The molecular mechanisms that LT-HSCs use to migrate from the FL to the FBM are not well known, even when these cells follow an established pattern of migration during development 
. The work presented here shows that we can isolate and determine the expression of eight migration-related genes from a single fetal LT-HSC by multiplex single cell RT-qPCR, accurately quantifying the single cell gene expression distribution within desired populations. We also begin to elucidate a pattern of adhesion molecules that might mediate homing of FL LT-HSC to the nascent BM vasculature as well as coordinate the migration of LT-HSC to the endosteal niche within the developing bone.
Miyamoto showed that single LSK (Lineage−/low
) cells had a promiscuous expression of multilineage-affiliated genes 
, indicating that cells within a population with similar cell surface phenotypes might have divergent genetic expression patterns leading to different cell fates. We examined LT-HSC (LSK CD150+
) from different anatomical locations and stages of development to quantitatively determine if a similar divergent gene expression pattern could be observed in migration-related genes. Our results demonstrate that single cells within the better-defined and more immature LT-HSC population still show a variable gene expression pattern. Nonetheless, we cannot rule out that the differences observed in our results might reflect expression differences among discrete sub-types within the LT-HSC population that inherently express these genes at different levels and not to transcriptional variability among phenotypically identical cells. Determining the single cell expression pattern of multiple housekeeping genes (for example Actb
, and Hprt
), believed to have more stable and uniform expression irrespective of the cells studied, could assess the latter possibility.
Studies on bone development have shown that successful migration of FL hematopoiesis to the FBM is dependent on normal embryonic bone formation 
and endochondral ossification 
. Initially, the primary skeleton is entirely cartilaginous 
, and the deposition of cartilage is orchestrated by the sequential maturation of chondrocytes (reviewed in 
). As chondrocytes mature, they transition from a prehypertrophic state to a hypertropic state, secreting extracellular matrix that later becomes mineralized. The death of hypertrophic chondrocytes coincides with the initiation of the ossification process, which includes the vascularization of the bone, and the invasion of hematopoietic progenitors 
Our results support the idea that invasion of the fetal bone marrow by LT-HSC and other hematopoietic progenitors is a multi-step process involving chemotactic signals and multiple adhesion molecules 
(). Bone vascular endothelial cells constitutively express E-selectin, P-selectin, VCAM-1 and ICAM-1 in the absence of inflammation, making the BM endothelial microenvironment unique in its ability to recruit LT-HSC expressing PSGL-1, VLA-4 (α4
) and LFA-1 (α4
. Indeed, our data shows that VLA-4 ( and ) and PSGL-1 () are expressed in LT-HSC and that their expression does not vary significantly from anatomical location or developmental time.
Model for the migration of LT-HSCs from the vascular niche to the endosteal niche.
Circulating LT-HSCs engage E- and P-selectins 
on the BM vascular endothelium through PSGL-1, tethering the LT-HSC to the vascular endothelium and allowing them to roll along the vessel wall. This initial contact facilitates CXCR4-SDF-1α engagement on the extravascular space. Signals from CXCR4 are required for the migration of LT-HSC into the fetal and adult BM 
. Our data shows that FL17.5 LT-HSCs uniformly express Cxcr4
( and ), allowing them to respond to SDF-1α gradients. CXCR4 signals increase the affinity of the integrins LFA-1 and VLA-4 for their ligands (ICAM-1 and VCAM-1, respectively) 
. Engagement of VCAM-1 by VLA-4 stabilizes the interaction of the vascular endothelium and the LT-HSC, causing the latter to stop rolling 
. Together, CXCR4 and integrin signaling induces polarization and diapedesis of LT-HSC across the BM vascular endothelium, a process mediated by LFA-1 
. Our RT-qPCR data show that all LT-HSC tested, regardless of their tissue of origin or developmental time, express comparable levels of Lfa1
(). Surface LFA-1 expression was corroborated by flow cytometric analysis on all the populations tested. However, a lower percentage of FBM17.5 LT-HSCs were LFA-1+
(). Overall, our data suggests that PSGL-1, VLA-4, and LFA-1 are expressed at a constant level in all LT-HSC, independent of developmental time or anatomical location, and that these molecules might allow the LT-HSC to migrate into hematopoietic organs during development via vascular endothelial cell recognition.
After diapedesis, fetal LT-HSCs encounter the immature marrow microenvironment, where they interact with the basal lamina composed of extracellular matrix (ECM) proteins collagen type I, laminin and fibronectin 
. They also interact via VLA-4 with CXCL12+
-reticular (CAR) cells surrounding the vessel 
, or to vascular endothelial niche cells via VE-cadherin 
. Furthermore, LT-HSCs encounter both free Ca+2 
and SDF-1α gradients 
that will direct them to the osteoblastic niche.
Our results show that most LT-HSC express levels of Cxcr4
near the median of the FL14.5 sample. The large distribution and down-regulation of Cxcr4
at this time reflects the fact that many LT-HSC are able to enter and leave the FL during this period of development. LT-HSCs with expression near the median might be retained in the FL since hematopoiesis is occurring in this organ at this developmental time. In contrast, Cxcr4
expression in the FL17.5 shows a tight distribution near the median. At this time, the liver is transitioning from a hematopoietic to a hepatic organ 
and many LT-HSCs are entering the circulation 
. The tight distribution of Cxcr4
at this time suggests that emigration of LT-HSC from the FL17.5 is mediated by a process other than down-regulation of CXCR4, such as decreased production of SDF-1α by FL stromal cells or proteolytic degradation of either SDF-1α or CXCR4 during the FL transition from a hematopoietic to a hepatic organ. In the FBM17.5, the distribution of Cxcr4
expression resembles that of FL14.5 with a lower mean. SDF-1α plays a critical role in the attraction and retention of HSC in the BM during development 
. CASR signaling increases the sensitivity of CXCR4 to SDF-1α in the presence of free Ca+2 
, which results from the ossification of the bone at 17.5 dpc. In this way, the lower expression of Cxcr4
that may have impaired the LT-HSC's ability to colonize the FBM17.5 environment is mitigated by increased receptor sensitivity. Cxcr4
is upregulated in the adult BM LT-HSC where it is expressed in a tight distribution around the medium, resembling that of the LT-HSCs in the FL17.5. In the case of the adult BM, the presence of high concentrations of SDF-1α helps retain the HSCs in the bone, stabilizing hematopoiesis. Outliers in the adult BM Cxcr4
expression might represent cells that have down-regulated CXCR4 in order to leave the BM to populate other niches ().
displays high expression variability but similar median values in all the fetal tissues tested by single cell multiplex RT-qPCR, with its expression being more uniform in adult BM LT-HSC. This finding was confirmed by flow cytometry. Our data supports and complements studies where FL Thy-1low
cells, a population that includes LT-HSC and more mature progenitors, show decreased VE-cadherin expression by 16.5 dpc, and absence in adult BM cells 
. We show that VE-cadherin expression is maintained in both FL and FBM samples at day 17.5 of gestation in Lineage−/low
, a more defined LT-HSC population. We propose that homotypic VE-cadherin interactions might play an important role in engaging LT-HSCs with both FL and FBM vascular endothelial niche cells during development. As the bone marrow develops into adulthood, the expression of Vecad
diminishes (). This loss of VE-cadherin expression might reflect the emergence of endosteal and reticular niches as the bones are completely formed.
The role played by N-cadherin in LT-HSC interaction with its BM niches is controversial. It was previously described that N-cadherin does not play a role in adult LT-HSC adhesion to or maintenance by osteoblasts 
. Recent transplantation studies have shown that loss of N-cadherin suppresses LT-HSC engraftment 
. However, these studies did not evaluate the role of N-cadherin during fetal LT-HSC development, migration or maintenance. Our results showed that single FBM17.5 LT-HSCs express high Ncad
levels. This suggests that N-cadherin might be required for the initial adhesion or maintenance of LT-HSCs in the FBM, an activity that is diminished when the adult bone is completely formed. Interestingly, CAR cells express low levels of N-cadherin 
, possibly facilitating their interaction with N-cadherin+
FBM LT-HSC. Moreover, studies in fetal bone development show that N-cadherin is required for proper bone formation 
, and that early osteoblast development from mesenchymal stem cells can be induced through N-cadherin 
. This raises the interesting possibility that N-cadherin expressing LT-HSC can positively influence the ontogeny of bone marrow osteoblasts during fetal development, in a manner analogous to fetal thymocytes being required for the development of the thymic medullary stroma 
Our data suggest that initial migration and colonization of the nascent bone marrow requires the coordinated expression of the chemokine receptor CXCR4 and adhesion molecules (). Bone marrow vascular endothelial cells express VCAM-1, E-Selectin, P-Selectin and ICAM-1 constitutively. The PSGL-1/Selectin interaction slows LT-HSC and facilitates rolling on the vascular sinus. Stable LT-HSC adhesion to endothelial cells occurs via VLA-4 binding VCAM-1, which also facilitates CXCR4/SDF-1α engagement and initiates endothelial transmigration. Once inside the BM, LT-HSCs interact with SDF-1α-producing reticular cells or with vascular endothelial cells. They are also exposed to free Ca2+
and SDF-1α, which are significantly less abundant in the developing BM that in the adult BM 
. Expression of CASR, the receptor for free Ca2+
, is increased and its signals render CXCR4 significantly more sensitive to SDF-1α 
. Together, Ca+2
and SDF-1α gradients guide the LT-HSC to the endosteal niche, where they can interact with SNO (Spindle-shaped N-cadherin+
osteoblastic) cells via N-cadherin. This migration might occur via the VLA-5 interaction with fibronectin in the extracellular matrix or by reticular cells directing the LT-HSC within the BM. As the BM develops and nears adulthood, Casr
is downregulated, allowing maintenance of hematopoiesis since SDF-1α and free Ca2+
are abundant and BM hematopoiesis has been set into motion.
We have characterized the fetal single cell expression pattern of molecules important in migration of adult LT-HSC, and showed how this pattern fluctuates according to their developmental stage and anatomical location. Recently, embryonic stem cells have been differentiated into hematopoietic progenitors (ES-HP), which could be used in regenerative medicine of blood disorders 
. However, the therapeutic capacity of ES-HP may be limited by their ability to home and engraft to the BM. We consider that the expression pattern of migration related genes shown in this study could be useful in predicting the migration potential of hematopoietic progenitors. The present study enhances our understanding of stem cell fate decisions during migration and has potential clinical relevance that could be applied to hematopoietic stem cell or ES-HP transplantation.