Cell-cell interactions are known to regulate transcriptional machinery for proliferation and development in multiple systems 
. However, the contributions of cell surface mediated protein signaling in ESC fate decisions are incompletely understood. Understanding the molecular signaling that influences commitment decisions is important in developing strategies to guide ESC commitment for regenerative therapies. Since AM are known to have major roles in cell differentiation during embryonic development 
, we explored the contributions of AM in ESC commitment decisions to hematopoietic/endothelial lineages. We describe expression of 21 genes encoding extracellular matrix proteins, cell adhesion molecules (CAM), hematopoietic lineage markers, junction molecules, receptors and other membrane associated proteins in ESC proliferation/differentiation and the functional consequences of manipulating AM expression levels on hematopoietic and endothelial commitment. Previous studies of expression profiling, using microarray and proteomic analysis, of murine ESC identified expressed AM 
in ESC. While these data demonstrate the AM are expressed during ESC culture, the functional role of individual AM in influencing ESC commitment decisions was not investigated. The ability to effectively direct differentiation of ESC for therapy will require understanding the complex cell-environment signaling pathways that influence commitment decisions. ESC differentiation towards the hematopoietic lineage is challenging, typically generating only low numbers of hematopoietic cells 
. While hematopoietic commitment can be increased by addition of cytokines 
, hematopoietic differentiation is still low and, addition of cytokines often drives differentiation beyond stem/progenitor stages, thus reducing usefulness for sustained therapy. Since AM are differentially expressed after LIF removal, we explored whether manipulation of AM expression influenced ESC commitment to hematopoietic and endothelial lineages.
EB are comprised of multiple transitional stages and lineages 
, thus reduction of heterogeneity by subpopulation isolation facilitates exploration of AM contributions to early hematopoietic/endothelial lineage commitment. Early hematopoietic/endothelial differentiation proceeds through defined stages of commitment. ESC undergoing commitment to the mesoderm lineage express the TF Bry 
. A VEGF receptor, Flk-1, is expressed in a subpopulation of Bry+ cells which are progenitors to early endothelial and hematopoietic cells 
and Flk-1+ cells that express Scl are committed to hematopoiesis 
. We used Bry, Flk-1 and Scl expression to discriminate subpopulations in developing EB. We show that temporal oscillations in Bry, Flk-1 and Scl expression during EB development are consistent with those reported previously 
Junction molecules, including E-cad, Cx43, ZO-1 and ZO-2, were among the 21 AM whose levels changed preferentially in EB subpopulations expressing Bry, Flk-1 or Scl. E-cad is present in adherens junctions, which indirectly link the cytoskeletons of adjacent cells 
; ZO-1 and ZO-2 are associated with tight junction complexes, which generate a permeability barrier and activate intracellular signaling responses that suppress cellular proliferation 
; Cx43 forms gap junctions, which allow second messengers to pass between cells through the cytosol 
. The temporal and quantitative variation of E-cad, Cx43, ZO-1 and ZO-2 expression during EB formation in culture conditions that support hematopoietic and endothelial commitment suggest a role for these proteins in ESC differentiation.
The effects of modulating E-cad, Cx43, ZO-1 and ZO-2 expression on hematopoietic and endothelial commitment potential of manipulated ESC were investigated to establish genotype-phenotype relationships of these AM. Knockdown ESC lines were engineered using RNAi stably introduced into the ESC genome using lentiviral transduction. Although clonal analysis is preferable in determining the role of AM in hematopoietic/endothelial commitment, culture of ESC at low density often results in loss of pluripotency. To reduce the heterogeneity in lentiviral transduction efficiency, transduced ESC were sorted based on GFP expression to generate ESC lines with greater than 95% of cells containing incorporated shRNA sequences. Using engineered ESC lines, we demonstrated that reduced levels of E-cad, Cx43, ZO-1 and ZO-2 had minimal influence on the frequency of SSEA-1 expressing cells or level of Oct-4 expression, markers associated with ESC pluripotency. These data suggest that pluripotency (as assessed by SSEA-1 and Oct-4 expression) is unaffected by manipulation of these AM.
If AM modulations affect the decision of ESC to differentiate into a Flk-1+ progenitor, then the number and/or timing of Flk-1 expressing engineered ESC during EB development would be altered. A reduction in E-cad, Cx43 and ZO-1 expression did not appear to affect the frequency of Flk-1+ cells. However, these same AM appeared to have a more prominent role in influencing intermediate and later stages of hematopoietic commitment because Kd-E-cad, Kd-Cx43 and Kd-ZO1 ESC showed reduced expression of hematopoietic TF, as well as a lower frequency of cells expressing CD45, present on cells committed to hematopoiesis 
(). These data suggest that Gata1, Runx1 and Scl expression, associated with intermediate stages of hematopoietic commitment are modulated by E-cad, Cx43 and ZO-1 expression. The reduction in TF expression in Kd-E-cad, Kd-Cx43 and Kd-ZO1 was paralleled with a decrease in the number of CFU, particularly CFU-M. Hematopoietic commitment decreased in engineered Kd-Cx43 ESC by approximately 2-fold, as evidenced by frequency of CD45+ cells and CFU. The reduction in hematopoietic commitment in Kd-Cx43 is consistent with data reported by Cancelas, et al. 
who observed a reduction in hematopoiesis in fetal liver of Cx43−/−
embryos particularly CFU-GM and BFU-e (40–50% decrease). Hematopoiesis is not completely inhibited in Cx43−/−
embryos, suggesting interactions between multiple proteins and/or pathways in hematopoietic commitment. Since the knockdown of AM with shRNA sequences does not completely abrogate gene expression in ESC, a more extensive knockdown AM in ESC would be predicted to have a larger impact on hematopoietic/endothelial commitment. Although our knockdown was not complete (33% of control expression), we observed decreased frequency of CD45+ cells (48% of control), CFU-GM (48% of control) and BFU-e (68% of control), which is comparable to data reported by Cancelas et al. 
Fok, et al reported that levels of E-cadherin, in heterozygous and homozygous knockout ESC, correlated with size of EB 
. Similarly, ZO-1 knockdown in mouse morulas reduced blastocyst formation in a concentration dependent manner 
. Overall, our ESC data suggest that E-cad, Cx43, and ZO-1 do not play substantial roles in hematopoietic commitment prior to the Flk-1+ progenitor stage (), but may be involved in differentiation decisions at or after the Flk-1+ expression stage.
Adhesion molecules expression and influence in hematopoietic and endothelial differentiation.
Flk-1+ cells are also capable of differentiating to endothelial cells 
. ESC propagated in a collagen matrix containing endothelial-specific cytokines differentiate along the endothelial lineage, forming EB with endothelial sprouts that invade the matrix and express the endothelial marker CD31 (PECAM) 
. Invasion is used as a surrogate endpoint for angiogenesis 
. Activators of angiogenesis, VEGF and FGF-2, increase the number of EB with endothelial sprouts, while inhibitors of angiogenesis decrease the length of endothelial sprouts on EB 
. We show that reduced E-cad, Cx43 and ZO-1 expression increases the frequency of EB with endothelial sprouting and number of sprouts on each EB. These data suggest that E-cad, Cx43 and ZO-1 expression may act in a negative regulatory manner to inhibit both endothelial differentiation (more EB with sprouts) and proliferation (more sprouts per EB). Interestingly, disruption of ZO-1 patterning in human ESC has been reported to increase vasculogenesis in EB 
. Expression of endothelial genes, Tie1 and Tie2, was not consistently altered in engineered ESC (data not shown). Tie1 and Tie2 levels in Kd-E-cad, Kd-Cx43 and Kd-ZO1 were similar to controls. Tie1 expression inhibits and Tie2 expression promotes angiogenesis 
. The Tie1 results, which were not consistent with inhibition of angiogenesis, may be complicated by expression in lineages other than endothelial cells present in the EB 
. Overall, however, the hematopoietic and endothelial data suggest that E-cad, Cx43 and ZO-1 expression influence lineage commitment at the point of endothelial and hematopoietic divergence ().
We demonstrate that ZO-2 levels are important for maintenance of early, intermediate and late stages of hematopoietic/endothelial differentiation in ESC. Kd-ZO2 ESC generated fewer Flk-1+ progenitors and hematopoietic cells than D3-ESC. Although endothelial sprouting in Kd-ZO2 ESC was similar to control ESC, there was an increase in number of sprouts on EB, suggesting increased proliferation of endothelial cells. These data suggest that low ZO-2 levels induce a block in ESC differentiation to Flk-1+ cells, but do not perturb endothelial commitment (). Although Flk-1 expression is necessary for hematopoietic and endothelial differentiation, Flk-1 expression has been observed in smooth muscle cells 
, cardiomyocytes 
, retinal progenitors 
and other neural lineages 
. Thus, a reduction in the frequency of Flk-1+ cells without alterations in endothelial commitment may suggest that endothelial cells can be generated through alternative non-Flk-1+ populations.
Overall, our data suggest that expression of junction molecules and their downstream components is associated with ESC commitment decisions between hematopoietic and endothelial lineages. Reduced expression of transmembrane junction molecules, E-cad and Cx43, increased endothelial commitment and decreased hematopoietic development, suggesting that these AM influence a common progenitor and activate divergent pathways towards lineage commitment (). Intracellular components, ZO-1 and ZO-2 possibly regulate differentiation decisions through additional interactions (Figure S4
). For example, ZO-1 associates with components of adherens, gap and tight junction pathways, including direct interactions with α-catenin and connexins 
as well as claudins, ZO-2, JAM1, ZONAB, occludens and actin 
. Expression of ZO-2 may be necessary for commitment to Flk-1+ progenitors; whereas, ZO-1 expression regulates later development decisions through ZONAB and α-catenin interactions. Modulation of cell fate commitment decisions between hematopoietic and endothelial lineages may occur through activation of intracellular signaling pathways, proliferation of endothelial progenitors or apoptosis of hematopoietic progenitors during EB development. Future analysis will require identification of downstream targets of these AM pathways in regulating hematopoietic/endothelial cell fate decisions and proliferation/apoptosis of hematopoietic/endothelial cells during EB development.
Finally, our data also suggest the AM may be useful as discriminants of transitional subpopulations within the Bry, Flk-1 and Scl expressing () populations. Undifferentiated ESC express E-cad, Cx43, ZO-1 and ZO-2. As Bry transcription was up-regulated in mesoderm cells, E-cad and ZO-2 expression was down-regulated in the Bry+ cells, while Cx43 and ZO-1 levels were constant with the Bry- population of cells. Flk-1+ hematopoietic and endothelial progenitors had higher levels of ZO-1 and ZO-2, retained similar levels of Cx43 and reduced levels of E-cad than Flk-1- subpopulation. Scl+ cells, which have committed to the hematopoietic lineage, expressed ZO-1 in addition to the up-regulation of E-cad, Cx43 and ZO-2. These data not only suggest that E-cad, Cx43, ZO-1 and ZO-2 expression is associated with commitment decisions, but also that these markers identify subpopulations within the Bry+, Flk-1+ and Scl+ populations.