Hematopoietic Stem and Progenitor Cell Maintenance and Multiple Lineage Differentiation Is an Integral Function of NFATc1

Hematopoietic stem and progenitor cell (HSPC) maintenance and the differentiation of various lineages is a highly complex but precisely regulated process. Multiple signaling pathways and an array of transcription factors influence HSPC maintenance and the differentiation of individual lineages to constitute a functional hematopoietic system. Nuclear factor of activated T cell (NFAT) family transcription factors have been studied in the context of development and function of multiple mature hematopoietic lineage cells. However, until now their contribution in HSPC physiology and HSPC differentiation to multiple hematopoietic lineages has remained poorly understood. Here, we show that NFAT proteins, specifically NFATc1, play an indispensable role in the maintenance of HSPCs. In the absence of NFATc1, very few HSPCs develop in the bone marrow, which are functionally defective. In addition to HSPC maintenance, NFATc1 also critically regulates differentiation of lymphoid, myeloid, and erythroid lineage cells from HSPCs. Deficiency of NFATc1 strongly impaired, while enhanced NFATc1 activity augmented, the differentiation of these lineages, which further attested to the vital involvement of NFATc1 in regulating hematopoiesis. Hematopoietic defects due to lack of NFATc1 activity can lead to severe pathologies such as lymphopenia, myelopenia, and a drastically reduced lifespan underlining the critical role NFATc1 plays in HSPC maintenance and in the differentaion of various lineages. Our findings suggest that NFATc1 is a critical component of the myriad signaling and transcriptional regulators that are essential to maintain normal hematopoiesis.


Introduction
Hematopoiesis is a complex but precisely regulated process through which hematopoietic stem and progenitor cells (HSPC) give rise to mature blood cells of all lineages [1,2]. Hematopietic stem cells (HSCs) remain at the top of the hierarchy, which have the unique ability to self-renew in a controlled manner and also, to differentiate to the immediate downstream progenitor cells of specific lineages. Because of the regulated proliferation, HSCs are mostly quiescent, which is a characteristic feature of the stemness of these cells. In the bone marrow (BM), HSCs reside in a very specialized environment, called the hematopoietic stem cell niche [3][4][5]. There, an array of intrinsic and extrinsic signaling pathways and multiple transcription factors (TFs) regulate HSPC maintenance [6][7][8]. HSPCs are identified as Lin − Sca1 + c-Kit + (LSK) cells, which are lineage marker negative and express both the stem cell antigen-1 (Sca1) and stem cell factor receptor (c-Kit/CD117). Further evaluation of LSK cells based on the expression of the signaling lymphocyte activation molecule (SLAM) family receptors defines HSCs as LSKCD150 + cells [9]. Based on their functional properties, HSCs consist of two distinct populations. LSK cells lack the expression of fetal liver kinase-2 (Flk-2) and are the long-term repopulating stem cells (LT-HSCs), which are mostly quiescent and undergo limited cell cycle for self-renewal [10]. LSK cells have high expression of Flk-2 and are the short-term repopulating stem cells (ST-HSCs), which have limited ability to self-renew but they differentiate to give rise to the common lymphoid progenitor (CLP), common myeloid progenitor (CMP), and the megakaryocyte-erythrocyte progenitor (MEP) populations. These progenitor populations occupy specific hematopoietic niches to give rise to mature blood cells of all lineages.
CLP-derived T cells develop in the thymus, whereas B cells and NK cells are produced in the BM. All myeloid cells such as dendritic cells (DCs), macrophages, monocytes, granulocytes, and neutrophils are derived from the CMPs in the BM. Whereas lymphocytes are the major components of the adaptive immune system, cells of the myeloid origin constitute the innate immune system. In addition, in the BM, MEPs differentiate to produce erythrocytes and megakaryocytes, which are vital for the survival of an organism. All these components that are responsible for specific functions, together, form the hematopoietic system. Although various TFs have been implicated to regulate HSPC differentiation and maintenance [6,11], the role of the nuclear factor of activated T cells (NFAT) family TFs in this process has not been explored yet. The NFAT family consists of five members, i.e., NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. Except for NFAT5, the other family members are activated in a calcium-and serine/threonine phosphatase calcineurin-dependent manner [12,13]. We recently reported a calcium and calcineurin-independent mechanism of NFAT activation, which played a critical role in T cell development [14]. Previous studies have shown the critical involvement of NFAT proteins in the development, differentiation, and function of various mature lineage-positive cells [15][16][17]. However, whether NFAT proteins together or any specific family member individually play any role in the maintenance of HSPCs, and whether they can influence hematopoiesis in addition to their roles in the already differentiated lineage-positive cells is an interesting aspect to be investigated. Using a murine model where NFATc1 activity was specifically ablated in a hematopoietic system (Vav-CreNfatc1 fl/fl mice), we recently showed that it played an indispensable role in T and B cell development in the thymus and BM, respectively [14,18,19]. Mice deficient in the activity of other NFAT family members did not show a similar phenotype. In addition, in Vav-CreNfatc1 fl/fl mice, the presence of other NFAT proteins did not compensate for the loss of NFATc1 activity in lymphocyte development [14]. NFATc1-deficiency in these mice also impaired erythropoiesis [20], suggesting that NFATc1 is a key component of the regulatory mechanism that contributes towards normal development of various hematopoietic lineages. These observed strong influences of NFATc1 deficiency on multiple hematopoietic lineages led us to investigate whether it also regulates the maintenance of HSPCs. Here, we show that NFATc1 is the key family member that exerts vital control over HSPCs, and also on the differentiation of other lineage-positive cells, which, so far, has been unknown. We demonstrate that NFATc1 is expressed right from the HSCs down to all progenitor populations and in lineage-positive cells, and NFATc1 deficiency leads to severe abnormalities in HSPC maintenance, which strongly affects hematopoiesis.

Immunofluorescence Staining
Sorted HSPC (LSK), Sca1 + (Lin − Sca1 + c-Kit − ), c-Kit + (Lin − Sca1 − c-Kit + ), L − S − K − (Lin − Sca1 − c-Kit − ), LT-HSCs, or ST-HSCs from WT mice were immunostained with NFATc1 (Santa Cruz, sc14034) antibodies following a previously published protocol [21]. Nuclear NFATc1 was confirmed by co-staining the cells with DAPI. Image acquisition and analysis were done with a TCS SP2 Leica confocal microscope and software. Nuclear NFATc1 in each immunofluorescence experiment was estimated on individual cells in each focus using the Adobe Photoshop software. The nuclear area was demarcated for each cell and the mean relative fluorescence was determined for the channel used using the histogram option. This was done for a number of cells in each focus and multiple foci from each experiment were analyzed. The mean for each group in each experimental replicate were calculated. These mean values in each group from three experimental replicates were used for the figures and for statistical analysis.

Adoptive Cell Transfer
Splenocytes (5 × 10 6 ) depleted of RBCs, from CD45.2 + Nfatc1 fl/fl or Vav-CreNfatc1 fl/fl donors, were transferred to lethally (9 Gy) irradiated CD45.1 + congenic WT recipients by retro-orbital injection into the venous sinus. Post-adoptive transfer, recipient mice were maintained with antibiotic-supplemented drinking water and were observed carefully. Hematopoietic reconstitution in recipient mice was analyzed at 11 weeks after transfer by gating on donor-derived cells.

Photographs
Photographs of Nfatc1 fl/fl , Vav-CreNfatc1 +/fl , and Vav-CreNfatc1 fl/fl mice for body size, tooth eruption, and the bones from hind limbs were taken using a Nikon Coolpix 4500 digital camera, and the photographs were processed using the Adobe Photoshop software.

NFAT Expression in HSPC Population
To investigate whether NFAT family transcription factors play any roles in HSPC development and differentiation, we analyzed for Nfat expression in WT LSK cells (Supplementary Figure S1a). Expressions of Nfatc1, Nfatc2, and Nfatc3, the three major members of NFAT family, were readily detectable in HSPCs (Figure 1a). In addition to HSPCs, strong Nfat expression was also detectable in Lin − Sca1 − c-Kit + (c-Kit + ) and Lin − Sca1 + c-Kit − (Sca1 + ) cells, suggesting that Nfat expression is not only present in HSPCs but also in all hematopoietic lineage cells (Figure 1a). A weak Nfat expression in Lin − Sca1 − c-Kit − (L − S − K − ) cells further suggests that there is a differential expression of Nfat genes in various Lin − BM cell populations ( Figure 1a). To determine whether NFAT proteins combinatorially or individually regulate HSPC physiology, and to identify the key NFAT family member regulating HSPC function, we analyzed various Nfat gene ko mice. Nfatc1 ko mice are embryonic lethal [22]. The analysis of mice deficient in NFATc2 (Nfatc2 −/− ), NFATc3 (Nfatc3 −/− ), or both (Nfatc2 −/− Nfatc3 −/− ) revealed comparable proportion of HSPCs as compared with littermate control mice (Figure 1b,c). Further, the relatively normal phenotype of these ko mice suggests that NFATc1 is most likely the key family member that plays a role in HSPC biology.
In line with the strong Nfatc1 expression, high levels of NFATc1 proteins were also detected in HSPC, c-Kit + , and Sca1 + cells (Figure 1d,e and Supplementary Figure S1b). The analysis of Nfatc1-eGfp-Bac tg reporter mice [23] further supported Nfatc1 expression in HSPCs (Figure 1f), and also in HSCs ( Figure 1g). Again, Nfatc1 expression was not restricted to HSCs, but was also detected in the downstream common lymphoid progenitor (CLP, Lin − IL-7R + Sca1 − c-Kit + ) and common myeloid progenitor (CMP;,Lin − IL-7R − Sca1 − c-Kit + ) cells, suggesting that NFATc1 is widely expressed in the hematopoietic system ( Figure 1h). Further, analysis of long-and short-term HSCs (Lin − Sca1 + c-Kit + Flk2 − (LT-HSCs) and Lin − Sca1 + c-Kit + Flk2 + (ST-HSCs)) revealed the presence of a higher level of NFATc1 proteins in ST-HSCs as compared with that in the LT-HSCs (Figure 1i,j).

NFATc1 Critically Regulates HSPC Development
To unravel the role of NFATc1 in HSPC generation, we analyzed Vav-CreNfatc1 fl/fl mice, in which NFATc1 activity was ablated in the hematopoietic system [14]. The flow cytometry analysis of BM cells immediately revealed gross anomalies in the distribution of cells according to their FSC and SSC patterns. Whereas, in Nfatc1 fl/fl mice, both FSC lo SSC lo and FSC hi SSC hi cells were evident; in Vav-CreNfatc1 fl/fl mice, the BM cells were mostly FSC lo SSC lo , suggesting a defective hematopoiesis in absence of NFATc1 ( Figure 2a). Due to the lack of NFATc1, Vav-CreNfatc1 fl/fl total BM cells as well as the Lin − cells, which contain the HSPCs, were much smaller in size, although they were similar in their granularity as compared with the control cells (Figure 2b,c). Defective hematopoiesis in generating enough blood cells was evident as the BM cellularity in the absence of NFATc1 reduced drastically in Vav-CreNfatc1 fl/fl mice (Figure 2d). Accordingly, the analysis of the HSPC population in Vav-CreNfatc1 fl/fl mice revealed a strong decrease as compared with that in the littermate control mice (Figure 2e-g). Simultaneously, c-Kit + and Sca1 + cells were also severely reduced in Vav-CreNfatc1 fl/fl mice ( Figure 2e) indicating a severe defect in hematopoiesis in general in the absence of NFATc1 activity. In the BM, self-renewing LT-HSCs, which undergo limited cell cycle and are mostly quiescent, give rise to the ST-HSCs, which divide actively and differentiate to produce the progenitor populations of all hematopoietic lineages. Surprisingly, in Vav-CreNfatc1 fl/fl HSPCs, we observed a strong decrease in the expression of Cd150 (Slamf1) and an increased expression of Flk2 indicating an increased prevalence of ST-HSCs and a paucity of LT-HSCs as compared with littermate Nfatc1 fl/fl mice ( Figure 2h). Hematopoietic loss of NFATc1 had a severe consequence on the life of Vav-CreNfatc1 fl/fl mice. These mice were not born with normal frequency (2.022% actual frequency against 12.5% expected). As compared with age-and gender-matched littermate control mice, the Vav-CreNfatc1 fl/fl mice were significantly smaller in their body size and weight (Figure 2i,j). This phenotype was already evident in Vav-CreNfatc1 fl/+ mice (Figure 2j), suggesting that the effects of NFATc1 deficiency in the hematopoietic system, on the overall phenotype of these animals are profound. Interestingly, none of the Vav-CreNfatc1 fl/fl mice survived beyond 4 weeks after birth (Figure 2k). The number of HSPCs was reduced in Vav-CreNfatc1 fl/fl mice, and their gene expression patterns were also severely affected in the absence of NFATc1 activity. Gene expression of several of the key transcription factors and other molecules essential for HSC quiescence, survival, and function was not lacking, rather, it was strongly upregulated in Vav-CreNfatc1 fl/fl HSPCs (Figure 2l). The severe phenotype of Vav-CreNfatc1 fl/fl mice suggests a general failure of the hematopoietic system in the absence of NFATc1 activity, which has not been observed in the case of mice deficient in any other NFAT family members. In support of this hypothesis, Nfatc1 expression was easily detected in HSPCs, various progenitor populations, as well as in mature hematopoietic cells (Figure 2m).
Nfatc1 expression is directed from two distinct promoters, a distal P1 promoter directing the synthesis of the inducible NFATc1α isoforms, and a proximal P2 promoter initiating the synthesis of the constitutively expressed NFATc1β isoforms [24]. We recently reported the critical role of this promoter-specific Nfatc1 expression during T and B cell differentiation in a developmental stage-dependent manner [18,19]. The analysis for any promoter-specific preferential Nfatc1 expression in hematopoietic stem and progenitor cells, as well as in lineage-positive mature cell populations, revealed a distinct P2 promoterinduced Nfatc1b activity in all hematopoietic cells starting from the HSCs downwards ( Figure 2m). P1-derived Nfatc1a isoform was detectable only in mature T and B cells, which express the TCR and BCR, respectively, along with Nfatc1b isoform (Figure 2m). We previously reported NFATc1 expression in lymphocytes and erythrocytes [14,[18][19][20]25]. Analysis of various myeloid and erythroid populations from Nfatc1-eGfp-Bac tg mice further confirmed NFATc1 expression in these lineage-positive cells (Supplementary Figure S2), suggesting a pan-hematopoietic system-specific function of NFATc1. We recently reported severely impaired T and B cell development in Vav-CreNfatc1 fl/fl mice due to defects in the thymus and BM, respectively, at the ealiest stages of differentiation. These defects invariably led to strong T and B cell lymphopenia in these mice (14,18,19). The development of similar pathological conditions in two distinct lymphoid lineage cells lacking NFATc1 activity led us to investigate whether additional defects at the HSPC level also contribute to these phenotypes in Vav-CreNfatc1 fl/fl mice. To delineate any HSPCspecific defects in T and B lymphopoiesis, we adoptively transferred CD45.2 + Nfatc1 fl/fl or Vav-CreNfatc1 fl/fl splenocytes to lethally irradiated congenic CD45.1 + hosts. Eleven weeks after transfer, we observed a strongly reduced cellularity in the thymus, LNs, and spleen of the CD45.1 + recipient mice receiving CD45.2 + Vav-CreNfatc1 fl/fl splenocytes, indicating defects in lymphopoiesis (Figure 3a). An evaluation of the CD45.2 + donor-derived cells in these organs showed a strongly reduced reconstitution capacity of Vav-CreNfatc1 fl/fl splenocytes as compared with Nfatc1 fl/fl cells (Figure 3b). Further analysis revealed that Vav-CreNfatc1 fl/fl splenocytes gave rise to very few CD4 + and CD8 + T cells in the NFATc1sufficient recipient mice (Figure 3c). Similarly, the analysis of B cell populations in the BM and spleen of the recipient mice revealed a much lower reconstitution of IgM + and IgD + B cells in the case of Vav-CreNfatc1 fl/fl donors as compared with Nfatc1 fl/fl donors (Figure 3d). These defects in T and B cell development recapitulate the severe lymphopenia in Vav-CreNfatc1 fl/fl mice we have reported recently and suggest that HSPC-specific defects also contribute to this lymphopenia in addition to other signaling defects.

NFATc1 Deficiency Severely Impairs Myelopoiesis
Owing to the impaired lymphopoiesis, we investigated whether other branches of hematopoiesis were also affected due to lack of NFATc1 activity in Vav-CreNfatc1 fl/fl mice. Strikingly, the phenotypic analysis revealed a lack of teeth eruption in Vav-CreNfatc1 fl/fl mice as compared with littermate controls (Figure 4a). This was consistent in all Vav-CreNfatc1 fl/fl mice analyzed, suggesting a possible defect in myeloid cell development. The complete absence of teeth in Vav-CreNfatc1 fl/fl mice as compared with the normal teeth eruption in Vav-CreNfatc1 +/fl mice indicates an absolute requirement of NFATc1 activity in this process. In addition to the lack of teeth eruption, bone formation in Vav-CreNfatc1 fl/fl mice was also defective, as bones from the hind limbs were short and more solid as compared with the littermate control mice (Figure 4b), indicating osteopetrosis. The role of NFATc1 in osteoblast differentiation and bone formation has been reported [26,27], and their involvement in bone-resorbing osteoclast development has also been suggested [28,29]. Osteoclasts differentiate from the CD11b + myeloid cells. Interestingly, the flow cytometry analysis to explore whether the teeth eruption problem was due to a defect in osteoblast or osteoclast activity in Vav-CreNfatc1 fl/fl mice, revealed a complete lack of CD11b + cells in the BM (Figure 4c,d). This suggested that the defect in Vav-CreNfatc1 fl/fl mice was in the osteoclast differentiation. Combined with the loss of CD11b + cells, severely reduced Gr1 + cells in Vav-CreNfatc1 fl/fl mice (Figure 4e (Figure 4h,i). The impaired myelopoiesis in Vav-CreNfatc1 fl/fl mice was specific to NFATc1 deficiency, as Nfatc2 −/− or Nfatc3 −/− mice did not show these abnormalities in myelopoiesis (Supplementary Figure S3a-d).
In addition to the lack of myeloid cells, we recently reported an enhanced production of Ter119 + erythroid cells in Vav-CreNfatc1 fl/fl mice, which suggested that the process of erythropoiesis was also dysregulated in the absence of NFATc1 activity [20]. Taken together, the lack of NFATc1 activity in the hematopoietic system severely impairs development and differentiation of multiple hematopoietic lineages.

NFATc1 Activity Is Indispensable for Normal Hematopoiesis
Ssince the NFATc1 in hematopoietic stem and progenitor cells was mainly of the P2 promoter-derived NFATc1β isoforms, next, we investigated if NFATc1β was critical for HSPC development and hematopoiesis. To check if HSPC development was affected in the absence of NFATc1β, we analyzed Vav-CreNfatc1P2 fl/fl mice in which Nfatc1 P2 promoter activity was ablated in the hematopoietic system [18]. We observed normal levels of HSPCs in Vav-CreNfatc1P2 fl/fl mice as compared with littermate control mice (Figure 5a-c). This was quite intriguing considering the grossly impaired HSPC population in Vav-CreNfatc1 fl/fl mice (Figure 2e-g). The RT-PCR analysis revealed a complete absence of P2 promoter activity in Vav-CreNfatc1P2 fl/fl mice (Figure 5d). However, in the absence of P2 activity, a strong P1 promoter-derived Nfatc1a isoform expression was detected in HSPCs and in all hematopoietic lineage cells (Figure 5d), which suggested that NFATc1 activity was present in these cells. As a result of normal HSPC numbers in Vav-CreNfatc1P2 fl/fl mice, the development of lymphoid, myeloid, and erythroid cells remained unaffected. The distribution of various T and B cell populations in peripheral lymphoid organs was comparable to that of littermate control mice (Figure 5e-g). Interestingly, the stark defects in myelopoiesis in terms of the development of CD11b + and Gr1 + cells in Vav-CreNfatc1 fl/fl mice were not observed in Vav-CreNfatc1P2 fl/fl mice (Figure 5h,i). In addition, the alterations in erythropoiesis in the absence of NFATc1 activity were rescued in Vav-CreNfatc1P2 fl/fl mice (Figure 5j,k). As a result of normal hematopoiesis, Vav-CreNfatc1P2 fl/fl mice did not exhibit any of the defects observed in the case of Vav-CreNfatc1 fl/fl mice ( Figure 2) and had a normal lifespan. Altogether, the analysis of Vav-CreNfatc1P2 fl/fl mice established that NFATc1 activity irrespective of the α or β isoforms was indispensable for HSPC maintenance and normal hematopoiesis.

Increase in NFATc1 Activity Impairs Hematopoiesis
Next, we investigated if the loss of NFATc1 activity resulted in a loss of HSPCs and a defective hematopoiesis; an increase in NFATc1 activity would probably augment HSPC development and differentiation of various lineages. In Il2 −/− mice, the frequency of HSPCs is increased several times as compared with that in littermate control mice [30]. This holds true in the BM, spleen, and in the blood from all Il2 −/− mice irrespective of gender (Figure 6a,b). We hypothesized that most likely there is an increase in NFAT activity in Il2 −/− HSPCs, which facilitates enhanced levels of HSPC development. The RT-PCR analysis revealed a strongly increased Nfat expression in Il2 −/− HSPCs as compared with that in WT mice (Figure 6c). In addition, the analysis of Il2 −/− Nfatc1-eGfp-Bac tg reporter mice further demonstrated increased NFATc1 levels in HSPCs and HSCs as compared with control cells (Figure 6d,e). Increased NFATc1 levels were also observed in the c-Kit + and Sca1 + cells from Il2 −/− Nfatc1-eGfp-Bac tg reporter mice (Supplementary Figure S4a). We previously showed that, in Il2 −/− mice, there is an increase in LT-HSCs and a corresponding decrease in the ST-HSCs [30], which was exactly the opposite of what we have observed in the case of Vav-CreNfatc1 fl/fl mice (Figure 2h). This further suggests that alterations in the level of NFATc1 activity will certainly have an adverse influence on HSPC maintenance.
The increased Nfat expression is most likely due to enhanced integrin-cAMP signaling in the Il2 −/− HSPCs. HSPCs generally express several integrins (Supplementary Figure S4b), and our analysis revealed that Il2 −/− HSPCs express much higher levels of various integrins as compared with WT HSPCs (Figure 6f). Integrin signaling has been reported to induce cAMP signaling [31][32][33], and recently, we showed that integrin-cAMP signaling was a critical regulator of Nfatc1 expression [18]. Complementing the increased integrin expression, we observed decreased phosphodiesterase (Pde4b) expression in Il2 −/− HSPCs, which further supported the enhanced cAMP activity in these cells as compared with WT HSPCs (Figure 6f). We analyzed Il2 −/− mice for various lineage-positive cells to explore if enhanced HSC numbers in Il2 −/− mice also enhanced hematopoiesis. Interestingly, in Il2 −/− mice, we observed strongly increased myeloid cell populations as compared with littermate WT mice. CD11b + and Gr1 + myeloid cell differentiation was increased in Il2 −/− mice (Figure 6g,h), which was in stark contrast to the situation in Vav-CreNfatc1 fl/fl mice. We previously showed that there was an increased expression of genes responsible for myelopoiesis in Il2 −/− mice [30], which was suppressed in Vav-CreNfatc1 fl/fl mice (Csf1r, Csf3r, and Mpl, Figure 4g). In addition, contrary to the enhanced Ter119 + erythroid cell differentiation in Vav-CreNfatc1 fl/fl mice, differentiation of these cells in Il2 −/− mice was drastically reduced, which we reported earlier [20]. The enhanced T cell population in Il2 −/− mice was also in contrast to the lymphopenic situation in Vav-CreNfatc1 fl/fl mice. Taken together, hematopoietic phenotype in Il2 −/− mice with an increased NFATc1 activity in HSPCs completely reverses the phenotype in NFATc1-deficient Vav-CreNfatc1 fl/fl mice. Although the hematopoiesis in Il2 −/− mice is also severely dysregulated due to additional factors, it clearly suggests that alteration in NFATc1 activity is certainly detrimental for HSPC maintenance and differentiation of multiple hematopoietic lineages.
Increased HSPC population due to enhanced NFATc1 activity was also observed in Vav-creNfatc1αA fl/fl mice (Figure 6i), in which NFATc1 activity in the hematopoietic cells was enhanced due to the expression of a constitutively active form of NFATc1 in a Cre-dependent manner [18].

Discussion
The roles of NFAT factors in lymphocyte development and function have been studied in detail [12,13,15,[34][35][36]. However, so far, the influence of NFAT proteins in HSC maintenance has not been properly investigated. A previous report suggested the involvement of NFAT in the maintenance of LT-and ST-HSCs [37], and a more recent report suggested suppression of NFAT activity was essential to maintain lymphoid-primed HSCs [38]. However, in both of these studies, NFAT activity was measured as a readout for an increase in intracellular calcium ion. In addition, the model systems used in these studies ruled out the the possibility that the observed effects on HSCs were solely because of altered NFAT activity. Adopting multiple approaches, we showed that NFAT family TFs were expressed in HSPCs, which might play critical roles in their maintenance and in the differentiation of various lineage-positive cells. Our observations show that, among the NFAT family members, NFATc1 activity plays a key role in HSPC maintenance and is indispensable for normal hematopoiesis. NFATc1 activity in maintaining the quiescence of skin stem cells has been reported [39], but its role in HSPC maintenance has not yet been explored in detail. The fact that HSPC phenotype and the hematopoiesis in Vav-CreNfatc1 fl/fl mice were severely affected resulting in a drastic reduction in multiple lineage-positive cells, suggests NFATc1 activity is intimately associated with hematopoiesis, and thereby the survival of an organism. Accordingly, Vav-CreNfatc1 fl/fl mice lacking NFATc1 activity in the hematopoietic system die at a very early age (Figure 2k). This severe phenotype in HSPC maintenance and hematopoiesis in Vav-CreNfatc1 fl/fl mice occurs despite them having intact NFATc2 and NFATc3 activity. Nfatc2 −/− , Nfatc3 −/− , or Nfatc2 −/− Nfatc3 −/− mice did not show the hematopoietic phenotype that we observed in Vav-CreNfatc1 fl/fl mice, suggested that they were unable to substitute for NFATc1 activity in HSPC maintenance and hematopoiesis.
NFATc1 expression in HSCs, and also in all subsequent stages that are derived from them and in the lineage-positive cells, is an interesting observation. NFATc1 activity in various hematopoietic cells have been reported [15,16,[40][41][42]. We showed that NFATc1 was also expressed in the progenitor populations of all hematopoietic lineages (Figure 2m), which attests that, at every stage, NFATc1 plays an important role that guides normal hematopoiesis. The early death of Vav-CreNfatc1 fl/fl mice also shows how critical NFATc1 activity is for the development of hematopoietic cells. Recently, we reported severe defects in T and B cell development in Vav-CreNfatc1 fl/fl mice [14,18,19]. Although these defects were characterized at a later stage during development, they suggested that, most likely, there were additional defects at an earlier stage, which exacerbated the situation at the later stages. Our findings of defective HSPC maintenance in Vav-CreNfatc1 fl/fl mice support this notion. In fact, the low expression of Cd150 and a high expression of Flk2 in Vav-CreNfatc1 fl/fl HSCs (Figure 2h) suggests a paucity of LSKCD150 + Flk2 − LT-HSCs and an increase in the LSKCD150 − Flk2 + ST-HSCs in the absence of NFATc1 activity. This could be a major contributor to the defects at the subsequent stages of hematopoiesis in these mice. It is quite intriguing that, despite stronger expression of many genes that are involved in maintaining HSC quiescence, survival, and proliferation (Figure 2i), there is such a strong impairment in the HSPC numbers in Vav-CreNfatc1 fl/fl mice. HSPCs in the absence of NFATc1 activity were also functionally defective as in adoptive transfer experiments, Vav-CreNfatc1 fl/fl HSPCs failed to efficiently repopulate the hematopoietic system of the recipient mice (Figures 3 and 4h,i).
We have previously shown severe defects in lymphopoiesis and erythropoiesis due to altered NFATc1 activity (14,(18)(19)(20). In addition, lack of or enhanced NFATc1 activity have been shown to be involved in many pathologies associated with the hematopoietic system [43,44]. We show that, as compared with the littermate control mice, Vav-CreNfatc1 fl/fl mice completely lack teeth eruption (Figure 4a). NFATc1 activity in bone formation is well documented as it plays critical roles in osteoblast and osteoclast differentiation [26][27][28][29]. However, in Vav-CreNfatc1 fl/fl mice, the lack of teeth eruption is unlikely to be due to a defect in osteoblast activity, as the bones in these mice are well developed although not normal as compared with the control mice ( Figure 4b). Rather, it could be due to a lack of activity in the case of bone resorbing osteoclast cells, which leads to the failure in teeth eruption. Osteoclasts are myeloid lineage cells that are differentiated from the CD11b + precursor cells [45,46]. Our observation regarding the complete lack of CD11b + cells in Vav-CreNfatc1 fl/fl mice provides evidence that NFATc1 activity is indispensable for their development, and thereby in osteoclast differentiation. Thus, due to lack of osteoclast activity, teeth failed to erupt in the jawbones when NFATc1 activity was absent. In addition to the lack of CD11b + cells, Vav-CreNfatc1 fl/fl mice also had a severe problem in the development of Gr1 + cells, which gave rise to granulocytes such as neutrophils [47,48]. The Gr1 + cell number in the BM of Vav-CreNfatc1 fl/fl mice was very low as compared with the littermate control mice (Figure 4e,f). These developmental defects were due to impaired expression in these mice of several genes related to the myeloid cell development (Figure 4g). NFATc1mediated defects in DC development have been reported previously [17,42,49]. The defects in the development of various myeloid cells that we have shown in Vav-CreNfatc1 fl/fl mice suggest that NFATc1 activity is not only involved in lymphopoiesis or erythropoiesis, but also critically regulates myelopoiesis. Thus, NFATc1 is a key regulator of the differentiation of multiple hematopoietic lineage cells.
The differential expression of two promoters regulating Nfatc1 expression provides further evidence that NFATc1 activity is vital for HSPC maintenance and normal hematopoiesis. The prevalence of P2 promoter-derived Nfatc1b expression in hematopoietic stem and progenitor cells as well as in lineage-positive cells (Figure 2m) suggests that a constitutive NFATc1 activity is required for normal hematopoiesis. The substitution of NFATc1β activity in hematopoiesis by P1 promoter-derived NFATc1α in Vav-CreNfatc1P2 fl/fl mice further underlines the indispensability of NFATc1 activity, irrespective of the isoforms in HSPC maintenance and lineage differentiation ( Figure 5). Additionally, a reverse phenotype to that of Vav-CreNfatc1 fl/fl mice in HSPC maintenance and in the differentiation of various lineage-positive cells, in Il2 −/− mice, reveals the association of NFATc1 activity in hematopoiesis. The increase in HSPC numbers, the accumulation of LT-HSCs, hugely increased numbers of CD11b + and Gr1 + cells, as well as the lack of Ter119 + erythroid cells in Il2 −/− mice ( Figure 6 and [20,30,50]) were completely in contrast to the phenotype we observed in Vav-CreNfatc1 fl/fl mice. In addition, due to enhanced NFATc1 activity in Vav-creNfatc1αA fl/fl mice, the HSPC population was significantly increased (Figure 6i), suggesting an essential role of NFATc1 in HSPC physiology. The contrasting hematopoietic phenotype due to the lack of (Vav-CreNfatc1 fl/fl mice), or increased (Il2 −/− and Vav-creNfatc1αA fl/fl mice), NFATc1 activity in the HSPCs underlines the essentiality of an optimal level of NFATc1 activity in facilitating hematopoiesis, and thereby the survival of an organism. Altogether, our study delineates a novel parameter in terms of NFATc1 activity in HSPC maintenance and in lineage differentiation, manipulation of which could help in multiple clinical conditions of dysregulated hematopoiesis in humans.
Author Contributions: C.B.d.B. maintained mice colonies, isolated cells, and performed experiments; S.K.-H. generated the Nfatc1-eGfp-Bac tg mice; E.S. contributed in organizing the study, data interpretation, and manuscript writing; A.K.P. designed the project, performed experiments, supervised the study, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.