Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.

The mammalian heart is formed from distinct sets of first (FHF) and second (SHF) heart field progenitors. Although multipotent progenitors have been previously shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. Herein, we have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome wide profiling of coding and non-coding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue. These results represent a novel approach combining tissue-engineering with stem cell biology for the generation of functional ventricular tissue.The mammalian heart is composed of a diversified set of muscle and non-muscle cells that arise from multipotent progenitors in the FHF and SHF (1,2). Defining the precise progenitor identity and the pathways that lead to ventricular myogenesis is critical for understanding cardiogenesis, with major implications for regenerative cardiovascular medicine.Accordingly, we generated a transgenic mouse with the red florescent protein dsRed under the control of an Isl1-dependent SHF specific enhancer of the transcription factor Mef2c (3,4). We then bred this mouse line with the previously described transgenic mouse line in which eGFP is under the control of the cardiac specific Nkx2.5 enhancer (5,6). Fluorescence microscopy of double transgenic embryos on embryonic day (ED) 9.5 revealed that the entire primitive heart tube was eGFP+, but only the right ventricle (RV) and the outflow tract (OFT) were also dsRed+. Further, the pharyngeal mesoderm (PM) which contributes to the RV and OFT (7,8) was dsRed+ but eGFP-( Figure 1A-1C). To delineate the local expression of eGFP and dsRed in the developing heart, we performed immunohistochemistry on ED9.5 embryos and found that eGFP+/dsRed+ cells (R+G+) were restricted to the RV and OFT, dsRed-/eGFP+ cells (R-

We recently observed that specific inhibitors of postproline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26؊ Jurkat T cells. The protein was identified by labeling with [ 3 H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.

A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell proline dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N-glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.

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