Potential conflict of interest: Nothing to report.
This work was supported by grants from the Ministerio de Ciencia e Innovación (MICINN; grant nos. SAF2007-65265 and SAF2009-12596), the Instituto de Salud Carlos III (grant no.: ISCIII08/1374), and the Comunidad Autónoma de Madrid (grant no.: SAL-0304-2006). N.S. and I.C. are recipients of fellowships from the Centro de Biología Molecular Severo Ochoa (CBMSO) and the MICINN, respectively. The CBMSO receives institutional funding from the Fundación Ramón Areces.
In the mouse embryo, hematopoietic progenitor cells migrate to the fetal liver (FL) between gestational days (E) 9.5 and 10.5, where they rapidly expand to form the main fetal reservoir of hematopoietic cells. The embryonic megakaryocyte progenitors (MKPs) in the E11.5 FL were identified as CD49fHCD41H (and c-KitDKDR+CD42+CD9++CD31+) cells, expressing several hepato-specific proteins. Unlike adult bone marrow megakaryocytes (MKs), embryonic MKPs were CD45− and represent an abundant population in the FL. The CD49fHCD41H MKPs purified by cytometry differentiated in vitro to produce proplatelets, independent of thrombopoietin stimulation, and they responded to stimulation with adenosine diphosphate, thrombin, and the PAR4 thrombin receptor-activating peptide. Moreover, after removing CD49fHCD41H MKPs from purified E11.5 FL hepatoepithelial-enriched cell preparations (c-KitDCD45−Ter119−), the remaining CD49fD cells neither differentiated nor survived in vitro. Indeed, direct cell-to-cell contact between the CD49fHCD41H and CD49fD populations was required to promote the hepatocyte differentiation of CD49fD cells. The addition of vascular endothelial growth factor A (VEGF-A) and medium conditioned by E11.5 CD49fHCD41H MKPs produced a partial effect on CD49fD cells, inducing the formation of hepatoepithelial layers. This effect was abolished by anti-VEGF-A antibodies. Together, these findings strongly suggest that CD49fHCD41H MKPs are fundamental to promote FL development, as proposed in adult liver regeneration. Conclusion: The cells of the MK lineage present in the developing mouse embryo liver promote the growth of hepatoepithelial cells in vitro through VEGF-A signaling and may play a role in liver development in vivo. (HEPATOLOGY 2012;56:1934–1945)
After gastrulation, genetic prepatterns are established in discrete areas of the embryo related to cell-lineage specification, cell differentiation, and morphogenesis. Hematopoiesis occurs in two phases in the embryo (primitive and definitive). Primitive hematopoiesis involves embryonic erythrocytes and myeloid cells, commencing in the yolk sac (YS) and proceeding as a self-limiting process throughout gestation. By contrast, definitive lymphohematopoiesis begins in the YS and, in an autonomous manner, in the para-aortic splanchnopleura/aorta-gonads-mesonephros (P-Sp/AGM) niche, which later becomes the source of all lymphoid and hematopoietic cell lineages.1-3
Megakaryocytes (MKs) are a particular blood cell type that share common features with hematopoietic stem cells (HSCs). In the adult, CD45+CD9+ CD41++ MKs are found primarily in the bone marrow (BM) as a scattered polyploid population of large cells. These MKs are responsible for the production of platelets, subcellular fragments involved in coagulation and the regulation of angiogenesis.4 In the mouse embryo, clonogenic bipotential megakaryocyte/erythroid progenitors (MEPs) appear in the YS at embryonic days 7.25 (E7.25) and E9.5, participating in primitive and definitive megakaryopoiesis, respectively.5, 6 At E10.5, large, immature reticulated platelets have been found in the bloodstream, and CD45−CD41H cells can be observed in vascular hematopoietic clusters.5, 7 With the discovery of thrombopoietin (TPO), MKs could be cultured, and platelets were generated in vitro from mature MKs, isolated by density purification, that produce long, pseudopodial cytoplasmic processes (i.e., proplatelets).8
After E10.5, the fetal liver (FL) represents the central compartment of hematopoiesis during gestation and receives extrinsic progenitors derived from the primary hematopoietic niches (YS, P-Sp/AGM, and the placenta), although it also harbors endodermal progenitors derived from the gut evagination that later give rise to the adult liver parenchyma. It has been suggested that cross-talk between the hematopoietic and hepatoepithelial compartments plays a central role in liver development.9 Bipotential hepatocyte and cholangiocyte progenitors (HeP) have been identified in the mouse embryo.10-12 Indeed, we identified an embryonic HeP population that was negative for hematopoietic markers (CD45−Ter119−), but that weakly expressed the stem cell factor receptor (c-KitD) and which could be separated into two subpopulations based on the level of α6 integrin chain expression (CD49f). The amount of CD49f expression in HeP remains unclear, with some studies describing HeP as CD49f negative11 and others describing postnatal liver progenitor cells as CD49fH.13 In the present study, we demonstrate that, at E11.5, the CD49fH subpopulation of c-KitD cells are functional MK precursors (MKPs) that are CD41HCD42a,b,c+CD9++. Furthermore, unlike the precursors from the adult BM, this population lacks the conventional hematopoietic tracer (CD45) and these cells express vascular endothelial growth factor A (VEGF-A). When cultured in vitro in the absence of TPO, these embryonic MKPs produce proplatelets, which are also clearly evident directly among the cells isolated from FL. Finally, we show that the CD49fHCD41H MKPs present in the FL of E11.5 embryos establish numerous contacts with albumin (ALB)+ cells in vivo and stimulate the development of CD49fD HeP in vitro in response to direct cellular contacts and VEGF-A.
BALB/c and C57BL/6 mice were maintained at the animal facility of the Instituto de Salud Carlos III (Madrid, Spain). Mice were mated overnight, and the day the vaginal plug was detected was considered day 0.5 of gestation (E0.5). Mice were sacrificed, exsanguinated to collect peripheral blood lymphocytes (PBLs), and the desired tissues (FL and AGM) were obtained as described in the Supporting Methods. All animal studies were approved by the institutional review boards of the Instituto de Salud Carlos III and the Centro de Biología Molecular Severo Ochoa (Madrid, Spain).
Flow Cytometry and Cell Purification.
Cells were treated with Fc-Block (BD Biosciences, San Diego, CA) and 10% normal mouse serum in phosphate-buffered saline before incubation (4°C, 30 minutes) with fluorescein isothiocyanate (FITC), biotin-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated antibodies (Abs) (as indicated in Supporting Table 1). Cell debris and dead cells were excluded by light-scattering parameters and propidium iodide staining, and cell suspensions were analyzed in a FACSCalibur with the CellQuest (BD Biosciences) and FlowJo (Tree Star Inc., Stanford University, Stanford, CA) software packages. Cells were purified under sterile conditions by fluorescence-activated cell sorting (FACS) on a FACSAria (BD Biosciences), and the purity of the cells recovered was over 98%. For cell-culture experiments, staining with Abs directed against c-Kit/FITC, CD49f/PE, CD45/biotin and Ter-119/biotin (visualized with streptavidin/APC) allowed c-KitDCD45−Ter119−, c-KitDCD45−Ter119−CD49fH, and c-KitDCD45− Ter119−CD49fD cells to be purified (forthwith referred to as c-KitDCD45−, CD49fH, and CD49fD, respectively). Because purified CD49fH and CD49fD cells were CD41HCD42c+CD45− and CD41− CD42c−CD45−, respectively, for some experiments, CD49fHCD41H and CD49fD cells were purified after staining with Abs directed against CD41/FITC, CD45/APC, and CD49f/PE.
Platelet and MKP Functional Assays.
Cells were collected, activated with adenosine diphosphate (ADP), thrombin, and the PAR4 thrombin receptor-activating peptide, and analyzed by flow cytometry (as indicated in the Supporting Methods).
Polymerase Chain Reaction.
RNA was extracted and oligo(dT)-primed complementary DNA (cDNA) was prepared as previously described.14 Polymerase chain reaction (PCR) amplification was performed with the primers and conditions indicated in Supporting Table 2 and Supporting Methods. For quantification of ALB and kinase domain region (KDR) expression, quantitative real-time PCR was performed as previously described.15 The relative amount of specific cDNA on each sample was determined by the 2−ΔΔCt method using G-protein subunit αs (GαS) expression as an internal control.
Purified E11.5 FL cells (1-2 × 105 cells/cm2) were cultured for 1-7 days under the general conditions detailed in the Supporting Methods. In some experiments, the following soluble factors were added: murine TPO (50 ng/mL; PeproTech, London, UK); VEGF-A (1-10 ng/mL; PeproTech); serotonin (1 μM; Sigma-Aldrich, St. Louis, MO); and cytochalasin B (CytoB; 20 μM; Sigma-Aldrich). When indicated, purified Leaf antimouse VEGF-A or Leaf isotype-matched Abs (ISO; clones 2G11-2A05 and RTK2758, respectively; BioLegend, San Diego, CA) were added to cultures. For coculture assays, CD49fD purified cells were plated in the upper transwell chamber of Col I/III-coated membranes (0.4-μm pore size) in 24-well plates (Costar, Cambridge, MA). CD49fH purified cells were plated either in the lower chamber or in combination with CD49fD cells in the upper chamber. After 7 days, cells in the upper chambers were collected for RNA extraction. Representative images from cultures were captured on a Leica DMI3000B microscope equipped with a DFC420 camera (Leica, Wetzlar, Germany). For scanning electron microscopy, cultures were treated as indicated in the Supporting Methods.
The slides that were stained contained the following: (1) cytospin preparations of sorted or unpurified FL cells prepared by centrifugation in a Cytospin-4 (65 G, 5 minutes; Shandon Southern Products, San Jose, CA); (2) cells cultured on Col I-coated slide chambers; and (3) E11.5 frozen tissue sections (7 μm thick). Samples were treated and stained as indicated in the Supporting Methods. The resulting images were processed using the ImageJ software (v1.43; National Institutes of Health, Bethesda, MD).
Calculation of Contact Frequencies Per Cell-Surface Area.
Contact frequency of CD49fHCD41H MKPs per cell surface area was calculated as described previously,16 dividing the number of contacts observed between MKPs (as CD41H cells) and ALB+ cells or MKPs, and with c-Kit+ cells, by the total surface area of these populations. Confocal immunofluorescence (IF) images were used to measure the corresponding cell radius and to determine frequencies in each population and cell contacts observed between them. Total surface area of each population is the product of the mean surface area of single cells by the total cell numbers of this population. The number of FL cells counted at E11.5 was 95,690 ± 7,110/organ (n = 10).
All data are presented as the means ± standard error of the mean (SEM) that were calculated with GraphPad Prism 4.0 software (GraphPad Software, Inc., La Jolla, CA), and the unpaired t test and the chi-square test were applied.
A Heterogeneous c-KitDCD45− HeP Population Is Present in the FL at E11.5.
CD49f expression in the c-KitDCD45− cell subpopulation of E11.5 FL was characterized by performing a detailed flow-cytometry phenotypic study. Expression of CD45 and either the VEGF receptor 2 (VEGFR2; recognized by the KDR marker) or the integrin αIIb chain (GPIIb/CD41) was quantified in electronically gated FL c-KitDCD49fH and c-KitDCD49fD cells (Fig. 1A-C). A large number of CD49fH cells were either CD45−KDR+/CD41++ or CD45++KDR−/CD41− (CD49fHCD41H and CD49fHCD45H, respectively), whereas a small proportion were CD45+KDR+CD41+. By contrast, most CD49fD cells did not express CD45, CD41, or KDR. The panhematopoietic marker (CD45) labels myeloid-derived cells in the early embryo, and indeed the majority of CD49fHCD45H cells were positive for CD11b/Mac1 (Fig. 1D). High levels of CD41 expression in adult BM is characteristic of MKs, whereas low levels are typical of HSCs. Analysis of the c-Kit receptor, the tetraspanin molecule (CD9), and of the CD42a, CD42b, and CD42c chains of the von Willebrand factor (VWF)/thrombin receptor (VWFR) revealed that CD49fHCD41H cells were c-KitD, CD9++, and VWFR+, a phenotype more characteristic of MKPs and not HSCs. These cells were large (mean forward-scattered light [FSC] intensity, 545 ± 24; n = 5), did not correspond to cellular fragments or platelets, and were abundant, representing up to 8 × 103 ± 282 (n = 10) of viable cells in the FL at E11.5. This initial analysis indicated that the E11.5 FL c-KitDCD49fH cell subset identified had a surface phenotype compatible with CD41H (and CD9+VWFR+) MK cells. In functional analyses performed on FL cell suspensions stimulated with ADP, thrombin, and the PAR4 peptide, CD41H cells up-regulated the active form of the CD41/CD61 fibrinogen receptor and fibrinogen binding as well as inducing actin polymerization (Table 1).
Table 1. Functional Assays in Resting and Activated MKP and Platelets
When the expression of other integrin and receptor molecules was analyzed (Fig. 1E,F), most of these electronically gated CD49fHCD41H cells expressed different levels of α4 (CD49d), α5 (CD49e), αV (CD51), αL (LFA1/CD11a), β1 (CD29), β2 (CD18), and β3 (CD61) chains as well as the endothelial marker CD31 and the intercellular adhesion molecule-1 (ICAM1, CD54). Approximately half of the CD49fHCD41H cells were positive for the integrin α2 chain (CD49b), and these cells had no αM (CD11b/Mac1) and β4 (CD104) chains nor the vascular cell adhesion molecule-1 (VCAM1, CD106), receptors for fms-related tyrosine kinase 3 (Flt3), or the lymphohematopoietic marker AA4.1. Taken together, these findings demonstrate the presence of significant numbers of cells with a surface phenotype and a functional behavior characteristic of MKPs in the E11.5 FL. These cells display several integrin receptors and are easily identified by flow cytometry as CD49fHCD41H (and CD9++CD42c+) cells.
E11.5 CD49fHCD41H Cells Spontaneously Develop Proplatelets In Vitro.
We used flow cytometry to purify and subsequently culture E11.5 CD49fHCD41H cells to analyze their differentiation in vitro. Cells with proplatelets were visible after 24 hours in culture in the absence of TPO. These cells had large cytoplasmic pseudopodia (mainly unbranched, with bulges along the shaft and a swelling at the tip), expressed CD41 and CD42c, and became prominent after 48 hours (Fig. 2A,B). In these conditions, few cells were attached to the surface of the plates, although the addition of TPO to cultures resulted in an increase in cell adherence (Fig. 2C) without affecting the number of MKs with proplatelets. On Col I-coated plates, attachment occurred earlier and the number of proplatelet-bearing MKs was higher. Staining with anti-CD41 and phalloidin after 48 hours in culture revealed different patterns of expression (Fig. 2D and Supporting Fig. 1), with some cells exhibiting a punctuate distribution of CD41, which colocalized with F-actin in podosome-like structures in adherent cells, and with F-actin clusters in the swellings along the shaft membrane of proplatelets. By contrast, in some cells, there were strong cytoplasmic accumulations of CD41, whereas others expressed CD41 primarily in microvilli and in membranes (indicative of the demarcation membrane system). When CytoB was added to recently seeded cultures, the formation of proplatelets was inhibited without affecting cell viability, and CD41 accumulated in the cytoplasm independently of F-actin (Fig. 2E). In conclusion, these in vitro data confirmed that embryo-derived CD49fHCD41H cells were MKPs capable of producing proplatelets in culture independently of TPO by an actin-dependent process.
CD49fHCD41H MKPs From E11.5 FL Display Hepatoepithelial and Endothelial Proteins.
Purified embryonic CD49fHCD41H MKPs exhibited a characteristic, punctuate VWF expression pattern in the cytoplasm (Fig. 3A) and were positive for ALB and nestin (NES; an intermediate filament expressed by endothelial and neural stem cells; Fig. 3B and Supporting Fig. 2). By contrast, CD49fD cells were ALB++ and were negative for NES. Isolated CD49fHCD41H MKPs were binucleated (and, less frequently, multinucleated) cells, some of which contained cytoplasmic protrusions, even after the mechanical stress produced by the FACS procedure (Fig. 3D). These proplatelets were more clearly observed when slides from unpurified E11.5 FL cells stained for CD41 were overexposed (Fig. 3E), indicating that fully developed proplatelets were not merely an in vitro differentiation product, but that they also existed in the E11.5 FL in vivo. The proplatelet-bearing CD41H cells present in unpurified FL were also ALB+ (Fig. 3F and Supporting Fig. 2).
To determine whether these expression patterns were the result of NES and ALB synthesis by FL MKPs, we performed PCR analyses on total FL and YS cells, purified CD49fHCD41H MKPs and CD49fD cell populations from E11.5 FL, and adult tissues, including immature c-Kit+Lin−CD9+CD41+ MK (iMKs) isolated from BM.4 These analyses confirmed that VWF and the glycoprotein Ibα (GPIbα) chain of its receptor were expressed more strongly in CD49fHCD41H MKPs than in CD49fD cells. Moreover, CD49fH CD41H MKPs expressed VEGF-A and its receptor (KDR/VEGFR2), as well as NES, VIM, and several hepato-specific transcripts, such as ALB, alpha-fetoprotein (AFP), and transthyretin (TTR), although they did not express α1-antitrypsin (AAT) (Fig. 4A). IFs on tissue sections of E11.5 indicated that 60% ± 13% of CD41H cells express VEGF-A, and 27% ± 3% of these CD41HVEGF+ cells displayed the highest VEGF-A signal in FL (Fig. 4B and Supporting Fig. 3). There was a 20-fold increase in the expression of ALB transcripts in CD49fD cells when determined by quantitative real-time PCR (Fig. 4C). Expression of hepatoepithelial genes seemed to be specific to CD49fHCD41H MKPs of FL origin, because none were expressed in CD45−CD41H MKPs isolated at E11.5 from other locations (such as the YS, AGM, and PBLs; data not shown) nor were they expressed in hematopoietic CD45HCD41− cells or in adult iMKs (Fig. 4D and Supporting Fig. 4). However, although dual-sorted purified CD45−CD41H liver cells expressed ALB, AFP, and transthyretin, but not AAT, it cannot be fully ruled out that the CD49fH liver preparations were contaminated with a small number of CD49fD cells that could account for the hepato-specific signals.
We next verified by IF whether CD41H MKPs from FL expressed the hepatocyte nuclear factors (HNFs), HNF-1, HNF-3β, and HNF-4α, which are essential for the expression of most hepatocyte genes. In preparations from unpurified E11.5 FL cells, and from purified c-KitDCD45− and CD49fHCD41H cells, there was only a weak punctuate nuclear HNF-4α and HNF-1 signal in CD41H cells (Fig. 5A and Supporting Fig. 5), and no staining for HNF-3β was observed (not shown). By contrast, brighter homogeneous signals were detected in the nuclei of CD49fDCD41− cells. In addition, no surface expression of hepatic glucose transporter type 2 (GLUT2) was detected in CD49fHCD41H MKPs (Fig. 5B). Therefore, the ALB protein detected in CD49fHCD41H MKPs from the E11.5 FL is most probably accumulated by endocytosis.
To further clarify the relationship between FL MKPs and HeP, the Dlk/CD13 markers used to define liver stem/progenitor cells17 were analyzed on electronically gated CD49fHCD41H and CD49fD cells from FL (Fig. 5C,D). We found that CD49fD cells contained most Dlk+CD13+ cells (1,291 ± 389 cells/FL), whereas CD49fHCD41H MKPs contained only 62.5 ± 9.8 cells/FL (n = 10) of Dlk+ cells. Taken together, these results reinforce the idea that FL CD49fHCD41H MKPs are distinct to HeP, even though they share some characteristics of hepatoepithelial and endothelial cells.
CD49fHCD41H MKPs From E11.5 FL Drive the In Vitro Development of Hepatoepithelial Layers in Culture.
The c-KitDCD45− population contained HeP that can establish hepatoepithelial layers in vitro.10 Because the subpopulation of CD49fH CD41H cells present in the c-KitDCD45− HeP appear to belong to the MK lineage, and the remaining CD49fD cells express hepatoepithelial transcripts and contain Dlk+CD13+ cells, we reasoned that these CD49fD cells may represent the true HeP present in the FL at E11.5. To investigate this hypothesis, we cultured purified c-KitDCD45−CD49fD (CD49fD) cells after removing c-KitDCD45−CD49fH (CD49fH) cells by FACS. In the absence of the CD49fH population, CD49fD cells could not grow in culture on any of the substrates tested (uncoated, collagen I, laminin, or fibronectin), and after 3 days in culture, most of them adopted a small, round appearance of apoptotic cells (Supporting Fig. 6). When CD49fH cells were seeded along with CD49fD cells, the mix of the purified subpopulations formed hepatoepithelial layers, as did cultures of total purified c-KitDCD45− cells (Fig. 6A). These cultured cells expressed HNF-4α (Supporting Fig. 6). We concluded that the presence of CD49fHCD41H MKPs was required for CD49fD HeP to grow in vitro. To determine whether this process was mediated by direct cell-to-cell contacts or by soluble factors, we cultured the purified CD49fH and CD49fD populations in transwells (Fig. 6B). Again, epithelial layers developed when both subpopulations were grown together in the upper chamber of transwell plates. By contrast, the growth of CD49fD HeP seeded in the upper chamber was poorer when the CD49fH MKPs were plated in the bottom chamber, despite the normal in vitro development of proplatelets in these cultures. The addition of conditioned medium from CD49fHCD41H cells to CD49fD cultures promoted a limited growth of hepatoepithelial layers (Supporting Fig. 6), in agreement with the fact that supernatants from complete FL cell cultures or growth-promoting factors need to be added to adult or FL-derived liver progenitors to generate hepatoepithelial layers in vitro.11-13, 18
The induction of ALB and AAT expression was considered evidence of hepatocyte differentiation in our cultures (Fig. 6C and Supporting Fig. 6). The greatest increase in ALB expression was induced when both CD49fH MKP and CD49fD HeP cells were grown together in the same chamber (4.9-fold). Conversely, when these two populations were separated by a membrane, ALB expression increased similar to that induced in CD49fD cultures to which conditioned medium was added (2.1- and 2.5-fold, respectively). Serotonin and VEGF were both detected in MKs and platelets and may play a role in hepatocyte growth and regeneration after liver injury.19, 20 Indeed, FL CD41H cells express the highest levels of VEGF-A in the FL (Fig. 4B). It has also been reported that maternal serotonin promotes embryonic FL growth.21 Although serotonin neither induced hepatoepithelial layer formation nor increased ALB expression in our system, VEGF-A induced both effects to a similar extent to that observed after the addition of conditioned medium, as well as inducing an increase in VEGFR2/KDR expression (Fig. 6D). By contrast, the addition of anti-VEGF Abs to c-KitDCD45− cells reduced ALB levels in cells of these cultures. Thus, in addition to the cell-to-cell contacts required for complete development of hepatoepithelial layers, our data indicate that soluble factors derived from MK and, in particular, VEGF-A are involved in the growth of ALB-producing cells.
Finally, the involvement of MKPs in establishing the hepatoblast niche in vivo was suggested by the close localization of both MKPs (as CD41H) and HeP (as ALB++) in vivo at E11.5, as demonstrated by the contact observed between MKPs and ALB++ cells (Fig. 7A,C) and between MKPs and the more-abundant c-Kit+ subpopulation or other MKPs (Fig. 7B,C). These data show that direct cellular contacts between MKs and HeP occur physiologically, and strongly suggest that MKs may facilitate the development of the hepatoepithelial liver compartment.
During FL morphogenesis in the postgastrulation embryo, a liver-specific progenitor (the hepatoblast) can be identified by its capacity to differentiate to both hepatocytes and cholangiocytes.10, 11 The phenotype of the early HeP at E11.5 has been defined as c-KitD/−CD45−Ter119−, with variable levels of CD49f expression, together with other markers, such as the hepatocyte growth factor (HGF) receptor (c-Met) and Dlk.10-12, 18 However, postnatal liver progenitors have been described as CD49fH.13 Our results demonstrate that at E11.5, the c-KitDCD45−Ter119− liver progenitors contain two subsets of cells: one defined as CD49fDCD41− that could represent HePs and another phenotype defined as CD49fHCD41H, suggesting an MK lineage. These CD49fHCD41H FL cells respond to ADP and thrombin stimulation, rapidly differentiating in vitro, as described for embryonic MEPs in semisolid assays.5, 6 Indeed, after 24 hours in culture, cytoplasmic elongations develop and proplatelets are generated in a process dependent upon the reorganization of the actin cytoskeleton, similar to that described in mature MKs.8, 22 Our observation that proplatelets are present in vivo under physiological conditions in isolated cellular FL preparations is particularly relevant until, as recently, proplatelet development by MKs was demonstrated to occur in vivo after TPO treatment.23
The CD49fHCD41H MKPs found in the FL represent a potential source of pure MKPs that are readily isolated by FACS or immunomagnetic methods, in contrast to the BM clonogenic MK population that is relatively small.4, 16 As observed in adult BM MKs, c-KitDCD49fHCD41H cells from E11.5 FL express several integrin receptors. The engagement of α4β1, and not αVβ3, has been proposed to enhance TPO-induced megakaryopoiesis.24 In FL c-KitDCD49fH CD41H cells, we observed weaker α4 expression in relation to the αV chain, which may be related to the TPO-independent maturation of these cells in vitro. This observation is consistent with findings from c-Mpl-deficient mice, in which MK generation occurs in a TPO-independent manner before E9.5 in the YS and before E14.5 in the FL.6, 25 The α6 integrin chain (CD49f) associates with either β1 (CD29) or β4 (CD104) integrin chains to form receptors for laminin and kalininis, respectively, and has been implicated in adhesion and in vivo homing. This chain is expressed by HSCs and myeloid progenitors in E14.5 FL and BM,26 among other cells, and by MKs and platelets generated in vitro.27 However, to the best of our knowledge, CD49f has not commonly been associated with MKs ex vivo. In contrast to the adult BM MK-lineage cells (including mature MKs) and other embryonic myeloid cells, embryonic CD49fHCD41H MKPs do not express the hematopoietic marker (CD45).
Several of the cell-surface markers presented by the CD49fHCD41H MKPs present in the FL at E11.5 differ from those in other hematopoietic niches (manuscript in preparation). Specifically, these cells express endothelial and hepatoepithelial proteins, the latter probably being taken up by endocytosis in the liver (where they would be produced by the HeP), because CD49fHCD41H MKPs appear to only weakly or not at all express HNF-1, HNF-4α, and HNF-3β. Similarly, most CD49fHCD41H MKPs are Dlk−CD13−, indicating that they are not liver stem/progenitor cells. The possible relationship between the few CD49fHDlk+ cells and the more-abundant CD49fD Dlk+CD13+ cells will require further analysis. These embryonic CD49fHCD41H MKPs preferentially contact ALB+ hepatoblasts in vivo and may contribute to specific hepatocyte-developmental niches, as proposed for adult MKs in the establishment of plasma cell niches in the BM.16In vitro, CD49fHCD41H MKPs stimulate the development of CD49fD HeP. Moreover, in transwell cultures, hepatospecific genes are up-regulated in immature CD49fD HeP in response to direct cell contact and CD49fHCD41H cell-derived soluble factors, in particular VEGF-A, which is produced most strongly by CD49fHCD41H cells. In fact, although VEGFR2/KDR is weakly expressed at E11.5 ex vivo by CD49fD HeP, its expression is up-regulated in vitro after the addition of VEGF-A. MKs produce VEGF,28 which participates in the endothelial organization of the vasculature, vasculogenesis, and blood island formation, and fulfils other nonvascular roles in the morphogenesis of adult organs and stem cell niches.16, 29-31 In addition to their role in hemostasis, platelets, the end product of MK differentiation, are involved in liver regeneration and hepatocyte proliferation through direct contact as well as the release of HGF, insulin growth factor, and VEGF.32, 33 Indeed, they are also involved in several other biological processes, including the spread of hematogenic tumor cells,34 vessel remodeling in the newborn,35 and the separation of blood and lymphatic circulation during development.36, 37 In conclusion, the data presented here describe the precise phenotypic identification of embryonic CD49fHCD41H MKPs. Our findings propose interesting new tools to study the role of MKs in tissue regeneration and strongly support a role new for CD49fHCD41H MKPs in the development of the FL, involving the action both of cellular contacts and VEGF-A.
The authors thank Beatriz Palacios, Fernando Martínez, and Carmen Prado for their technical assistance and help with the animal care and Mark Sefton for his editorial assistance.