Phosphatidylinositol-3 kinase activity controls survival and stemness of zebrafish hematopoietic stem/progenitor cells

Hematopoietic Stem and Progenitor Cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multi-potent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs. 2 Key points Loss of Pten and inhibition of PI3K induced apoptosis of hematopoietic stem/progenitor cells upon endothelial to hematopoietic transition Surviving hematopoietic stem/progenitor cells committed to all blood lineages but displayed reduced stemness


Introduction
Stem cells define a particular type of cells that maintain self-renewal capacity and may differentiate into multiple cell types at the same time. HSPCs are multipotent cells giving rise to all blood lineages during life [1][2][3] . In all vertebrates, an initial primitive wave of hematopoiesis occurs in the embryo, giving rise to primitive erythrocytes and myeloid cells. A definitive wave follows in which HSPCs are generated that will found multi-lineage hematopoiesis in developmentally successive hematopoietic organs up to adulthood. Our understanding of the emergence of HSPCs during the definitive wave is derived primarily from pioneer live in vivo imaging [4][5][6] . HSPCs emerge in a process whereby cells in the ventral wall of the dorsal aorta (VDA) undergo an endothelial to hematopoietic transition (EHT) 5 and then transiently colonize the fetal liver in mammals 7 , or the caudal hematopoietic tissue (CHT) in zebrafish 8 . There, HSPCs expand and differentiate into all blood lineages and supply the developing embryos with mature blood cells. Subsequently, HSPCs migrate again to colonize the thymus and the bone marrow in mammals 7 or whole kidney marrow in fish 8 to produce blood cells in the adult.
HSPCs are tightly regulated in terms of dormancy, self-renewal, proliferation and differentiation. Disrupting this balance can have pathological consequences such as bone marrow failure or hematologic malignancy. The tumor suppressor, PTEN, has an important role in hematologic malignancies, particularly T-lineage acute lymphoblastic leukemia (T-ALL). Deleterious mutations in PTEN appear in 5-10% of T-ALL cases and about 17% of patients lack PTEN expression in the hematopoietic lineage 9,10 . PTEN counteracts phosphatidylinositol-3 kinase (PI3K) and hence acts upstream in the PI3K-Akt (also known as Protein kinase B, PKB) pathway 11 . Loss of PTEN function results in hyperactivation of the PI3K-Akt signaling pathway. Clonal evolution of leukemia-propagating cells in zebrafish highlights the role of Akt signaling in the process 12 . Conditional deletion of Pten in mice in hematopoietic stem cells (HSCs) in adult bone marrow promotes HSC proliferation, resulting in depletion of long-term HSCs, indicating that Pten is essential for the maintenance of HSCs 13,14 .
The zebrafish genome encodes two pten genes with redundant function designated ptena and ptenb 15 . Single mutants display no morphological phenotype and are viable and fertile, but mutants that retain only a single wild type copy of pten develop hemangiosarcomas during adulthood 16 . Ptena -/ptenb -/mutants lack functional Ptena and Ptenb, are embryonic lethal at 5-6 days post fertilization (dpf) and display hyperplasia and dysplasia 15 . We reported that double mutant zebrafish larvae lacking functional Pten display increased numbers of HSPCs in the CHT at 4-5 dpf. Whereas these HSPCs commit to different blood lineages, they fail to differentiate into mature blood cells. Inhibition of PI3K using LY294002, which compensates for the loss of Pten, restores differentiation of HSPCs into mature blood cells. Hence loss of Pten enhances HSPCs proliferation and arrests differentiation 17 .
The past decades have led to an increase in our knowledge of hematopoiesis, but we are still far from a complete understanding of how HSPCs are established. Likewise, the role of Pten in steady-state hematopoiesis has been studied, but its potential role in the ontogeny of HSPCs is not fully understood. We have addressed these questions in zebrafish larvae. We imaged the emergence of HSPCs from the VDA in vivo in ptena -/ptenb -/embryos and in PI3K-inhibitor treated wild type embryos, which showed surprisingly similar defects. Furthermore, we performed single cell RNA sequencing (scRNA-seq) during the onset and at the end of the definitive wave. Our results indicate that elevated and reduced PI3K signaling had opposite effects on HSPCs at the end of the definitive wave.

Ethics statement
All animal experiments described in this manuscript were approved by the local animal experiments committee (Hubrecht Institute: Koninklijke Nederlandse Akademie van Wetenschappen-Dierexperimenten commissie protocol HI180701 and University Montpellier: Direction Sanitaire et Vétérinaire de l'Hérault and Comité d'Ethique pour l'Expérimentation Animale under reference CEEA-LR-13007) and performed according to local guidelines and policies in compliance with national and European law.
Confocal, fluorescence, brightfield microscopy and time-lapse imaging Fluorescence images of transgenic embryos were acquired using TCS-SPE and time-lapse imaging using TCS-SP2 as described 26 and processed with ImageJ 27 . For all live imaging embryos were anesthetized with tricaine 21 , mounted on a glass cover dish with 0.7% low melting agarose and covered with standard E3 medium. Whole mount bright field images were taken with a Leica DC 300F stereomicroscope.

In situ hybridization
Whole mount in situ hybridization was performed according to standard protocols 28 and images were taken using a Zeiss Axioplan microscope connected to a Leica DFC480 camera.

Acridine orange staining and whole mount immunohistochemistry
Embryos were incubated with 5 µg/ml acridine orange 29 for 20 minutes between 35 and 40 hpf and subsequently washed with standard E3 medium. Embryos were then imaged as described above. Immunohistochemical labeling performed using fixed (40 hpf) embryos to detect apoptosis using an activated caspase-3-specific antibody (BD Pharmingen) 30 . After confocal images were collected embryos were genotyped.

Photoconversion
Fluorescent tracing of VDA-derived HSPCs colonizing the CHT was done using the tg(kdrl:Dendra2) line as described before 31,32 with a Leica SP5 confocal microscope with a 20x dry objective. At 28 hpf an area of approximately 40x750 nm around the VDA, parallel to the yolk sac extension was photoconverted. The 405nm UV laser intensity and exposure time were optimized for strong Dendra2-conversion without cell damage. After photoconversion embryos were transferred to E3 medium and at 50-60 hpf their CHT areas were imaged on a Leica SPE Live confocal microscope using a 20x dry objective. To exclude bleedthrough of Dendra-green, red channel detection was set stringently (630-680nm).
Quantification of GFP low progenitor cells using tg(cd41:eGFP) GFP low and GFP high expressing cells were quantified in the CHT at 48 hpf ,50 hpf or 4 dpf using confocal imaging and Volocity and Imaris software. Ptena +/ptenb -/mutants on tg(cd41:eGFP) background were crossed and offspring was mounted at 48 hpf. Wild type tg(cd41:eGFP) embryos were treated with 5 µM LY294002 as described above and mounted and imaged at 50 hpf or 4 dpf. All GFP low expressing cells were counted in the entire CHT.

Flow cytometry
The AGMs of approximately 4000 36 hpf and 400 CHTs of 5 dpf old tg(kdrl:mCherry/cd41:eGFP) embryos were dissected and collected in Leibovitz-medium. After washing with PBSO the AGMs were deyolked using calcium-free Ringer's solution (116mM NaCl, 2.9mM KCl and 5mM HEPES) and then AGMs and CHTs were dissociated in TrypLE Express (Gibco) for 45 minutes at 32°C. The resulting cell suspension was washed in PBSO and passed through a 40-µm filter after resuspension in PBS supplemented with 2mM EDTA, 2% FCS and 0.5µg/ml DAPI. DAPI staining was used to exclude dead cells. Cells with kdrland cd41 low -positive signal were subjected to fluorescence-activated cell sorting (FACS) using a BD FACSAriaII and BD FACSFusion.
ScRNA-seq with SORT-seq ScRNA-seq was performed by Single Cell Discoveries BV (Utrecht, the Netherlands), according to an adapted version of the SORT-seq protocol 33,34 , with adapted primers described in 35 .

Data analysis
During sequencing, Read1 used for identification of the Ilumina library barcode, cell barcode and UMI. Read2 was used to map to the reference transcriptome of Zv9 Danio rerio. Data was demultiplexed as described 36 . Single cell transcriptomics analysis was done using the RaceID3 algorithm, following an adapted version of the RaceID manual (https://cran.rproject.org/web/packages/RaceID/vignettes/RaceID.html). Cells that had less than 1500 UMIs and genes that were detected in less than 5 UMIs in 5 cells were discarded. The number of initial clusters was set at 5. Differential gene expression analysis was done as described in 33 with an adapted version of the DESseq2 algorithm 37 .

Data sharing statement
For original data, please contact j.denhertog@hubrecht.eu. scRNA data are avaliable at GEO under accession number XXXX

Results
The onset of the definitive wave of hematopoiesis is independent of Pten The onset of the definitive wave starts at 32 hours post fertilization (hpf) with the specification of endothelial cells that will become HSPCs in the floor of the dorsal aorta (DA) in the aorta-gonadmesonephros (AGM) region ( Figure 1a), a conserved process between mammals and zebrafish 4-6 . Runx1 expression from 32 hpf onwards and c-myb expression from 35 hpf onwards mark the hemogenic endothelium of the VDA and its HSPC progeny 8,38 . We found that ptena -/ptenb -/mutant embryos expressed runx1 and c-myb along the VDA during the period that HSPCs emerge (between 30 and 44 hpf) just like their siblings ( Figure S1), indicating that loss of Pten does not affect initiation of definitive hematopoiesis.
Loss of Pten results in apoptosis of HSPCs during EHT in ptena -/ptenb -/mutant embryos.
In zebrafish, endothelial cells from the VDA transform into HSPCs in a process called EHT 5 . Subsequently, HSPCs join the blood flow in the underlying posterior cardinal vein (PCV) to transiently seed the CHT 4,5,8 . We monitored EHT events in the AGM by time-lapse confocal imaging of an area spanning two adjacent intersegmental vessels in the tg(kdrl:eGFP) transgenic background from 35 to 48 hpf (Figure 1a,b). The vasculature of ptena -/ptenb -/mutants and siblings was indistinguishable at this stage 39 . The floor of the aorta in ptena -/ptenb -/mutant embryos displayed the characteristic contraction then bending of cells towards the subaortic space 5 , indicating that the initiation of EHT was normal in ptena -/ptenb -/mutant embryos. However, half of the EHT events in ptena -/ptenb -/mutant embryos were abortive, in that 13 out of 24 HSPCs (54% in 9 embryos) failed to detach and disintegrated (Figure 1c-h, Movie S1). In contrast, siblings or wild type embryos did not display abortive EHT (n=75 in total). Live imaging using acridine orange 29  The number of HSPCs that colonize the CHT is reduced in ptena -/ptenb -/mutant embryos Following EHT, HSPCs transiently colonize the CHT 8 . We generated a tg(kdrl:Dendra2) transgenic line. The Dendra2 protein along the entire VDA was photoconverted green-to-red between 26 and 28 hpf, i.e. before the onset of EHT events (Figure 2a). Photoconverted HSPCs in ptena -/ptenb -/mutant embryos colonized the CHT between 50 and 60 hpf, albeit less HSPCs were detected than in the CHT of siblings (Figure 2b,c). We quantified the number of HSPCs that colonized the CHT at 48 hpf, i.e. by the peak of HSPC emergence from the VDA, using tg(cd41:eGFP) embryos, which express low GFP (GFP low ) in HSPCs 20,40 . Consistent with the initial apoptosis of half of the EHT derived HSPCs, 51% less GFP low HSPCs were detected in the CHT of ptena -/ptenb -/mutant embryos at 48 hpf compared to siblings (Figure 2d-f, Figure S2).
PI3K inhibition rescues EHT events in ptena -/ptenb -/mutant embryos but is detrimental for HSPCs in wild type embryos.
To address whether apoptosis of half of the EHT-derived HSPCs was due to enhanced PI3K signaling, embryos were treated with the PI3K inhibitor LY294002 from the onset of EHT (32 hpf) onwards. Inhibition of PI3K prevented apoptosis of HSPCs in ptena -/ptenb -/mutant embryos, in that none of the HSPCs that we imaged disintegrated (table 1) (Fisher's exact test, p=0.0013) (Figure 3a-c). Surprisingly, in wild type and sibling embryos that were treated with LY294002 in parallel with the ptena -/ptenb -/mutant embryos, disintegrating HSPCs in the VDA were observed (n=6) (Figure 3d-g, Movie S2) (Fisher's exact test, p=0.021).
Consistent with abortive EHT events upon LY294002 treatment, significantly less GFP low HSPCs in tg(cd41:eGFP) transgenic embryos colonized the CHT of LY294002-treated wild type embryos at 50 hpf (Figure 3h-i,l), comparable to the reduction observed in ptena-/-ptenb-/mutant embryos (Figure 2d). This reduction of HSPCs persisted through 4 dpf in LY294002-treated embryos (Figure 3j-k,m). These data suggest that normal, i.e. not too high and not too low PI3K activity levels are essential for emergence of HSPCs.

PI3K inhibition in wild type embryos results in HSPCs that engage in all blood lineages
In situ hybridization was performed using a panel of blood progenitor markers. LY294002-treatment reduced expression of the HSPC marker c-myb at 4 dpf (Figure 4a,b). The lineage markers globin (erythroid lineage), ikaros (lymphoid lineage) and l-plastin (pan-leukocytic, including myeloid lineage) were expressed, but reduced in LY294002-treated larvae compared to controls (Figure 4c-h). LY294002treated HSPCs also committed to the thrombocytic lineage as demonstrated by GFP high cells in tg(cd41:eGFP) embryos at 5 dpf 20 (Figure 4i,j). The number of GFP low cells in the definitive hematopoietic organs, thymus and kidney of at 8 and 12 dpf tg(cd41:eGFP) larvae was reduced in response to LY294002 (Figure 4k-n). These results show that reduced PI3K signaling did not block specification of particular blood lineages, but that the reduction in HSPC numbers affected founding of the definitive hematopoietic organs by HSPCs.
Singe cell RNA sequencing reveals two types of HSPCs, one of which is preferentially lost upon inhibition of the PI3K-pathway.
Cells from LY294002-treated embryos had an uneven distribution over all clusters. In HSPCs II and myeloid/neutrophil progenitors, cells from LY294002-treated embryos were overrepresented compared to control embryos (Fisher's Exact test, p<0.001). In HSPCs I, EHT-and myeloid/monocyte progenitors, cells from LY294002-treated embryos were underrepresented (Fisher's Exact test, p<0.001) (Figure 5f,i). These data indicate that LY294002-treatment led predominantly to loss of cells from HSPCs I cluster, which is consistent with the imaging data where half of the HSPCs fail to complete EHT (Figure 3d-g). The loss of HSPCs I in response to PI3K inhibition is accompanied by an increase in HSPCs II and myeloid/neutrophil progenitors.

More HSPCs upon inhibition of PI3K and less HSPCs in pten mutants
The CHTs of approximately 100 control and 100 LY294002-treated embryos were processed for scRNAseq. Of the 928 cd41 low cells that were analyzed, 684 remained after filtering. RaceID3 separated the cells in distinct clusters (Figure 6a). Cells in cluster 2 expressed erythrocyte progenitor-related genes (hbbe2, alas2 and cahz) (Figure 6b). Cluster 3 is characterized by cells expressing genes related to thrombocyte/erythrocyte progenitors (gata1a, klf1 50,54,55 ) (Figure 6c). Cells in cluster 1 express genes indicative of HSPCs, including c-myb (Figure 6d). Cluster 4 represents early myeloid progenitors, as runx3, pu.1 (also known as spi1b) and cebpb 54 are highly expressed (figure 6e). Cluster 5 is characterized by neutrophil progenitor-related gene expression (mpx) (figure 6f). Analysis of the distribution of hematopoietic cells, using a Fisher's exact test indicated that the thrombocyte/erythrocyte progenitor cells were underrepresented in the LY294002-treated embryos (p<0.01) and HSPCs were significantly overrepresented (p<0.001) (Figure 6g,h).These results indicate a significant shift towards HSPCs at the expense of the thrombocyte/erythrocyte progenitor cluster in response to LY294002 treatment.
Likewise, we assessed transcriptomic differences by scRNA-seq in HSPCs from the CHT between ptena -/ptenb -/mutant embryos and their siblings at 5 dpf. Approximately 100 ptena -/ptenb -/mutant embryos and siblings were selected based on phenotype 15 , which yielded 614 cd41 low cells after filtering. RaceID3 indicated that clusters emerged representing the same hematopoietic lineages as described for the wild type and LY294002-treated data (cf. Figure 6 and 7). Analysis of the distribution of hematopoietic cells from pten mutants and their siblings over the five clusters indicated that the erythrocyte-and neutrophil progenitor cells were overrepresented in the pten mutant (p<0.001 and p<0.05) and that HSPCs were significantly underrepresented (p<0.001) ( Figure 7g-h, S5). These results indicate a significant shift in ptena -/ptenb -/mutant embryos towards erythrocyte progenitor and neutrophil progenitors at the expense of HSPCs.

Discussion
We used zebrafish mutant embryos lacking functional Pten to investigate how loss of Pten affects the ontogeny of hematopoiesis. Characterization of zebrafish ptena -/ptenb -/mutant embryos led to the unexpected finding that half of the HSPCs undergo apoptosis upon emergence from the VDA during EHT at the onset of the definitive wave ( Figure 1). Loss of function of Pten is usually linked to enhanced cell survival, such as for instance in Pten knock-out mice 56 . We reported that ɣ-irradiation reduces apoptosis in ptena -/ptenb -/mutant embryos 15 . Apoptosis of zebrafish HSPCs has been reported before, in that grechetto mutants display decreasing numbers of HSPCs due to apoptosis 51 . Runx1 knockdown also induced abortive EHT events due to apotosis 5 . Runx1 expression was not affected in the VDA of ptena -/ptenb -/mutants ( Figure S1), suggesting that the mechanism underlying EHT defects in ptena -/ptenb -/mutant embryos and Runx1 morphants are distinct. Apoptosis of HSPCs in pten mutants is due to enhanced PI3K-mediated signaling, because treatment with a PI3K inhibitor rescued apoptosis of HSPCs. Surprisingly, treatment of wild type embryos with the PI3K inhibitor induced death of half of the HSPCs upon emergence from the VDA as well (Figure 3). These results suggest that upon emergence from the VDA, HSPCs require a moderate level of PI3K signaling, as hyperactivation of PI3K signaling in Pten mutants as well as inhibition of PI3K signaling induced apoptosis of emerging HSPCs.
After emerging from the VDA, the surviving HSPCs enter circulation and seed the CHT, as demonstrated by photoconversion of endothelial cells prior to EHT in ptena -/ptenb -/mutant embryos and siblings ( Figure 2). Half of the HSPCs of ptena -/ptenb -/mutant embryos and LY294002-treated embryos colonized the CHT, compared to wild type embryos (Figure 2,3, Table 1). In LY294002-treated embryos the decrease in HSPCs remained, whereas in ptena -/ptenb -/mutant embryos the surviving HSPCs hyperproliferate leading to an increase in HSPCs at later stages 17 . Surviving HSPCs from ptena -/ptenb -/mutants engage in all blood lineages 17 . However, definitive differentiation of major blood lineages is arrested in the ptena -/ptenb -/mutants, consistent with the inverse correlation of proliferation and differentiation of stem cells 57 . The surviving HSPCs of LY294002-treated embryos also engaged in all blood lineages (Figure 4), demonstrating pluripotency of the HSPCs.
Using scRNA-seq at the onset of the definitive wave (36hpf) of hematopoiesis two HSPC clusters were identified, that both expressed HSPC markers. In control embryos equal numbers of cells populated the HSPCs I and HSPCs II clusters. Predominantly the cells from the HSPCs I cluster were lost upon PI3Kinhibition ( Figure 5). Our imaging data indicated that half of the HSPCs disintegrated upon treatment with LY294002 ( Figure 3). It is tempting to speculate that the surviving half of the HSPCs all belong to the HSPCs II cluster. Whereas both HSPCs clusters expressed HSPC markers, expression of ENSDARG00000080337_ACO24175.4 and to a lesser extent tmed1b distinguished the HSPCs II cluster from the HSPCs I cluster. In situ hybridization using an ENSDARG00000080337_ACO24175.4-specific probe indicated high expression throughout the embryo, which did not allow validation of the difference in expression in HSPCs I and HSPCs II cells ( Figure S3). Little is known about ENSDARG00000080337_ACO24175.4, except that it is a mitochondrial ribosomal gene (mt rDNA). Interestingly, HSCs have significantly lower rates of protein synthesis than other hematopoietic cells 58 . The protein product of ENSDARG00000080337_ACO24175.4 may have a role in protein synthesis. Hence, the difference in expression levels may indicate that the HSPCs II cells that survive PI3K-inhibition are less stem cell-like and more progenitor-like, poised to differentiate.
In response to LY294002-treatment the number of cd41 low HSPCs was reduced in the CHT at 4 dpf and in the definitive hematopoietic organs at 8 and 12 dpf (Figure 3,4). scRNA-seq of putative HSPCs (cd41 low , kdrl + cells) at the end of the definitive wave (5dpf) indicated initiation of differentiation in different blood lineages (Figure 6), consistent with in situ hybridization ( Figure 4). Yet, inhibition of PI3K arrested differentiation, i.e. increased HSPC fate, predominantly at the expense of thrombocyte/erythrocyte progenitor fate ( Figure 6). Overall, it is evident that there is a significant reduction in hematopoietic cell number (Figure 4, 6), which may be caused by preferential loss of HSPCs with more stem cell-like properties ( Figure 5).
ScRNA-seq at the end of the definitive wave showed a significant increase in erythrocyte-and neutrophil-progenitors in ptena -/ptenb -/mutant embryos (Figure 7, S5), consistent with earlier in vivo data 17 . However, we reported an overall increase in HSPCs, due to hyperproliferation, whereas here, we observed a decrease in HSPCs in the scRNA-seq data. An explanation for this apparent discrepancy is that the hyperproliferating HPSCs we observed earlier 17 actually have initiated differentiation already and are scored as erythrocyte and neutrophil progenitors by scRNA-seq.
Conditional knock-out of Pten in HSCs in mouse adult bone marrow, drives HSCs into the cell cycle, resulting in transient expansion of the spleen and eventually in depletion of HSCs in the bone marrow. These conditional PTEN-deficient mice die of a myeloproliferative disorder that resembles acute myeloid/lymphoid leukemia, indicating that PTEN is required for maintenance of HSCs 13,14 . It is noteworthy that there are differences between the conditional mouse models and the zebrafish model we used. In the mouse, Pten is deleted in adult bone marrow cells, well after HSCs have formed, whereas in zebrafish, Pten is systemically deleted and therefore effective prior to the emergence of HSPCs. Studies in mice showed that regardless of cell state, HSCs and multi-potent progenitors had a lower protein synthesis rate than more restricted hematopoietic progenitors. Loss of PTEN in HSCs caused depletion of HSCs, due to a higher rate of protein synthesis 58 , which is consistent with our observation that loss of Pten in zebrafish caused HSPCs to hyperproliferate and become less stem-cell like.
Long-term HSCs are quiescent, whereas short-term HSCs proliferate more 2 . It would be tempting to speculate that the HSPCs that undergo apoptosis upon loss of Pten or upon PI3K-inhibition are involved in long-term colonization of definitive hematopoietic organs. The surviving HSPCs in pten mutants at the onset of the definitive wave would then represent multi-potent progenitors that only have limited potential for self-renewal. Investigating the regulatory network underlying the surviving and disintegrating HSPCs will further expand our understanding of short-and long-lived HSCs and will eventually contribute to the development of efficient stem cell based therapies 59,60 . Tables   Table 1. Number of apoptotic cells during EHT.
The number of cells undergoing apoptosis during EHT was determined in control and LY294002 (5µM from 32 hpf onwards) treated wild type and ptena -/ptenb -/embryos (35-48 hpf) by confocal time lapse imaging. The number of embryos that was imaged is given as well as the number of apoptotic HSPCs or surviving HSPCs. The percentages of emerging or apoptotic HSPCs, relative to the total number of HSPCs are also indicated.         Figure 1