementary information Development • Supplementary information inhibitor. Development: doi:10.1242/dev.193037: Supplementary information