Ability to form hematopoietic stem/progenitor cell-containing cell clusters weakens during the fetal-to-adult transition in hematopoietic development

Ability to form hematopoietic stem/progenitor cell-containing cell clusters weakens during the fetal-to-adult transition in hematopoietic development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ability to form hematopoietic stem/progenitor cell-containing cell clusters weakens during the fetal-to-adult transition in hematopoietic development Ayumi Itabashi, Yuki Yokoi, Kiyoka saito, Ryota Tsukahara, Gerel Melig, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7069210/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Apr, 2026 Read the published version in Inflammation and Regeneration → Version 1 posted 5 You are reading this latest preprint version Abstract Background Definitive hematopoietic stem cells (HSCs) emerge within intra-aortic hematopoietic cell clusters (IAHCs) located in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation in the mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and finally settle in the bone marrow (BM). We previously showed that the transcription factor Sox17 is expressed in IAHCs. Transduction of the Sox17 gene in IAHC cells induces the formation of cell clusters in vitro that resemble IAHCs and retain hematopoietic potential. In addition, a previous report showed that Sox17 -transduced hematopoietic stem/progenitor cells (HSPCs) in the BM maintained pluripotency. However, the relationship between Sox17-induced cluster formation and the developmental transition from fetal to adult hematopoiesis has not been clarified. Methods We examined whether viral transduction of the Sox17 gene in HSPCs leads to the formation of cell clusters. To identify the candidate genes involved in cluster formation, we performed RNA-sequencing (RNA-seq) analysis on Sox17-ER T-transduced HSPCs from the AGM region and the BM cultured with or without tamoxifen. We further analyzed the ability of one candidate gene, the Procr gene, to support cluster formation and hematopoietic function. Results A large number of multilineage colonies were observed in Sox17-ERT -transduced tamoxifen-treated HSPCs prepared from the AGM region, the FL, and the BM. However, the ability to form cell clusters was lower in BM-derived HSPCs compared to those from the AGM region and the FL. RNA-seq analysis revealed several genes that were highly expressed in Sox17-ERT -transduced tamoxifen-treated HSPCs from the AGM region. The Procr gene, one of these genes, was expressed in IAHCs and was found to contribute to both cluster formation and the maintenance of hematopoietic capacity in Sox17 -transduced cells of the AGM region. Conclusions Our results revealed that the Sox17-induced cluster-forming ability is attenuated in BM HSPCs compared to the AGM and the FL HSPCs, suggesting that HSPC characteristics are developmentally altered during the transition from fetal to adult hematopoiesis. Moreover, the Procr gene plays a substantial role in cluster formation and supports hematopoietic capacity in the midgestation mouse embryos. hematopoiesis HSPC Sox17 IAHCs Procr Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background In mammals including mice, the sites of embryonic hematopoiesis change over the course of development [ 1 ]. Hematopoietic stem cells (HSCs), which possess the long-term repopulating ability, initially emerge in the aorta-gonad-mesonephros (AGM) region at embryonic day (E) 10.5 [ 2 , 3 ]. After the production of HSCs in the placenta around E12.5 [ 4 , 5 ], HSCs expand in the fetal liver (FL) and finally migrate to the bone marrow (BM). In the AGM region, HSCs are observed in the intra-aortic hematopoietic cell clusters (IAHCs), which arise from the hemogenic endothelium in the dorsal aorta [ 6 , 7 ]. The hemogenic endothelium is known to give rise to both hematopoietic cells and endothelial cells [ 8 ]. Whole-mount immunohistochemical analysis revealed that c-Kit, a marker of HSCs, is expressed in IAHCs. The protein expression of CD31 and vascular-endothelial cadherin (VEC), both of which are endothelial markers, and that of CD45, a hematopoietic cell marker, varies depending on the position of the cells within IAHCs. CD31 and VEC are strongly expressed in basal cells in IAHCs, supporting their emergence from hemogenic endothelium [ 6 ], whereas CD45 is expressed in apical cells [ 6 ], indicating that hematopoietic differentiation of blood cells occurs within IAHCs. Moreover, the expression levels of CD31, VEC, and CD45 differ among the HSCs in the IAHCs, the FL, and the BM [ 9 ]. Embryonic HSCs have a high proliferative capacity to supply blood cells. In contrast, BM HSCs, which exhibit altered expression of several proteins compared to IAHCs and/or FL HSCs, enter a quiescent state upon interaction with niche cells in the BM [ 10 ]. Sox17 is a transcription factor, known as an endodermal marker [ 11 ]. Sox17-deficient mice exhibit defects in gut tube formation and die during midgestation in mouse embryos [ 12 – 14 ]. Mice heterozygous for Sox17 show a biliary atresia-like phenotype, hepatitis with aberrant cell wall formation in the gallbladders [ 15 – 18 ], and female subfertility associated with implantation failure [ 19 ]. Furthermore, conditional knockout analyses have revealed that the deletion of Sox17 in the immediate postpartum period significantly reduces the absolute number of HSCs, although Sox17 does not affect the HSC numbers in the BM [ 20 ]. Whole-mount immunohistochemistry of the AGM region has shown that Sox17 is expressed in endothelial cells of the dorsal aorta and IAHC cells from the E10.5 AGM region, which are positioned close to endothelial cells in E10.5 embryos [ 21 – 23 ]. When Sox17 is introduced into IAHC cells from the E10.5 AGM region, HSC-containing Sox17 -transduced cells can be maintained through multiple passages in the co-culture of stromal cells [ 21 ]. Moreover, the transcription factor Sox17 directly induces the Notch1 expression, followed by increased expression of the Notch1-downstream molecule Hes1 to sustain the hematopoietic activity [ 23 ]. Sox17 also directly enhances the expression of VEC and endothelial cell-selective adhesion molecules (ESAM), promoting the formation of hematopoietic cell clusters with hematopoietic potential [ 24 ]. Although Sox17 is not expressed in BM HSCs, the ectopic expression of Sox17 in BM HSCs maintains the stemness of HSCs and induces the expression of fetal HSC marker proteins [ 25 ]. These results suggest that the Sox17-regulated molecular mechanisms active in fetal HSCs may retain latency but function in the BM HSCs [ 26 ]. We previously showed that the introduction of Sox17 into IAHC cells from the AGM region induces the formation of cell clusters with hematopoietic potential [ 21 , 23 , 24 , 27 , 28 ]. During the fetal-to-adult transition, the Sox17 expression in HSCs decreases. However, it remains unclear whether Sox17-induced cluster formation in HSPCs is functionally linked to the fetal-to-adult transition. In the present study, we examined the formation of cell clusters and the maintenance of hematopoietic capacity following Sox17 transduction into hematopoietic stem/progenitor cells (HSPCs) derived from the AGM region, the FL, and the BM. Whereas the hematopoietic activity is maintained in Sox17 -transduced HSPCs, their cell cluster-forming ability decreases during the fetal-to-adult transition. We compared the gene expression profiles of Sox17-ERT -transduced tamoxifen-treated BM HSPCs with those of Sox17-ERT -transduced tamoxifen-treated IAHC HSPCs. We also compared IAHC HSPCs in which Sox17 was located to the nucleus with those in which Sox17 remained in the cytoplasm. From these comparisons, we identified candidate genes that were more highly expressed in Sox17-ERT -transduced cells where Sox17 was localized in the nucleus. One such candidate gene, the Procr gene (the encoding endothelial protein C receptor, EPCR), was found to be expressed in IAHC cells of the dorsal aorta at E10.5 embryos. Knockdown of the Procr gene in Sox17 -transduced AGM cells reduced both the cluster-forming ability and the hematopoietic capacity. Methods Isolation of HSPCs prepared from the AGM region, the FL, and the BM CD45 low c-Kit high cells, which are a component of IAHCs, were isolated from the AGM region of E10.5 mouse embryos as described previously [ 21 ]. FLs were excised from E14.5 ICR mouse embryos and suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% (v/v) fetal calf serum (FCS). FL cells were incubated on ice for 15 min with biotin-conjugated anti-mouse CD8a, CD4, Ly6G/Ly6C, CD45R/B220, CD11b, and Ter119 antibodies (lineage antibodies, BioLegend, San Diego, CA). After washing, the cells were stained with PE-conjugated anti-mouse Sca-1 antibody (BioLegend), APC-conjugated anti-mouse c-Kit antibody (eBioscience, San Diego, CA), and PE-Cy7-conjugated streptavidin. BM cells from C57BL/6N mice were also suspended in DMEM containing 2% (v/v) FCS. After BM cells were treated with biotinylated lineage antibodies, they were stained with the APC-Cy7-conjugated anti-mouse Sca-1 antibody, the APC-conjugated anti-mouse c-Kit antibody, and the R-PE-conjugated avidin. To separate lineage-positive cells, BM cells were treated with anti-PE Microbeads (Miltenyi Biotec Inc., Auburn, CA) and passed through a magnetic-activated cell sorting column (Miltenyi Biotech). Both FL cells and lineage-negative BM cells were stained with 1 µg/ml propidium iodide (Calbiochem, San Diego, CA), and lineage − c-Kit + Sca-1 + (KLS) cells were recovered by fluorescence-activated cell sorting (BD Biosciences, San Diego, CA). All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Care Committee of Tokyo Medical and Dental University (approval numbers: A2018-265C2, A2019-108C4, and A2021-177). Enforced expression of genes in HSPCs prepared from the AGM region, the FL, and the BM Retroviruses encoding IRES-GFP , Sox17-IRES-GFP , or Sox17-ERT-IRES-GFP genes were used to infect CD45 low c-Kit high cells from the E10.5 AGM region and KLS cells from the E14.5 FL for 3 hrs. The infected cells were then cultured with OP9 stromal cells in α-minimal essential medium (α-MEM) supplemented with 10% (v/v) FCS, 25 ng/ml stem cell factor (PeproTech, Rocky Hill, NJ), 10 ng/ml interleukin-3 (PeproTech), and 10 ng/ml thrombopoietin (PeproTech). Sox17-ERT-IRES-GFP -transduced cells were cultured with or without 1 µM tamoxifen citrate. Additionally, retroviruses encoding these genes were used to infect KLS cells from the BM of 10-week-old mice on the RetroNectin (Takara Bio., Kyoto, Japan)-coated dishes for 4 days. After washing, the cells were cultured with OP9 cells in the same media Analyses of the cluster-forming ability and the hematopoietic ability in Sox17 -transduced cells The exogenous gene-transduced CD45 low c-Kit high AGM cells and FL KLS cells described above were sorted based on the GFP expression on days 4 and 7 of culture, while the gene-transduced BM KLS cells were sorted on day 7. The sorted cells were then cultured on OP9 cells. After 11 days of culture, the number of clusters formed by the gene-transduced cells was counted. The cells were dissociated and sorted again based on the GFP expression (exogenous gene expression) for further analyses, such as RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR), Giemsa staining, and the colony-forming assays. RNA was extracted by ISOGEN (WAKO, Osaka, Japan), and cDNA wad synthesized by ReverTraAce (TOYOBO, Osaka, Japan). The same amounts of cDNA were subjected to PCR using TAKARA Taq (TAKARA). Primer sequences were as follows: 5’-ACCACCCGATACCCACCTAT-3’ and 5’-GCCATGGCAGTCACCATGCT-3’ (GATA-2), 5’-GAGAGGTGGCACAACCATTT-3’ and 5’-GGGAACGTGACTGGAGATGT-3’ (c-Myb), 5’-CCAGCAAGCTGAGGAGCGGCG-3’ and 5’-CCGACAAACCTGAGGTCGTTG-3’ (Runx-1), 5’-AGGTGCAGCCACAGAACTTA-3’ and 5’-TCGGACCAATCAGAGATGTT-3’ (Notch-1), 5’-GTCATGGCCATGGTCGAGTA-3’ and 5’-CTCCTCGGCATCTTGCTGAA-3’ (CD31), 5’-GACTGGAACCAGCACGCTAACC-3’ and 5’-CGCCGTCATTGTCTGCCTCTTC-3’ (VEC), 5’-TTTATGGTGTGGGCCAAAG-3’ and 5’-CCGCTTCATGCGCTTCACCT-3’ (Sox17), and 5’-CAGCCTGGCTGGCTACGTACA-3’ and 5’-CCAGGGTGTGATGGTGGGAA-3’ (β-actin). Sox17 -transduced cells were transferred onto glass slides using a cytospin (Shandon, Sewickley, PA), and their morphology was examined by May-Grünwald-Giemsa staining. Additionally, Sox17 -transduced cells were embedded in Methocult (M3434; StemCell Technologies, Vancouver, Canada) and individual colonies were counted based on morphology after 7 days of culture. RNA sequencing analysis For RNA sequencing analysis, CD45 low c-Kit high AGM cells and BM KLS cells transduced with the Sox17-ERT-IRES-GFP gene were cultured with or without tamoxifen. After 11 days, GFP + cells were sorted, and total RNA was extracted from each sample using RNeasy Plus Mini Kit (Qiagen, Germany). The RNA samples were then sent to AZENTA Japan Corp. (Tokyo, Japan) for RNA sequencing using the Illumina HiSeq/Nova seq platform and 2 × 150 bp configuration. All raw RNA sequencing data have been deposited in the DDBJ Sequence Read Archive (DRA) under the BioProject accession number PRJDB20795 and Run accession numbers DRR683968-DRR683971. Distribution analyses of Sox17 and c-Kit proteins in Sox17 -transduced cells After 11 days of the transduction with the Sox17-ERT-IRES-GFP gene into CD45 low c-Kit high AGM cells cultured with or without tamoxifen, the non-adherent Sox17-ERT-IRES-GFP -transduced cells were collected from the respective cultures under a microscope and transferred onto glass slides using a cytospin (Shandon, Sewickley, PA). Both the cells on the glass slides and the remaining adherent cells in the culture dishes were fixed in 2% paraformaldehyde for 10 min. They were then treated with PBS containing 1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100, 2% (w/v) bovine serum albumin (PBS-MT/BSA) for 1 hr. The cells were incubated overnight at 4 ˚C with a rat anti-mouse CD117 (c-Kit) antibody (2B8; eBioscience). After washing three times with PBS-MT, they were stained with Alexa Fluor® 488-conjugated donkey anti-rat IgG (Life Technologies, Carlsbad, CA) in PBS-MT. Imaging was performed using the BIOREVO microscopy (BZ-X810; KEYENCE, Osaka, Japan). Whole-mount immunohistochemistry Whole-mount immunohistochemistry was performed according to a previously reported protocol [ 23 , 24 , 29 ]. E10.5 mouse embryos were fixed in 2% PFA-PBS for 20 min and then dehydrated in methanol. After removal of the left body wall between the forelimb and hindlimb, these tissues were rehydrated in PBS and pretreated with PBS-MT at 4 ˚C for 1 hr. The tissues were stained overnight at 4˚C in PBS-MT with rat anti-mouse CD117 (c-Kit) antibody (2B8), goat anti-mouse EPCR (encoded by a Procr gene) antibody (R&D, Minneapolis, MN), and rabbit-anti CD31 antibody (ab28364, Abcam, Cambridge, UK), or a rabbit anti-SOX17 antibody (EPR20684, Abcam). After washing with PBS-MT three times, the tissues were further stained overnight at 4˚C in PBS-MT with Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (Life Technologies), Alexa Fluor® 546-conjugated donkey anti-goat IgG (Life Technologies), and Alexa Fluor® 647-conjugated donkey anti-rat IgG (Jackson ImmunoResearch, West Grove, PA). After washing with PBS-MT, the tissues were treated with Hoechst 33258 (Nacalai Tesque, Kyoto, Japan) and then dehydrated in methanol. These dehydrated tissues were incubated for 3 min in a 1:1 mixture of methanol and BABB (a 1:2 mixture of benzyl alcohol and benzyl benzoate), followed by a 3 min treatment with 100% BABB. The stained tissues were examined by confocal microscopy (LSM710, Carl Zeiss, Oberkochen, Germany). Loss-of-function analysis of the Procr gene in Sox17 -transduced cells The Sox17-IRES-mCherry gene was introduced into CD45 low c-Kit high AGM cells. After three passages in the coculture with OP9 stromal cells, retrovirus-mediated introduction of short hairpin RNA (shRNA) against the Luciferase (Luc) gene (shLuc) or the Procr gene (shProcr) driven by the U6 promoter, along with a GFP gene driven by the SV40 promoter, was performed in Sox17-IRES-mCherry -transduced cells. Following three additional passages of shRNA-transduced cells, the expression of the Procr gene in GFP + cells was analyzed by RT-PCR. The sorted GFP + cells were cocultured with fresh OP9 cells and after 4 days of the culture, the number of the non-adherent cell clusters was counted. Moreover, the colony-forming ability of GFP + cells was assessed by Methocult (M3434). shRNAs targeted the following sequences: 5'-ACTTACGCTGAGTACTTCG-3' (shLuc), and 5'-GTGTGGAGTTCCTGGAGAA-3'. (shProcr) Results The cluster-forming ability of Sox17-IRES-GFP -transduced HSPCs decreases during development We previously showed that the introduction of the transcription factor Sox17 into HSPC-containing CD45 low c-Kit high cells from the AGM region in midgestation mouse embryos maintained the formation of cell clusters with the hematopoietic ability [ 21 , 23 , 24 , 27 , 28 ]. It remained unclear whether the Sox17 overexpression could similarly sustain the cluster-forming activity in HSPCs from the FL and BM. We retrovirally introduced either the Sox17-IRES-GFP gene or the control IRES-GFP gene into CD45 low c-Kit high cells of the AGM region, as well as linage − c-Kit + Sca-1 + (KLS) HSPCs from the FL and BM, and these virus-infected cells cocultured with OP9 stromal cells. On day 11 after infection, cell clusters were observed in Sox17 -transduced cells from the FL and BM as well as from the AGM region (Fig. 1 A). The number of cell clusters formed by Sox17 -transduced cells decreased as embryonic development progressed (Fig. 1 B). The hematopoietic potential is maintained in Sox17-IRES-GFP -transduced cells from the FL and BM We assessed the hematopoietic potential of Sox17 -transduced cells using the colony-forming assay. Eleven days after retroviral infection, sorted GFP + cells were embedded in a methylcellulose medium. The number of total and mixed colonies containing three lineages (granulocytes, macrophages, and erythrocytes) was increased in Sox17 -transduced cells from the AGM region, the FL, and the BM, indicating enhanced multilineage differentiation potential (Fig. 1 C). By examination of May-Grünwald-Giemsa staining, IRES-GFP -transduced cells predominantly exhibited the morphology of granulocytes and macrophages. In contrast, Sox17-IRES-GFP -transduced cells showed a more blastic morphology characterized by the round nuclei and a small proportion of the cytoplasm (Fig. 1 D). Moreover, the expression of hematopoietic transcription factors (GATA-2, c-Myb, and Runx1) and adhesion molecules (CD31 and VEC) was maintained in Sox17 -transduced cells from FL and BM (Fig. 1 E). Increased expression of adhesion molecules in Sox17 -transduced BM cells was previously reported [ 25 ]. These findings indicate that the introduction of the Sox17 gene into HSPCs from the FL and BM supports both their hematopoietic ability and cluster formation. The cluster-forming ability of Sox17-ERT-IRES-GFP -transduced HSPCs in the presence of tamoxifen decreases with the development Next, to examine the importance of the Sox17 transcriptional activity in maintaining the cell cluster formation with hematopoietic ability, we introduced the Sox17-ERT-IRES-GFP gene into CD45 low c-Kit high AGM cells and HSPCs from the FL and BM. It was previously reported that Sox17-ERT-IRES-GFP -transduced cells exhibit tamoxifen-induced nuclear translocation of the fusion protein [ 24 , 30 ]. For the first 4 days of the culture, the cells were maintained in the presence of tamoxifen, after which they were cultured in the media with or without tamoxifen. After 11 days of culture, Sox17-ERT-IRES-GFP -transduced cells from the AGM region, the FL, and the BM formed cell clusters in the presence of tamoxifen (Fig. 2 A). However, the number of cell clusters formed in response to Sox17 nuclear translocation decreased with developmental stage, with only a few cell clusters observed in BM-derived cells (Fig. 2 B). We further examined the colony-forming ability of Sox17-ERT-IRES-GFP -transduced cells with or without tamoxifen. An increased number of total and mixed colonies was observed in Sox17-ERT-IRES-GFP -transduced cells from the AGM region, the FL, and the BM in the presence of tamoxifen (Fig. 2 C). The morphology of Sox17-IRES-GFP -transduced cells cultured with tamoxifen resembled that of cells transduced with Sox17-IRES-GFP gene, whereas cells cultured without tamoxifen showed morphologies similar to those of IRES-GFP -transduced cells (Fig. 1 D and Fig. 2 D). RT-PCR analysis demonstrated that the expression of GATA-2, c-Myb, and Runx1 was maintained in Sox17-ERT-IRES-GFP -transduced cells derived from the AGM region and the FL in the presence of tamoxifen. In BM-derived cells, the expression of GATA-2 and c-Myb was retained under the same conditions (Fig. 2 E). Tamoxifen also maintained the expression of adhesion molecules CD31 and VEC in Sox17-ERT-IRES-GFP -transduced cells (Fig. 2 E). These results indicate that the tamoxifen-inducible nuclear translocation of the Sox17-ERT fusion protein maintains the cluster formation with the hematopoietic ability and gene expression in HSPCs from the FL and BM, although this effect diminishes with development. The difference in the features of Sox17-ERT -transduced colonies, adherent colonies, and non-adherent colonies caused by the tamoxifen-inducible nuclear translocation Tamoxifen-dependent nuclear translocation of Sox17 in Sox17-ERT -transduced cells of the AGM region on the stromal cells found three types of cell morphology; colonies (the flat colony), adherent clusters (the cluster which is attached to OP9 stromal cells), and non-adherent clusters (the cluster which floats in the medium) (Fig. 3 A). The number of Sox17-ERT-IRES-GFP -transduced non-adherent clusters derived from the AGM region was increased in a tamoxifen concentration-dependent manner, whereas the number of colonies was decreased (Fig. 3 A). To examine the ratio of Sox17-nuclear translocation in a tamoxifen dependent fashion, the Sox17-ERT-IRES-GFP gene was introduced in NIH3T3 cells in medium containing various concentrations of tamoxifen. The Sox17-ERT protein was detected in the cytoplasm of NIH3T3 cells cultured in the absence of tamoxifen, whereas the Sox17-ERT protein was almost exclusively found in the nucleus of the NIH3T3 cells in the culture with 10 µM tamoxifen (Fig. 3 B). We showed a reduction of Sox17 in the cytoplasm at higher tamoxifen concentration, whereas the Sox17 nuclear translocation was found to increase at high concentrations of tamoxifen (Fig. 3 B). These results revealed the relationship between the Sox17 nuclear translocation and the cluster formation. To examine the c-Kit expression in colonies, adherent clusters, and non-adherent clusters of Sox17-ERT-IRES-GFP -transduced cells of the AGM region in the presence and absence of tamoxifen, we performed the immunostaining after 11 days of the culture. As shown in Fig. 3 C, the c-Kit expression was not observed in the colonies, while c-Kit was expressed in adherent and non-adherent clusters by the Sox17-ERT nuclear localization in the presence of tamoxifen (Fig. 3 C). We examined the colony-forming ability of non-adherent cells and adherent cells in Sox17-ERT -transduced cells with tamoxifen. After 11 days of the culture, the GFP + cluster cells were directly recovered under the microscope, and the medium, in which some GFP + clusters remained, was discarded from the dish. The new medium was added to the dish and the adherent cells containing adherent clusters and colony-forming cells were recovered after the pipetting. The colony-forming abilities of non-adherent cells and adherent cells in the presence of tamoxifen are high compared to cells in the absence of tamoxifen (Fig. 3 D). There was little difference in the colony-forming ability between Sox17 -transduced non-adherent cells and adherent cells. Identification of candidate genes responsible for reduced the cluster-forming ability of Sox17 -transduced AGM cells by RNA sequencing Our present results showed that the cluster-forming ability induced by the Sox17 introduction decreased during developmental progression. However, specific genes responsible for promoting cluster formation in the AGM region and the mechanisms by which Sox17 induced the process remain unclear. To identify candidate genes involved in this activity, we, conducted RNA sequencing (RNA-seq) analysis on four distinct cell populations: Sox17-ERT -transduced AGM cells cultured with tamoxifen (AGM Tam (+)) and without tamoxifen (AGM Tam (-)) and Sox17-ERT -transduced BM cells cultured with tamoxifen (BM Tam (+)) and without tamoxifen (BM Tam (-)). All RNA-seq data from this study have been deposited in the DDBJ Sequence Read Archive (DRA) under the BioProject accession number PRJDB20795, with individual Run accession numbers DRR683968-DRR683971. The number of significant differentially expressed genes (DEGs) identified in each pairwise comparison is shown in Fig. 4 A. Heatmaps illustrating gene expression patterns across the four cell populations are presented in Fig. 4 B. The Venn diagram revealed 318 overlapping genes that were differentially expressed both between AGM Tam (+) and AGM Tam (-), and between AGM Tam (+) and BM Tam (+) (Fig. 4 C). From the RNA-seq data, we selected 85 candidate genes that were up-regulated in Sox17-ERT -transduced AGM cells cultured with tamoxifen, compared to both the BM cells cultured with tamoxifen and AGM cells cultured without tamoxifen (Fig. 4 D). Involvement of one candidate gene, the Procr gene, in the cluster formation with hematopoietic potential in AGM-derived HSPCs To examine the role of these candidate genes in the cluster formation of Sox17 -transduced cells, we focused on the Procr gene, which encodes the endothelial protein C receptor protein (EPCR). EPCR is known as the transmembrane glycoprotein expressed on the endothelial cells and initiates the anticoagulant pathway by activating protein C [ 31 ]. A previous study reported that EPCR, along with CD31 and c-Kit, marks HSCs in the E11.5 AGM region [ 32 ]. We first confirmed the expression of the Procr gene by RT-PCR and found that the Procr1 expression was upregulated in Sox17 -transduced AGM cells cultured with tamoxifen (Fig. 5 A). Next, we analyzed the EPCR expression in IAHCs in the dorsal aorta of the E10.5 mouse embryo by the whole-mount immunohistochemistry. The EPCR expression partially overlapped with that of c-Kit + IAHCs and Sox17-expressing cells in the dorsal aorta (Fig. 5 B). To examine the functional role of EPCR in the cluster formation, we introduced shRNA against the Procr gene into Sox17 -transduced AGM cells. Specifically, Sox17-IRES-mCherry -transduced AGM cells were retrovirally infected with either control shRNA against control luciferase (shLuc) or shRNA against Procr (shProcr), both of which co-expressed the GFP gene. Eleven days after shRNA introduction, RT-PCR analysis confirmed that the Procr gene expression was lower in shProcr-transduced cells compared to shLuc-transduced cells (Fig. 5 C). The shProcr-transduced cells exhibited fewer cell clusters than shLuc-transduced cells (Fig. 5 D). Moreover, the colony-forming ability of shProcr-transduced cells was also significantly diminished relative to shLuc-transduced cells (Fig. 5 E). These results indicate that reduced expression of the Procr gene decreases both the cell cluster-forming ability and the hematopoietic ability in Sox17 -transduced cells from the AGM region. Discussion We previously showed that Sox17 -transduced cells from the E10.5 AGM region maintained both the cluster-forming ability and the hematopoietic ability in in vitro cultures [ 21 , 23 , 24 , 27 , 28 ]. The introduction of Sox17 or tamoxifen-inducible Sox17-ERT into HSPCs from the FL and BM revealed that the hematopoietic potential was maintained in both Sox17 -transduced cells and Sox17-ERT -transduced cells following a tamoxifen-induced nuclear translocation (Figs. 1 and 2 ). We found that Sox17 -transduced cells derived from IAHC cells in the AGM region and HSPCs in the FL and BM exhibit cluster-forming ability. This cluster-forming ability declined during development (Figs. 1 and 2 ). In midgestation mouse embryos, IAHC cells expressed the c-Kit, a marker also found in HSPCs of the FL and BM [ 6 ]. Mice deleted for Runx-1, which is an essential transcription factor for definitive hematopoiesis, showed no IAHCs in the dorsal aorta and embryonic lethality [ 6 , 33 ]. Conditional knockout mice of GATA2 in VEC + endothelial cells also results in a reduced number of IAHCs in the dorsal aorta [ 7 ]. These results indicate the requirement of IAHCs for the emergence of HSPCs in the dorsal aorta. Moreover, adherent molecules such as VEC and CD31 are expressed in basal cells of IAHCs in the dorsal aorta [ 6 , 24 ]. The expression of c-Kit was also observed in the cluster cells induced by nuclear translocation of Sox17, similarly to its expression in IAHC cells (Fig. 3 C). In the absence of the Sox17 nuclear translocation, flat colonies were formed, while the Sox17 nuclear translocation resulted in the formation of nonadherent and adherent clusters, which had the higher hematopoietic ability (Fig. 3 D). Thus, there were many similarities between IAHCs in the dorsal aorta and the cluster cells formed by the Sox17 nuclear transport. In this study, we found that the ability of Sox17 -transduced HSPCs to form cell clusters decreased as development progressed. This reduction likely reflects the developmental stage-specific requirement for Sox17 in HSC maintenance. The BM, but not the AGM, has niche cells that support this maintenance of quiescent HSCs [ 34 ]. We hypothesized that the differences in the cell cluster-forming ability were caused by an effect of Sox17 on the induced expression of adhesion molecules [ 24 ], even though the Sox17-induced molecules are known to function in the BM [ 25 ]. To identify the molecular basis of this phenomenon, RNA-seq was performed on Sox17-ERT -transduced tamoxifen-treated AGM HSPCs, which have the highest cluster-forming ability, and Sox17-ERT -transduced tamoxifen-treated BM HSPCs, which have the lowest cluster-forming ability. The candidate genes were highly expressed in the Sox17 -transduced cells containing AGM HSPCs, which had the highest cluster-forming capacity. The Procr gene, which is one of their candidate genes, is expressed in IAHC cells of the dorsal aorta and is associated with the colony-forming ability in the Sox17 -transduced cells in the AGM region (Fig. 5 ). The EPCR protein encoded by the Procr gene in HSPCs is first observed in the BM, where it marks a population enriched in LT-HSCs [ 35 ]. In the FL, the EPCR expression is also observed in HSPCs and the stem cell ability was maintained in EPCR-expressing HSPCs after a few days of in vitro culture [ 36 ]. In the AGM region of E11.5 mouse embryos, the EPCR expression level in CD31 + c-Kit + cells correlates with their long-term repopulation capacity [ 32 ]. In the present study, the Procr expression was elevated in Sox17-ERT -transduced AGM cells cultured with tamoxifen-induced nuclear translocation of Sox17, compared with Sox17-ERT -transduced BM HSPCs (Figs. 4 and 5 A). The Sox17 expression is significantly higher in EPCR-expressing FL HSPCs compared to EPCR-nonexpressing HSPCs, and the Sox17 expression markedly decreased in both EPCR-expressing and EPCR-nonexpressing HSPCs after 1 day of in vitro culture [ 36 ]. These findings suggest a functional association between Sox17 and Procr expression. Our data indicate that the Procr gene expression in Sox17 -transduced cells was involved in the cluster formation with the hematopoietic ability (Fig. 5 C and D). The molecular mechanism of the Procr gene will be analyzed in the future and other candidate genes will be examined for their effects on hematopoietic and cluster-forming potentials. Conclusion The introduction of a transcription factor Sox17 into HSPCs prepared from the AGM region, the FL, and the BM maintained the hematopoietic potential but led to a progressive decline in the cluster-forming ability during development. Comparative analysis of gene expression between Sox17 nuclear translocation in the AGM cells and the BM revealed that the Procr gene has a function in the cluster formation with hematopoietic ability in Sox17 -transduced AGM cells. Abbreviations HSCs hematopoietic stem cells IAHCs intra-aortic hematopoietic cell clusters AGM aorta-gonad-mesonephros FL fetal liver BM bone marrow HSPCs hematopoietic stem/progenitor cells E embryonic day VEC vascular-endothelial cadherin ESAM endothelial cell-selective adhesion molecules DMEM Dulbecco’s modified Eagles medium KLS lineage − c-Kit + Sca-1 + RT-PCR reverse transcription-polymerase chain reaction Tam tamoxifen EPCR endothelial protein C receptor protein shRNA short hairpin RNA Luc luciferase Declarations Ethics approval and consent to participate Not applicable to this review manuscript. Consent for publication Not applicable to this review manuscript. Competing interests Author Ayumi Itabashi is now an employee of Daiichi Sankyo. Funding This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant numbers 26440118 and 18K06249 (I.N.), 22130008, 15H04292, and 18H02678 (T.T.)) and Nanken-Kyoten (Grant numbers, H26-A39, H27-A35, H28-A11), Tokyo Medical Dental University. Authors contributions IA, YY, KS, RT, GM, KA, NI, and IN conducted the in vivo and in vitro experiments. IA and IN analyzed the data of the RNA sequence. IA, TT, and IN planned the experiments. IA, TT, and IN wrote the manuscript. All the authors read and approved the final manuscript. Acknowledgments We thank Dr. T. Nakano for the OP9 cells, Dr. K. Tabu for valuable discussions, and Ms E. Wada, Mr. K Nakamura, Dr. H. Takebayashi for their technical support. We also thank K. Inoue for secretarial assistance. Availability of data and materials. Further information and requests for resources and reagents should be directed to the author: Ikuo Nobuhisa ( [email protected] ). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7069210","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498270525,"identity":"7050abff-7af1-4968-94db-83b6ecfd90cd","order_by":0,"name":"Ayumi Itabashi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ayumi","middleName":"","lastName":"Itabashi","suffix":""},{"id":498270526,"identity":"6f054e11-0391-4fd3-a730-2334120a5a06","order_by":1,"name":"Yuki Yokoi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Yokoi","suffix":""},{"id":498270527,"identity":"7bbeedb7-9618-4496-8926-30787c7e000f","order_by":2,"name":"Kiyoka saito","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kiyoka","middleName":"","lastName":"saito","suffix":""},{"id":498270528,"identity":"60c97613-5492-4b79-8330-3b297cc04e9f","order_by":3,"name":"Ryota Tsukahara","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ryota","middleName":"","lastName":"Tsukahara","suffix":""},{"id":498270529,"identity":"577e96ac-a6c6-46e1-97be-c748545c2302","order_by":4,"name":"Gerel Melig","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gerel","middleName":"","lastName":"Melig","suffix":""},{"id":498270530,"identity":"a38bcb20-3d2f-4ab2-9be4-f97adfc80248","order_by":5,"name":"Koya Azuma","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Koya","middleName":"","lastName":"Azuma","suffix":""},{"id":498270531,"identity":"4b38aace-b300-41a7-abc8-a979d6d7c5d7","order_by":6,"name":"Naoki Iizuka","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Iizuka","suffix":""},{"id":498270532,"identity":"daf8f46b-7835-43c8-ac4f-bc41c4380e60","order_by":7,"name":"Tetsuya Taga","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tetsuya","middleName":"","lastName":"Taga","suffix":""},{"id":498270533,"identity":"fd0c9f5d-7254-464f-be75-968cfac6406b","order_by":8,"name":"IKUO NOBUHISA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYFACHgZmBgMGBn4GII0MmHGoR2iRbCBNCxAYHMCnCBmYs/ceky4oqJM3vt382OBnzuF8/gYeA4YfNQzs5ji0WPacS5OeYXDYcNudY8aJvdsOW844wGPA2HOMgdmyAbsWgxs5ZtI8BgcYt91IMD7Au+2wAcP9NwYMvECfAZ2KT0ud/eYZ6Z8P/gVqkQfZ8pewFubEDRI5xskgWwyAWpjx2nLmXLI1j8Hh5Bk3coqNZbelGxgeYCs4LHNMArdfjvcevM3zp862f0b6Zsm326wN5A4wb3z4psYmGVeIYQdAJ0kkG5CkBQTsSNcyCkbBKBgFwxQAAK4iVN1qyMHGAAAAAElFTkSuQmCC","orcid":"","institution":"Nakamura Gakuen University: Nakamura Gakuen Daigaku","correspondingAuthor":true,"prefix":"","firstName":"IKUO","middleName":"","lastName":"NOBUHISA","suffix":""}],"badges":[],"createdAt":"2025-07-08 00:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7069210/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7069210/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41232-026-00420-w","type":"published","date":"2026-04-23T15:59:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89094205,"identity":"c9eb9045-dc93-4c0e-b5dd-03f62a25177d","added_by":"auto","created_at":"2025-08-14 15:11:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131915,"visible":true,"origin":"","legend":"\u003cp\u003eA low ability to form cell clusters of \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced HSPCs from the BM. \u003cstrong\u003eA, B\u003c/strong\u003e \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced HSPCs prepared from the AGM region, the formed cell clusters. Four days after transduction with either the\u003cem\u003e IRES-GFP\u003c/em\u003e (Mock) or the \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e (Sox17), the GFP\u003csup\u003e+\u003c/sup\u003e cells were cocultured with OP9 stromal cells. After 7 days of the introduction, the GFP\u003csup\u003e+\u003c/sup\u003e cells (5 × 10\u003csup\u003e2\u003c/sup\u003e cells) were replated onto new OP9 stromal cells. After 11 days of the introduction, the number of cell clusters was counted. \u003cstrong\u003eA\u003c/strong\u003e Morphologies of \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells or \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells derived from IAHC cells in the AGM region and HSPCs from the FL and BM cocultured with OP9 stromal cells, after 11 days of the introduction. Bars = 100 mm. \u003cstrong\u003eB\u003c/strong\u003e Number of cell clusters in \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells or \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells (n = 4, the AGM region; n = 3, the FL; n = 4, the BM). \u003cstrong\u003eC\u003c/strong\u003e Colony-forming ability of \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells or \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells. After 11 days of the introduction, \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells or \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells (1 × 10\u003csup\u003e3\u003c/sup\u003e cells) were cultured in Methocult (M3434). The number of total and multilineage colonies was scored after 7 days of culture (n = 4, the AGM region; n = 5, the FL; n = 3, the BM). \u003cstrong\u003eD\u003c/strong\u003e Morphologies of \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells or \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells were shown by Giemsa staining. Bars = 50 mm. \u003cstrong\u003eE\u003c/strong\u003e RT-PCR analysis of marker gene expression in \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells and \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells derived from IAHCs in the AGM region and HSPCs from the FL, and the BM, cocultured with OP9 stromal cells.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/5918a0c423165e29fdb58483.jpg"},{"id":89096187,"identity":"1dc3207b-78eb-4f17-bde4-105a82e98882","added_by":"auto","created_at":"2025-08-14 15:27:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143937,"visible":true,"origin":"","legend":"\u003cp\u003eA low ability to form cell clusters of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced tamoxifen-treated HSPCs from the BM. \u003cstrong\u003eA, B\u003c/strong\u003e \u003cem\u003eSox17-ERT-IRES-GF\u003c/em\u003eP-transduced cells derived from IAHCs cells of the AGM region and HSPCs from the FL and BM formed cell clusters on tamoxifen-induced nuclear translocation of Sox17. Four days after the introduction of the \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e construct, the GFP\u003csup\u003e+\u003c/sup\u003e cells were co-cultured with OP9 stromal cells with tamoxifen. After 7 days of the introduction, the GFP\u003csup\u003e+\u003c/sup\u003e cells (1 × 10\u003csup\u003e3\u003c/sup\u003e cells from AGM, 3 × 10\u003csup\u003e3\u003c/sup\u003e cells from FL and BM) were cultured with new OP9 stromal cells in media with or without tamoxifen. After 11 days of the introduction, the number of cell clusters was counted. \u003cstrong\u003eA\u003c/strong\u003e Morphologies of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells from IAHCs in the AGM region and HSPCs from the FL and BM cocultured with OP9 stromal cells with or without tamoxifen, are shown, after 11 days of the introduction. Bars = 100 mm. \u003cstrong\u003eB\u003c/strong\u003e Number of cell clusters in \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells in the culture with or without tamoxifen (n = 3, the AGM region; n = 5, the FL; n = 5, the BM). \u003cstrong\u003eC\u003c/strong\u003e Colony-forming ability of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells cultured with or without tamoxifen. After 11 days of the introduction, \u003cem\u003eSox17-ERT-IRES-GF\u003c/em\u003eP-transduced cells in the culture with or without tamoxifen were cultured in Methocult (M3434). The number of total and multilineage colonies was scored after 7 days of culture (n = 3, the AGM region; n = 6, the FL; n = 5, the BM). \u003cstrong\u003eD\u003c/strong\u003e Morphologies of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells cultured with or without tamoxifen showed by Giemsa staining. Bars = 50 mm. \u003cstrong\u003eE\u003c/strong\u003e RT-PCR analysis of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells derived from IAHCs in the AGM region and HSPCs from the FL and BM cocultured with or without tamoxifen.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/745cbdbadc45211d6f219c15.jpg"},{"id":89094694,"identity":"ea700f2f-cc51-47fc-947c-bf3244e25017","added_by":"auto","created_at":"2025-08-14 15:19:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129032,"visible":true,"origin":"","legend":"\u003cp\u003eCluster cells resulting from Sox17 nuclear translocation in the presence of tamoxifen have a higher hematopoietic ability than the colony cells in the absence of tamoxifen. \u003cstrong\u003eA\u003c/strong\u003e The frequency of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced colonies, adherent clusters, and non-adherent clusters derived from the AGM region varied depending on the concentration of tamoxifen. Morphologies of \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced AGM cells, which are termed colony, adherent cluster, and non-adherent cluster were shown, after 4 days of the co-culture with OP9 stromal cells with 0.2, 0.5, 1.0 mM tamoxifen. Bars = 100 mm. The number of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced colonies, adherent clusters, and non-adherent clusters was counted, after 4 days of the co-culture with stromal cells with 0.2, 0.5, and 1.0 mM tamoxifen. \u003cstrong\u003eB\u003c/strong\u003e Subcellular localization of Sox17-ERT protein in NIH3T3 cells with or without tamoxifen. The proportion of cells exhibiting nuclear versus cytoplasmic Sox17 localization was assessed at various tamoxifen concentrations. Arrowheads in the left photograph showed the distribution of Sox17 in the cytoplasm. White arrowheads in the right photograph indicated the presence of Sox17 in the nucleus. \u003cstrong\u003eC\u003c/strong\u003e Expression of c-Kit in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced AGM cells forming adherent and non-adherent clusters in cultures with tamoxifen. Bars = 50 mm. \u003cstrong\u003eD\u003c/strong\u003e No significant difference was observed in colony-forming capacity between adherent and non-adherent clusters. After the last 4 days of the introduction of \u003cem\u003eSox17\u003c/em\u003e genes in the culture of the AGM region with or without tamoxifen, non-adherent clusters with tamoxifen were collected under microscopy, and the remaining adherent cells containing adherent clusters were collected by pipetting. Similarly, cells from the \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced colony were collected by pipetting. GFP\u003csup\u003e+\u003c/sup\u003e cells were embedded in Methocult (M3434). After 7 days of the culture, the number of total and multilineage colonies was counted after 7 days of culture (n = 3, AGM; n = 5, FL; n = 6, BM).\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/9a12ae6873940df16883a17d.jpg"},{"id":89094209,"identity":"9caab9c8-0adb-4aa9-aaa0-410b6eac1637","added_by":"auto","created_at":"2025-08-14 15:11:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":113168,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of genes that were highly expressed in AGM-derived cells with the Sox17 nuclear translocation\u003cstrong\u003e \u003c/strong\u003eby RNA-seq.\u003cstrong\u003eA \u003c/strong\u003eThe number of DEGs\u003cstrong\u003e \u003c/strong\u003efrom \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced AGM cells in the culture with tamoxifen (AGM Tam(+)) vs \u003cem\u003eSox17-ER\u003c/em\u003eT-transduced BM cells in the culture with tamoxifen (BM Tam(+)), \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced AGM cells in the culture without tamoxifen (AGM Tam(-)) vs AGM Tam(+), AGM Tam(-) vs S\u003cem\u003eox17-ERT\u003c/em\u003e-transduced BM cells in the culture without tamoxifen (BM Tam(-)), and BM Tam(-) vs BM Tam(+). Blue columns indicate upregulated genes, and red bars indicate downregulated genes in the comparison between two populations. Red columns showed genes whose expression was downregulated by comparison between two cell populations. \u003cstrong\u003eB \u003c/strong\u003eHeatmaps show the hierarchical clustering of DEGs among four cell populations. Each row represents a gene, and each column shows a sample. The color scale represents the raw Z-score of gene expression levels, ranging from blue (low expression) to red (high expression). \u003cstrong\u003eC\u003c/strong\u003e Venn diagram displaying the intersection of DEGs among the comparisons: \u0026nbsp;AGM Tam(+) vs BM Tam(+), AGM Tam(-) vs AGM Tam(+), AGM Tam(-) vs BM Tam(-), and BM Tam(-) vs BM Tam(+). The numbers in each overlapping region represent the number of shared DEGs. \u003cstrong\u003eD \u003c/strong\u003eGenes that were upregulated specifically in AGM Tam(+) cells compared to both AGM Tam(-) and BM Tam(+) cells were selected for further analysis.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/4efde619e7e381958448d3f3.jpg"},{"id":89094696,"identity":"deb3e2e1-45a4-414c-a680-b03ba1e384e4","added_by":"auto","created_at":"2025-08-14 15:19:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":72884,"visible":true,"origin":"","legend":"\u003cp\u003eInvolvement of the \u003cem\u003eProcr\u003c/em\u003e gene in the cluster-forming ability and the hematopoietic ability of \u003cem\u003eSox17\u003c/em\u003e-transduced AGM cells \u003cstrong\u003eA\u003c/strong\u003e RT-PCR analysis of the \u003cem\u003eProcr \u003c/em\u003egene expression in \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells derived from IAHC cells in the AGM region, cultured for 4 days with or without tamoxifen. \u003cstrong\u003eB\u003c/strong\u003e Expression of EPCR, encoded by the \u003cem\u003eProcr\u003c/em\u003e gene, in IAHCs of the dorsal aorta in E10.5 mouse embryos. Whole embryos were stained with antibodies against c-Kit (cyan), EPCR encoded by the \u003cem\u003eProcr\u003c/em\u003e gene (red), and either CD31 or Sox17 (green). Bars = 20 mm. \u003cstrong\u003eC\u003c/strong\u003e RT-PCR analysis of the \u003cem\u003eProcr\u003c/em\u003e gene expression in \u003cem\u003eSox17-IRES-mCherry\u003c/em\u003e-transduced cells co-transduced with control Luc-shRNA (shLuc) or Procr-shRNA (shProcr). PCR amplification was carried out with 32 and 35 cycles for the \u003cem\u003eProcr\u003c/em\u003e gene and 28 cycles for the \u003cem\u003eb-actin\u003c/em\u003e gene. \u003cstrong\u003eD\u003c/strong\u003e Number of cell clusters formed by \u003cem\u003eSox17\u003c/em\u003e-transduced cells after the introduction of either shLuc or shProcr. GFP\u003csup\u003e+\u003c/sup\u003e cells (1 × 10\u003csup\u003e3\u003c/sup\u003e), transduced with Sox17 and either shRNA, were co-cultured with OP9 stromal cells. The number of clusters was counted after 11 days of the culture. \u003cstrong\u003eE\u003c/strong\u003e Colony-forming ability of \u003cem\u003eSox17\u003c/em\u003e-transduced cells following the introduction of shLuc or shProcr. After 11 days of the introduction of shLuc or shProcr to \u003cem\u003eSox17\u003c/em\u003e-transduced cells, \u003cem\u003eSox17\u003c/em\u003e and shRNA-transduced GFP\u003csup\u003e+\u003c/sup\u003e cells (3 × 10\u003csup\u003e3\u003c/sup\u003e cells) were embedded in Methocult (M3434). The number of multilineage colonies was scored after 7 days of culture.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/ef510e536758a0f2d1302dbc.jpg"},{"id":107928579,"identity":"e3ab532f-c6ee-4de6-a462-aea959eb887a","added_by":"auto","created_at":"2026-04-27 16:11:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":982122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/efb04a5f-ecec-4794-a49e-77bc48d66e3e.pdf"},{"id":89094697,"identity":"479e3100-2108-4899-aab6-dbedc45172a8","added_by":"auto","created_at":"2025-08-14 15:19:47","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4377193,"visible":true,"origin":"","legend":"","description":"","filename":"RTPCROriginalphotos.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7069210/v1/efb2a2573fd064b4162b8db2.pptx"}],"financialInterests":"","formattedTitle":"Ability to form hematopoietic stem/progenitor cell-containing cell clusters weakens during the fetal-to-adult transition in hematopoietic development","fulltext":[{"header":"Background","content":"\u003cp\u003eIn mammals including mice, the sites of embryonic hematopoiesis change over the course of development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Hematopoietic stem cells (HSCs), which possess the long-term repopulating ability, initially emerge in the aorta-gonad-mesonephros (AGM) region at embryonic day (E) 10.5 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. After the production of HSCs in the placenta around E12.5 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], HSCs expand in the fetal liver (FL) and finally migrate to the bone marrow (BM). In the AGM region, HSCs are observed in the intra-aortic hematopoietic cell clusters (IAHCs), which arise from the hemogenic endothelium in the dorsal aorta [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The hemogenic endothelium is known to give rise to both hematopoietic cells and endothelial cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Whole-mount immunohistochemical analysis revealed that c-Kit, a marker of HSCs, is expressed in IAHCs. The protein expression of CD31 and vascular-endothelial cadherin (VEC), both of which are endothelial markers, and that of CD45, a hematopoietic cell marker, varies depending on the position of the cells within IAHCs. CD31 and VEC are strongly expressed in basal cells in IAHCs, supporting their emergence from hemogenic endothelium [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], whereas CD45 is expressed in apical cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], indicating that hematopoietic differentiation of blood cells occurs within IAHCs. Moreover, the expression levels of CD31, VEC, and CD45 differ among the HSCs in the IAHCs, the FL, and the BM [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Embryonic HSCs have a high proliferative capacity to supply blood cells. In contrast, BM HSCs, which exhibit altered expression of several proteins compared to IAHCs and/or FL HSCs, enter a quiescent state upon interaction with niche cells in the BM [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSox17 is a transcription factor, known as an endodermal marker [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Sox17-deficient mice exhibit defects in gut tube formation and die during midgestation in mouse embryos [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mice heterozygous for Sox17 show a biliary atresia-like phenotype, hepatitis with aberrant cell wall formation in the gallbladders [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e–\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and female subfertility associated with implantation failure [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, conditional knockout analyses have revealed that the deletion of Sox17 in the immediate postpartum period significantly reduces the absolute number of HSCs, although Sox17 does not affect the HSC numbers in the BM [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Whole-mount immunohistochemistry of the AGM region has shown that Sox17 is expressed in endothelial cells of the dorsal aorta and IAHC cells from the E10.5 AGM region, which are positioned close to endothelial cells in E10.5 embryos [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. When Sox17 is introduced into IAHC cells from the E10.5 AGM region, HSC-containing \u003cem\u003eSox17\u003c/em\u003e-transduced cells can be maintained through multiple passages in the co-culture of stromal cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, the transcription factor Sox17 directly induces the Notch1 expression, followed by increased expression of the Notch1-downstream molecule Hes1 to sustain the hematopoietic activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Sox17 also directly enhances the expression of VEC and endothelial cell-selective adhesion molecules (ESAM), promoting the formation of hematopoietic cell clusters with hematopoietic potential [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although Sox17 is not expressed in BM HSCs, the ectopic expression of Sox17 in BM HSCs maintains the stemness of HSCs and induces the expression of fetal HSC marker proteins [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These results suggest that the Sox17-regulated molecular mechanisms active in fetal HSCs may retain latency but function in the BM HSCs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe previously showed that the introduction of Sox17 into IAHC cells from the AGM region induces the formation of cell clusters with hematopoietic potential [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. During the fetal-to-adult transition, the Sox17 expression in HSCs decreases. However, it remains unclear whether Sox17-induced cluster formation in HSPCs is functionally linked to the fetal-to-adult transition. In the present study, we examined the formation of cell clusters and the maintenance of hematopoietic capacity following Sox17 transduction into hematopoietic stem/progenitor cells (HSPCs) derived from the AGM region, the FL, and the BM. Whereas the hematopoietic activity is maintained in \u003cem\u003eSox17\u003c/em\u003e-transduced HSPCs, their cell cluster-forming ability decreases during the fetal-to-adult transition. We compared the gene expression profiles of \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced tamoxifen-treated BM HSPCs with those of \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced tamoxifen-treated IAHC HSPCs. We also compared IAHC HSPCs in which Sox17 was located to the nucleus with those in which Sox17 remained in the cytoplasm. From these comparisons, we identified candidate genes that were more highly expressed in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced cells where Sox17 was localized in the nucleus. One such candidate gene, the \u003cem\u003eProcr\u003c/em\u003e gene (the encoding endothelial protein C receptor, EPCR), was found to be expressed in IAHC cells of the dorsal aorta at E10.5 embryos. Knockdown of the \u003cem\u003eProcr\u003c/em\u003e gene in \u003cem\u003eSox17\u003c/em\u003e-transduced AGM cells reduced both the cluster-forming ability and the hematopoietic capacity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eIsolation of HSPCs prepared from the AGM region, the FL, and the BM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e cells, which are a component of IAHCs, were isolated from the AGM region of E10.5 mouse embryos as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. FLs were excised from E14.5 ICR mouse embryos and suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% (v/v) fetal calf serum (FCS). FL cells were incubated on ice for 15 min with biotin-conjugated anti-mouse CD8a, CD4, Ly6G/Ly6C, CD45R/B220, CD11b, and Ter119 antibodies (lineage antibodies, BioLegend, San Diego, CA). After washing, the cells were stained with PE-conjugated anti-mouse Sca-1 antibody (BioLegend), APC-conjugated anti-mouse c-Kit antibody (eBioscience, San Diego, CA), and PE-Cy7-conjugated streptavidin. BM cells from C57BL/6N mice were also suspended in DMEM containing 2% (v/v) FCS. After BM cells were treated with biotinylated lineage antibodies, they were stained with the APC-Cy7-conjugated anti-mouse Sca-1 antibody, the APC-conjugated anti-mouse c-Kit antibody, and the R-PE-conjugated avidin. To separate lineage-positive cells, BM cells were treated with anti-PE Microbeads (Miltenyi Biotec Inc., Auburn, CA) and passed through a magnetic-activated cell sorting column (Miltenyi Biotech). Both FL cells and lineage-negative BM cells were stained with 1 µg/ml propidium iodide (Calbiochem, San Diego, CA), and lineage\u003csup\u003e−\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e (KLS) cells were recovered by fluorescence-activated cell sorting (BD Biosciences, San Diego, CA). All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Care Committee of Tokyo Medical and Dental University (approval numbers: A2018-265C2, A2019-108C4, and A2021-177).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnforced expression of genes in HSPCs prepared from the AGM region, the FL, and the BM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRetroviruses encoding \u003cem\u003eIRES-GFP\u003c/em\u003e, \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e, or \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e genes were used to infect CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e cells from the E10.5 AGM region and KLS cells from the E14.5 FL for 3 hrs. The infected cells were then cultured with OP9 stromal cells in α-minimal essential medium (α-MEM) supplemented with 10% (v/v) FCS, 25 ng/ml stem cell factor (PeproTech, Rocky Hill, NJ), 10 ng/ml interleukin-3 (PeproTech), and 10 ng/ml thrombopoietin (PeproTech). \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells were cultured with or without 1 µM tamoxifen citrate. Additionally, retroviruses encoding these genes were used to infect KLS cells from the BM of 10-week-old mice on the RetroNectin (Takara Bio., Kyoto, Japan)-coated dishes for 4 days. After washing, the cells were cultured with OP9 cells in the same media\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalyses of the cluster-forming ability and the hematopoietic ability in\u003c/b\u003e \u003cb\u003eSox17\u003c/b\u003e\u003cb\u003e-transduced cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe exogenous gene-transduced CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e AGM cells and FL KLS cells described above were sorted based on the GFP expression on days 4 and 7 of culture, while the gene-transduced BM KLS cells were sorted on day 7. The sorted cells were then cultured on OP9 cells. After 11 days of culture, the number of clusters formed by the gene-transduced cells was counted. The cells were dissociated and sorted again based on the GFP expression (exogenous gene expression) for further analyses, such as RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR), Giemsa staining, and the colony-forming assays.\u003c/p\u003e\u003cp\u003eRNA was extracted by ISOGEN (WAKO, Osaka, Japan), and cDNA wad synthesized by ReverTraAce (TOYOBO, Osaka, Japan). The same amounts of cDNA were subjected to PCR using TAKARA Taq (TAKARA). Primer sequences were as follows: 5’-ACCACCCGATACCCACCTAT-3’ and 5’-GCCATGGCAGTCACCATGCT-3’ (GATA-2), 5’-GAGAGGTGGCACAACCATTT-3’ and 5’-GGGAACGTGACTGGAGATGT-3’ (c-Myb), 5’-CCAGCAAGCTGAGGAGCGGCG-3’ and 5’-CCGACAAACCTGAGGTCGTTG-3’ (Runx-1), 5’-AGGTGCAGCCACAGAACTTA-3’ and 5’-TCGGACCAATCAGAGATGTT-3’ (Notch-1), 5’-GTCATGGCCATGGTCGAGTA-3’ and 5’-CTCCTCGGCATCTTGCTGAA-3’ (CD31), 5’-GACTGGAACCAGCACGCTAACC-3’ and 5’-CGCCGTCATTGTCTGCCTCTTC-3’ (VEC), 5’-TTTATGGTGTGGGCCAAAG-3’ and 5’-CCGCTTCATGCGCTTCACCT-3’ (Sox17), and 5’-CAGCCTGGCTGGCTACGTACA-3’ and 5’-CCAGGGTGTGATGGTGGGAA-3’ (β-actin).\u003c/p\u003e\u003cp\u003e\u003cem\u003eSox17\u003c/em\u003e-transduced cells were transferred onto glass slides using a cytospin (Shandon, Sewickley, PA), and their morphology was examined by May-Grünwald-Giemsa staining. Additionally, \u003cem\u003eSox17\u003c/em\u003e-transduced cells were embedded in Methocult (M3434; StemCell Technologies, Vancouver, Canada) and individual colonies were counted based on morphology after 7 days of culture.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA sequencing analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor RNA sequencing analysis, CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e AGM cells and BM KLS cells transduced with the \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e gene were cultured with or without tamoxifen. After 11 days, GFP\u003csup\u003e+\u003c/sup\u003e cells were sorted, and total RNA was extracted from each sample using RNeasy Plus Mini Kit (Qiagen, Germany). The RNA samples were then sent to AZENTA Japan Corp. (Tokyo, Japan) for RNA sequencing using the Illumina HiSeq/Nova seq platform and 2 × 150 bp configuration. All raw RNA sequencing data have been deposited in the DDBJ Sequence Read Archive (DRA) under the BioProject accession number PRJDB20795 and Run accession numbers DRR683968-DRR683971.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDistribution analyses of Sox17 and c-Kit proteins in\u003c/b\u003e \u003cb\u003eSox17\u003c/b\u003e\u003cb\u003e-transduced cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter 11 days of the transduction with the \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e gene into CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e AGM cells cultured with or without tamoxifen, the non-adherent \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells were collected from the respective cultures under a microscope and transferred onto glass slides using a cytospin (Shandon, Sewickley, PA). Both the cells on the glass slides and the remaining adherent cells in the culture dishes were fixed in 2% paraformaldehyde for 10 min. They were then treated with PBS containing 1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100, 2% (w/v) bovine serum albumin (PBS-MT/BSA) for 1 hr. The cells were incubated overnight at 4 ˚C with a rat anti-mouse CD117 (c-Kit) antibody (2B8; eBioscience). After washing three times with PBS-MT, they were stained with Alexa Fluor® 488-conjugated donkey anti-rat IgG (Life Technologies, Carlsbad, CA) in PBS-MT. Imaging was performed using the BIOREVO microscopy (BZ-X810; KEYENCE, Osaka, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWhole-mount immunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhole-mount immunohistochemistry was performed according to a previously reported protocol [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. E10.5 mouse embryos were fixed in 2% PFA-PBS for 20 min and then dehydrated in methanol. After removal of the left body wall between the forelimb and hindlimb, these tissues were rehydrated in PBS and pretreated with PBS-MT at 4 ˚C for 1 hr. The tissues were stained overnight at 4˚C in PBS-MT with rat anti-mouse CD117 (c-Kit) antibody (2B8), goat anti-mouse EPCR (encoded by a \u003cem\u003eProcr\u003c/em\u003e gene) antibody (R\u0026amp;D, Minneapolis, MN), and rabbit-anti CD31 antibody (ab28364, Abcam, Cambridge, UK), or a rabbit anti-SOX17 antibody (EPR20684, Abcam). After washing with PBS-MT three times, the tissues were further stained overnight at 4˚C in PBS-MT with Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (Life Technologies), Alexa Fluor® 546-conjugated donkey anti-goat IgG (Life Technologies), and Alexa Fluor® 647-conjugated donkey anti-rat IgG (Jackson ImmunoResearch, West Grove, PA). After washing with PBS-MT, the tissues were treated with Hoechst 33258 (Nacalai Tesque, Kyoto, Japan) and then dehydrated in methanol. These dehydrated tissues were incubated for 3 min in a 1:1 mixture of methanol and BABB (a 1:2 mixture of benzyl alcohol and benzyl benzoate), followed by a 3 min treatment with 100% BABB. The stained tissues were examined by confocal microscopy (LSM710, Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoss-of-function analysis of the\u003c/b\u003e \u003cb\u003eProcr\u003c/b\u003e \u003cb\u003egene in\u003c/b\u003e \u003cb\u003eSox17\u003c/b\u003e\u003cb\u003e-transduced cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eSox17-IRES-mCherry\u003c/em\u003e gene was introduced into CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e AGM cells. After three passages in the coculture with OP9 stromal cells, retrovirus-mediated introduction of short hairpin RNA (shRNA) against the \u003cem\u003eLuciferase (Luc)\u003c/em\u003e gene (shLuc) or the \u003cem\u003eProcr\u003c/em\u003e gene (shProcr) driven by the U6 promoter, along with a \u003cem\u003eGFP\u003c/em\u003e gene driven by the SV40 promoter, was performed in \u003cem\u003eSox17-IRES-mCherry\u003c/em\u003e-transduced cells. Following three additional passages of shRNA-transduced cells, the expression of the \u003cem\u003eProcr\u003c/em\u003e gene in GFP\u003csup\u003e+\u003c/sup\u003e cells was analyzed by RT-PCR. The sorted GFP\u003csup\u003e+\u003c/sup\u003e cells were cocultured with fresh OP9 cells and after 4 days of the culture, the number of the non-adherent cell clusters was counted. Moreover, the colony-forming ability of GFP\u003csup\u003e+\u003c/sup\u003e cells was assessed by Methocult (M3434). shRNAs targeted the following sequences: 5'-ACTTACGCTGAGTACTTCG-3' (shLuc), and 5'-GTGTGGAGTTCCTGGAGAA-3'. (shProcr)\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe cluster-forming ability of\u003c/b\u003e \u003cb\u003eSox17-IRES-GFP\u003c/b\u003e\u003cb\u003e-transduced HSPCs decreases during development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe previously showed that the introduction of the transcription factor Sox17 into HSPC-containing CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e cells from the AGM region in midgestation mouse embryos maintained the formation of cell clusters with the hematopoietic ability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It remained unclear whether the Sox17 overexpression could similarly sustain the cluster-forming activity in HSPCs from the FL and BM. We retrovirally introduced either the \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e gene or the control \u003cem\u003eIRES-GFP\u003c/em\u003e gene into CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e cells of the AGM region, as well as linage\u003csup\u003e\u0026minus;\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e (KLS) HSPCs from the FL and BM, and these virus-infected cells cocultured with OP9 stromal cells. On day 11 after infection, cell clusters were observed in \u003cem\u003eSox17\u003c/em\u003e-transduced cells from the FL and BM as well as from the AGM region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The number of cell clusters formed by \u003cem\u003eSox17\u003c/em\u003e-transduced cells decreased as embryonic development progressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe hematopoietic potential is maintained in\u003c/b\u003e \u003cb\u003eSox17-IRES-GFP\u003c/b\u003e\u003cb\u003e-transduced cells from the FL and BM\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe assessed the hematopoietic potential of \u003cem\u003eSox17\u003c/em\u003e-transduced cells using the colony-forming assay. Eleven days after retroviral infection, sorted GFP\u003csup\u003e+\u003c/sup\u003e cells were embedded in a methylcellulose medium. The number of total and mixed colonies containing three lineages (granulocytes, macrophages, and erythrocytes) was increased in \u003cem\u003eSox17\u003c/em\u003e-transduced cells from the AGM region, the FL, and the BM, indicating enhanced multilineage differentiation potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). By examination of May-Gr\u0026uuml;nwald-Giemsa staining, \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells predominantly exhibited the morphology of granulocytes and macrophages. In contrast, \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells showed a more blastic morphology characterized by the round nuclei and a small proportion of the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Moreover, the expression of hematopoietic transcription factors (GATA-2, c-Myb, and Runx1) and adhesion molecules (CD31 and VEC) was maintained in \u003cem\u003eSox17\u003c/em\u003e-transduced cells from FL and BM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Increased expression of adhesion molecules in \u003cem\u003eSox17\u003c/em\u003e-transduced BM cells was previously reported [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These findings indicate that the introduction of the \u003cem\u003eSox17\u003c/em\u003e gene into HSPCs from the FL and BM supports both their hematopoietic ability and cluster formation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe cluster-forming ability of\u003c/b\u003e \u003cb\u003eSox17-ERT-IRES-GFP\u003c/b\u003e\u003cb\u003e-transduced HSPCs in the presence of tamoxifen decreases with the development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, to examine the importance of the Sox17 transcriptional activity in maintaining the cell cluster formation with hematopoietic ability, we introduced the \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e gene into CD45\u003csup\u003elow\u003c/sup\u003ec-Kit\u003csup\u003ehigh\u003c/sup\u003e AGM cells and HSPCs from the FL and BM. It was previously reported that \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells exhibit tamoxifen-induced nuclear translocation of the fusion protein [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For the first 4 days of the culture, the cells were maintained in the presence of tamoxifen, after which they were cultured in the media with or without tamoxifen. After 11 days of culture, \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells from the AGM region, the FL, and the BM formed cell clusters in the presence of tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, the number of cell clusters formed in response to Sox17 nuclear translocation decreased with developmental stage, with only a few cell clusters observed in BM-derived cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eWe further examined the colony-forming ability of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells with or without tamoxifen. An increased number of total and mixed colonies was observed in \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells from the AGM region, the FL, and the BM in the presence of tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The morphology of \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e-transduced cells cultured with tamoxifen resembled that of cells transduced with \u003cem\u003eSox17-IRES-GFP\u003c/em\u003e gene, whereas cells cultured without tamoxifen showed morphologies similar to those of \u003cem\u003eIRES-GFP\u003c/em\u003e-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). RT-PCR analysis demonstrated that the expression of GATA-2, c-Myb, and Runx1 was maintained in \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells derived from the AGM region and the FL in the presence of tamoxifen. In BM-derived cells, the expression of GATA-2 and c-Myb was retained under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Tamoxifen also maintained the expression of adhesion molecules CD31 and VEC in \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results indicate that the tamoxifen-inducible nuclear translocation of the Sox17-ERT fusion protein maintains the cluster formation with the hematopoietic ability and gene expression in HSPCs from the FL and BM, although this effect diminishes with development.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe difference in the features of\u003c/b\u003e \u003cb\u003eSox17-ERT\u003c/b\u003e\u003cb\u003e-transduced colonies, adherent colonies, and non-adherent colonies caused by the tamoxifen-inducible nuclear translocation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTamoxifen-dependent nuclear translocation of Sox17 in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced cells of the AGM region on the stromal cells found three types of cell morphology; colonies (the flat colony), adherent clusters (the cluster which is attached to OP9 stromal cells), and non-adherent clusters (the cluster which floats in the medium) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The number of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced non-adherent clusters derived from the AGM region was increased in a tamoxifen concentration-dependent manner, whereas the number of colonies was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To examine the ratio of Sox17-nuclear translocation in a tamoxifen dependent fashion, the \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e gene was introduced in NIH3T3 cells in medium containing various concentrations of tamoxifen. The Sox17-ERT protein was detected in the cytoplasm of NIH3T3 cells cultured in the absence of tamoxifen, whereas the Sox17-ERT protein was almost exclusively found in the nucleus of the NIH3T3 cells in the culture with 10 \u0026micro;M tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We showed a reduction of Sox17 in the cytoplasm at higher tamoxifen concentration, whereas the Sox17 nuclear translocation was found to increase at high concentrations of tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results revealed the relationship between the Sox17 nuclear translocation and the cluster formation. To examine the c-Kit expression in colonies, adherent clusters, and non-adherent clusters of \u003cem\u003eSox17-ERT-IRES-GFP\u003c/em\u003e-transduced cells of the AGM region in the presence and absence of tamoxifen, we performed the immunostaining after 11 days of the culture. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the c-Kit expression was not observed in the colonies, while c-Kit was expressed in adherent and non-adherent clusters by the Sox17-ERT nuclear localization in the presence of tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe examined the colony-forming ability of non-adherent cells and adherent cells in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced cells with tamoxifen. After 11 days of the culture, the GFP\u003csup\u003e+\u003c/sup\u003e cluster cells were directly recovered under the microscope, and the medium, in which some GFP\u003csup\u003e+\u003c/sup\u003e clusters remained, was discarded from the dish. The new medium was added to the dish and the adherent cells containing adherent clusters and colony-forming cells were recovered after the pipetting. The colony-forming abilities of non-adherent cells and adherent cells in the presence of tamoxifen are high compared to cells in the absence of tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). There was little difference in the colony-forming ability between \u003cem\u003eSox17\u003c/em\u003e-transduced non-adherent cells and adherent cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of candidate genes responsible for reduced the cluster-forming ability of\u003c/b\u003e \u003cb\u003eSox17\u003c/b\u003e\u003cb\u003e-transduced AGM cells by RNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur present results showed that the cluster-forming ability induced by the Sox17 introduction decreased during developmental progression. However, specific genes responsible for promoting cluster formation in the AGM region and the mechanisms by which Sox17 induced the process remain unclear. To identify candidate genes involved in this activity, we, conducted RNA sequencing (RNA-seq) analysis on four distinct cell populations: \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced AGM cells cultured with tamoxifen (AGM Tam (+)) and without tamoxifen (AGM Tam (-)) and \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced BM cells cultured with tamoxifen (BM Tam (+)) and without tamoxifen (BM Tam (-)). All RNA-seq data from this study have been deposited in the DDBJ Sequence Read Archive (DRA) under the BioProject accession number PRJDB20795, with individual Run accession numbers DRR683968-DRR683971. The number of significant differentially expressed genes (DEGs) identified in each pairwise comparison is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Heatmaps illustrating gene expression patterns across the four cell populations are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The Venn diagram revealed 318 overlapping genes that were differentially expressed both between AGM Tam (+) and AGM Tam (-), and between AGM Tam (+) and BM Tam (+) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). From the RNA-seq data, we selected 85 candidate genes that were up-regulated in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced AGM cells cultured with tamoxifen, compared to both the BM cells cultured with tamoxifen and AGM cells cultured without tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eInvolvement of one candidate gene, the\u003c/b\u003e \u003cb\u003eProcr\u003c/b\u003e \u003cb\u003egene, in the cluster formation with hematopoietic potential in AGM-derived HSPCs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the role of these candidate genes in the cluster formation of \u003cem\u003eSox17\u003c/em\u003e-transduced cells, we focused on the \u003cem\u003eProcr\u003c/em\u003e gene, which encodes the endothelial protein C receptor protein (EPCR). EPCR is known as the transmembrane glycoprotein expressed on the endothelial cells and initiates the anticoagulant pathway by activating protein C [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A previous study reported that EPCR, along with CD31 and c-Kit, marks HSCs in the E11.5 AGM region [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We first confirmed the expression of the \u003cem\u003eProcr\u003c/em\u003e gene by RT-PCR and found that the \u003cem\u003eProcr1\u003c/em\u003e expression was upregulated in \u003cem\u003eSox17\u003c/em\u003e-transduced AGM cells cultured with tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Next, we analyzed the EPCR expression in IAHCs in the dorsal aorta of the E10.5 mouse embryo by the whole-mount immunohistochemistry. The EPCR expression partially overlapped with that of c-Kit\u003csup\u003e+\u003c/sup\u003e IAHCs and Sox17-expressing cells in the dorsal aorta (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To examine the functional role of EPCR in the cluster formation, we introduced shRNA against the \u003cem\u003eProcr\u003c/em\u003e gene into \u003cem\u003eSox17\u003c/em\u003e-transduced AGM cells. Specifically, \u003cem\u003eSox17-IRES-mCherry\u003c/em\u003e-transduced AGM cells were retrovirally infected with either control shRNA against control \u003cem\u003eluciferase\u003c/em\u003e (shLuc) or shRNA against \u003cem\u003eProcr\u003c/em\u003e (shProcr), both of which co-expressed the \u003cem\u003eGFP\u003c/em\u003e gene. Eleven days after shRNA introduction, RT-PCR analysis confirmed that the \u003cem\u003eProcr\u003c/em\u003e gene expression was lower in shProcr-transduced cells compared to shLuc-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The shProcr-transduced cells exhibited fewer cell clusters than shLuc-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Moreover, the colony-forming ability of shProcr-transduced cells was also significantly diminished relative to shLuc-transduced cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results indicate that reduced expression of the \u003cem\u003eProcr\u003c/em\u003e gene decreases both the cell cluster-forming ability and the hematopoietic ability in \u003cem\u003eSox17\u003c/em\u003e-transduced cells from the AGM region.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe previously showed that \u003cem\u003eSox17\u003c/em\u003e-transduced cells from the E10.5 AGM region maintained both the cluster-forming ability and the hematopoietic ability in in vitro cultures [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The introduction of Sox17 or tamoxifen-inducible Sox17-ERT into HSPCs from the FL and BM revealed that the hematopoietic potential was maintained in both \u003cem\u003eSox17\u003c/em\u003e-transduced cells and \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced cells following a tamoxifen-induced nuclear translocation (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We found that \u003cem\u003eSox17\u003c/em\u003e-transduced cells derived from IAHC cells in the AGM region and HSPCs in the FL and BM exhibit cluster-forming ability. This cluster-forming ability declined during development (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn midgestation mouse embryos, IAHC cells expressed the c-Kit, a marker also found in HSPCs of the FL and BM [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mice deleted for Runx-1, which is an essential transcription factor for definitive hematopoiesis, showed no IAHCs in the dorsal aorta and embryonic lethality [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Conditional knockout mice of GATA2 in VEC\u003csup\u003e+\u003c/sup\u003e endothelial cells also results in a reduced number of IAHCs in the dorsal aorta [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These results indicate the requirement of IAHCs for the emergence of HSPCs in the dorsal aorta. Moreover, adherent molecules such as VEC and CD31 are expressed in basal cells of IAHCs in the dorsal aorta [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The expression of c-Kit was also observed in the cluster cells induced by nuclear translocation of Sox17, similarly to its expression in IAHC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In the absence of the Sox17 nuclear translocation, flat colonies were formed, while the Sox17 nuclear translocation resulted in the formation of nonadherent and adherent clusters, which had the higher hematopoietic ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Thus, there were many similarities between IAHCs in the dorsal aorta and the cluster cells formed by the Sox17 nuclear transport.\u003c/p\u003e\u003cp\u003eIn this study, we found that the ability of \u003cem\u003eSox17\u003c/em\u003e-transduced HSPCs to form cell clusters decreased as development progressed. This reduction likely reflects the developmental stage-specific requirement for Sox17 in HSC maintenance. The BM, but not the AGM, has niche cells that support this maintenance of quiescent HSCs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We hypothesized that the differences in the cell cluster-forming ability were caused by an effect of Sox17 on the induced expression of adhesion molecules [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], even though the Sox17-induced molecules are known to function in the BM [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To identify the molecular basis of this phenomenon, RNA-seq was performed on \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced tamoxifen-treated AGM HSPCs, which have the highest cluster-forming ability, and \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced tamoxifen-treated BM HSPCs, which have the lowest cluster-forming ability. The candidate genes were highly expressed in the \u003cem\u003eSox17\u003c/em\u003e-transduced cells containing AGM HSPCs, which had the highest cluster-forming capacity. The \u003cem\u003eProcr\u003c/em\u003e gene, which is one of their candidate genes, is expressed in IAHC cells of the dorsal aorta and is associated with the colony-forming ability in the \u003cem\u003eSox17\u003c/em\u003e-transduced cells in the AGM region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The EPCR protein encoded by the \u003cem\u003eProcr\u003c/em\u003e gene in HSPCs is first observed in the BM, where it marks a population enriched in LT-HSCs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the FL, the EPCR expression is also observed in HSPCs and the stem cell ability was maintained in EPCR-expressing HSPCs after a few days of in vitro culture [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the AGM region of E11.5 mouse embryos, the EPCR expression level in CD31\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003e cells correlates with their long-term repopulation capacity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the present study, the Procr expression was elevated in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced AGM cells cultured with tamoxifen-induced nuclear translocation of Sox17, compared with \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced BM HSPCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The Sox17 expression is significantly higher in EPCR-expressing FL HSPCs compared to EPCR-nonexpressing HSPCs, and the Sox17 expression markedly decreased in both EPCR-expressing and EPCR-nonexpressing HSPCs after 1 day of in vitro culture [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These findings suggest a functional association between Sox17 and Procr expression. Our data indicate that the \u003cem\u003eProcr\u003c/em\u003e gene expression in \u003cem\u003eSox17\u003c/em\u003e-transduced cells was involved in the cluster formation with the hematopoietic ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). The molecular mechanism of the \u003cem\u003eProcr\u003c/em\u003e gene will be analyzed in the future and other candidate genes will be examined for their effects on hematopoietic and cluster-forming potentials.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe introduction of a transcription factor Sox17 into HSPCs prepared from the AGM region, the FL, and the BM maintained the hematopoietic potential but led to a progressive decline in the cluster-forming ability during development. Comparative analysis of gene expression between Sox17 nuclear translocation in the AGM cells and the BM revealed that the \u003cem\u003eProcr\u003c/em\u003e gene has a function in the cluster formation with hematopoietic ability in \u003cem\u003eSox17\u003c/em\u003e-transduced AGM cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehematopoietic stem cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIAHCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eintra-aortic hematopoietic cell clusters\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAGM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eaorta-gonad-mesonephros\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efetal liver\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebone marrow\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSPCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehematopoietic stem/progenitor cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eembryonic day\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVEC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003evascular-endothelial cadherin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eESAM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eendothelial cell-selective adhesion molecules\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDulbecco\u0026rsquo;s modified Eagles medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKLS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elineage\u003csup\u003e\u0026minus;\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRT-PCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereverse transcription-polymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTam\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etamoxifen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEPCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eendothelial protein C receptor protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eshRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eshort hairpin RNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLuc\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eluciferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eNot applicable to this review manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable to this review manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eAuthor Ayumi Itabashi is now an employee of Daiichi Sankyo.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant numbers 26440118 and 18K06249 (I.N.), 22130008, 15H04292, and 18H02678 (T.T.)) and Nanken-Kyoten (Grant numbers, H26-A39, H27-A35, H28-A11), Tokyo Medical Dental University.\u003c/p\u003e\u003ch2\u003eAuthors contributions\u003c/h2\u003e\u003cp\u003eIA, YY, KS, RT, GM, KA, NI, and IN conducted the in vivo and in vitro experiments. IA and IN analyzed the data of the RNA sequence. IA, TT, and IN planned the experiments. IA, TT, and IN wrote the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe thank Dr. T. Nakano for the OP9 cells, Dr. K. Tabu for valuable discussions, and Ms E. Wada, Mr. K Nakamura, Dr. H. Takebayashi for their technical support. We also thank K. Inoue for secretarial assistance.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials.\u003c/h2\u003e\u003cp\u003eFurther information and requests for resources and reagents should be directed to the author: Ikuo Nobuhisa ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDzierzak E, Speck NA. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol. 2008;9,:29\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMedvinsky A, Rybtsov S, Taoudi S. Embryonic origin of the adult hematopoietic system: Advances and questions. Development. 2011;138:1017\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKauts ML, Vink CS, Dzierzak E. Hematopoietic (stem) cell development \u0026mdash; how divergent are the roads taken? FEBS Lett. 2016;590:3975\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRhodes KE, Gekas C, Wang Y, Lux CT, Francis CS, Chan DN, Conway S, Orkin SH, Yoder MC. Mikkola HKA The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation. Cell Stem Cell. 2008;2:252\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDzierzak E, Robin C. Placenta as a source of hematopoietic stem cells. Trends Mol Med. 2010;16:361\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYokomizo T, Dzierzak E. Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development. 2010;137:3651\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Pater E, Kaimakis P, Vink CS, Yokomizo T, Yamada-Inagawa T, van der Linden R, Kartalaei PS, Camper SA, Speck N, Dzierzak E. Gata2 is required for HSC generation and survival. J Exp Med. 2013;210:2843\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoisset J-C, Clapes T, Klaus A, Papazian N, Onderwater J, Mommaas-Kienhuis M, Cupedo T. Robin C Progressive maturation toward hematopoietic stem cells in the mouse embryo aorta. Blood. 2015;125:465\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMazo IB, Massberg S, von Andrian. UH Hematopoietic stem and progenitor cell trafficking. Trends Immunol. 2011;32:493\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePietras EM, Warr MR. Passegu\u0026eacute; E Cell cycle regulation in hematopoietic stem cells. J Cell Biol. 2011;195:709\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTam PPL, Kanai-Azuma M, Kanai Y. Early endoderm development in vertebrates: lineage differentiation and morphogenetic function. Curr Opin Genet Dev. 2003;13:393\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, Sanai Y, Yonekawa H, Yazaki K, Tam PPL. Hayashi Y Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 2002;129:2367\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatsui T, Kanai-Azuma M, Hara K, Matoba S, Hiramatsu R, Kawakami H, Kurohmaru M, Koopman P. Kanai Y Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice. J Cell Sci. 2006;119:3513\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHara K, Kanai-Azuma M, Uemura M, Shitara H, Taya C, Yonekawa H, Kawakami H, Tsunekawa N, Kurohmaru M. Kanai Y Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis. Dev Biol. 2009;330:427\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUemura M, Hara K, Shitara H, Ishii R, Tsunekawa N, Miura Y, Kurohmaru M, Taya C, Yonekawa H, Kanai-Azuma M, Kanai Y. Expression and function of mouse Sox17 gene in the specification of gallbladder/bile-duct progenitors during early foregut morphogenesis. Biochem Biophys Res Commun. 2010;391:357\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUemura M, Ozawa A, Nagata T, Kurasawa K, Tsunekawa N, Nobuhisa I, Taga T, Hara K, Kudo A, Kawakami H, Saijoh Y, Kurohmaru M, Kanai-Azuma M, Kanai Y. Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice. Dev. 2013;140:639\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUemura M, Higashi M, Pattarapanawan M, Takami S, Ichikawa N, Higashiyama H, Furukawa T, Fujishiro J, Fukumura Y, Yao T, Tajiri T, Kanai-Azuma M, Kanai Y. Gallbladder wall abnormality in biliary atresia of mouse Sox17 (+/-) neonates and human infants. Dis Model Mech. 2020;13:dmm042119.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHigashiyama H, Ozawa A, Sumitomo H, Uemura M, Fujino K, Igarashi H, Imaimatsu K, Tsunekawa N, Hirate Y, Kurohmaru M, Saijoh Y, Kanai-Azuma M, Kanai Y. Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia. Development. 2017;144:1906\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHirate Y, Suzuki H, Kawasumi M, Takase HM, Igarashi H, Naquet P, Kanai Y. Kanai-Azuma M Mouse Sox17 haploinsufficiency leads to female subfertility due to impaired implantation. Sci Rep. 2016;6:24171.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim I, Saunders TL, Morrison SJ. Sox17 Dependence Distinguishes the Transcriptional Regulation of Fetal from Adult Hematopoietic Stem Cells. Cell. 2007;130:470\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNobuhisa I, Osawa M, Uemura M, Kishikawa Y, Anani M, Harada K, Takagi H, Saito K, Kanai-Azuma M, Kanai Y, Iwama A, Taga T. Sox17-Mediated Maintenance of Fetal Intra-Aortic Hematopoietic Cell Clusters. Mol Cell Biol. 2014;34:1976\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLizama CO, Hawkins JS, Schmitt CE, Bos FL, Zape JP, Cautivo KM, Borges Pinto H, Rhyner AM, Yu H, Donohoe ME, Wythe JD. Zovein AC Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition. Nat Commun. 2015;6:7739.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaito K, Nobuhisa I, Harada K, Takahashi S, Anani M, Lickert H, Kanai-Azuma M, Kanai Y, Taga T. Maintenance of hematopoietic stem and progenitor cells in fetal intra-aortic hematopoietic clusters by the Sox17-Notch1-Hes1 axis. Exp. Cell Res. 2018;365:145\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakahashi S, Nobuhisa I, Saito K, Gerel M, Itabashi A, Harada K, Osawa M, Endo TA, Iwama A, Taga T. Sox17-mediated expression of adherent molecules is required for the maintenance of undifferentiated hematopoietic cluster formation in midgestation mouse embryos. Differentiation. 2020;115:53\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim I, Lim MS, Morrison SJ. Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors. Genes Dev. 2011;25:1613\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChhabra A. Mikkola HKA Return to youth with Sox17. Genes Dev. 2011;25:1557\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarada K, Nobuhisa I, Anani M, Saito K, Taga T. Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region. Cytokine. 2017;95:35\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnani M, Nobuhisa I, Osawa M, Iwama A, Harada K, Saito K, Taga T. Sox17 as a candidate regulator of myeloid restricted differentiation potential. Dev Growth Differ. 2014;56:469\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYokomizo T, Yamada-Inagawa T, Yzaguirre AD, Chen MJ, Speck NA. Dzierzak E Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos. Nat Protoc. 2012;7:421\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakajima-Takagi Y, Osawa M, Oshima M, Takagi H, Miyagi S, Endoh M, Endo TA, Takayama N, Eto K, Toyoda T, Koseki H, Nakauchi H. Iwama A Role of SOX17 in hematopoietic development from human embryonic stem cells. Blood. 2013;121:447\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohan Rao LV, Esmon CT. Pendurthi UR Endothelial cell protein C receptor: a multiliganded and multifunctional receptor. Blood. 2014;124:1553\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng X, Zhang G, Gong Y, Ning X, Bai Z, He J, Zhou F, Ni Y, Lan Y, Liu B. Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan. J Genet Genomics. 2019;46:489\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA. Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature. 2009;457:887\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eComazzetto S, Shen B, Morrison SJ. Niches that regulate stem cells and hematopoiesis in adult bone marrow. Dev Cell. 2021;56:1848\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBalazs AB, Fabian AJ, Esmon CT, Mulligan RC. Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood. 2006;107:2317\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIwasaki H, Arai F, Kubota Y, Dahl M, Suda T. Endothelial protein C receptor-expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver. Blood. 2010;116:544\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-and-regeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ireg","sideBox":"Learn more about [Inflammation and Regeneration](http://inflammregen.biomedcentral.com/)","snPcode":"41232","submissionUrl":"https://www.editorialmanager.com/ireg/default2.aspx","title":"Inflammation and Regeneration","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hematopoiesis, HSPC, Sox17, IAHCs, Procr","lastPublishedDoi":"10.21203/rs.3.rs-7069210/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7069210/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eDefinitive hematopoietic stem cells (HSCs) emerge within intra-aortic hematopoietic cell clusters (IAHCs) located in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation in the mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and finally settle in the bone marrow (BM). We previously showed that the transcription factor Sox17 is expressed in IAHCs. Transduction of the \u003cem\u003eSox17\u003c/em\u003e gene in IAHC cells induces the formation of cell clusters in vitro that resemble IAHCs and retain hematopoietic potential. In addition, a previous report showed that \u003cem\u003eSox17\u003c/em\u003e-transduced hematopoietic stem/progenitor cells (HSPCs) in the BM maintained pluripotency. However, the relationship between Sox17-induced cluster formation and the developmental transition from fetal to adult hematopoiesis has not been clarified.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe examined whether viral transduction of the \u003cem\u003eSox17\u003c/em\u003e gene in HSPCs leads to the formation of cell clusters. To identify the candidate genes involved in cluster formation, we performed RNA-sequencing (RNA-seq) analysis on \u003cem\u003eSox17-ER\u003c/em\u003eT-transduced HSPCs from the AGM region and the BM cultured with or without tamoxifen. We further analyzed the ability of one candidate gene, the \u003cem\u003eProcr\u003c/em\u003e gene, to support cluster formation and hematopoietic function.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eA large number of multilineage colonies were observed in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced tamoxifen-treated HSPCs prepared from the AGM region, the FL, and the BM. However, the ability to form cell clusters was lower in BM-derived HSPCs compared to those from the AGM region and the FL. RNA-seq analysis revealed several genes that were highly expressed in \u003cem\u003eSox17-ERT\u003c/em\u003e-transduced tamoxifen-treated HSPCs from the AGM region. The \u003cem\u003eProcr\u003c/em\u003e gene, one of these genes, was expressed in IAHCs and was found to contribute to both cluster formation and the maintenance of hematopoietic capacity in \u003cem\u003eSox17\u003c/em\u003e-transduced cells of the AGM region.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur results revealed that the Sox17-induced cluster-forming ability is attenuated in BM HSPCs compared to the AGM and the FL HSPCs, suggesting that HSPC characteristics are developmentally altered during the transition from fetal to adult hematopoiesis. Moreover, the \u003cem\u003eProcr\u003c/em\u003e gene plays a substantial role in cluster formation and supports hematopoietic capacity in the midgestation mouse embryos.\u003c/p\u003e","manuscriptTitle":"Ability to form hematopoietic stem/progenitor cell-containing cell clusters weakens during the fetal-to-adult transition in hematopoietic development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 15:11:42","doi":"10.21203/rs.3.rs-7069210/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-10T11:13:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-10T11:02:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Inflammation and Regeneration","date":"2025-07-20T03:58:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-08T07:33:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation and Regeneration","date":"2025-07-07T20:37:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-and-regeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ireg","sideBox":"Learn more about [Inflammation and Regeneration](http://inflammregen.biomedcentral.com/)","snPcode":"41232","submissionUrl":"https://www.editorialmanager.com/ireg/default2.aspx","title":"Inflammation and Regeneration","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8ac36f0-d51f-4cef-992f-a6b62ab43252","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:07:46+00:00","versionOfRecord":{"articleIdentity":"rs-7069210","link":"https://doi.org/10.1186/s41232-026-00420-w","journal":{"identity":"inflammation-and-regeneration","isVorOnly":false,"title":"Inflammation and Regeneration"},"publishedOn":"2026-04-23 15:59:31","publishedOnDateReadable":"April 23rd, 2026"},"versionCreatedAt":"2025-08-14 15:11:42","video":"","vorDoi":"10.1186/s41232-026-00420-w","vorDoiUrl":"https://doi.org/10.1186/s41232-026-00420-w","workflowStages":[]},"version":"v1","identity":"rs-7069210","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7069210","identity":"rs-7069210","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}