This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

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Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed ␣, ␤, and ␥. The putative binding site for integrins has been mapped to the G domain of the ␣ chain, although trimerization with ␤ and ␥ chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of ␤ and ␥ chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the ␥ chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the ␥1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the ␥2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either ␥1 or ␥2 chains. However, the peptide segment modeled after the C-terminal region of ␥1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.Laminins are a family of glycoproteins present in the basement membrane (1-3). All laminins are large heterotrimeric glycoproteins composed of ␣, ␤, and ␥ chains that assemble into a cross-shaped structure. To date, five ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) have been identified, combinations of which yield at least 15 isoforms with distinct subunit compositions (4). Laminins contribute to basement membrane architecture and influence cell adhesion, spreading, and migration through binding to their cell surface receptors, particularly the integrin family of cell adhesion receptors (5-9).Integrins play important roles in cell-matrix adhesion and signaling events regulating proliferation and differentiation of cells. Among the various integrin family members, ␣6␤1, ␣6␤4, ␣3␤1, and ␣7␤1 have been shown to be the major laminin receptors expressed in many cell types (10). Binding sites for these integrins have been mapped to the C-terminal globular (G) 3 domain of the laminin ␣ chains (6, 11-15). The G domain consists of five tandemly repeated LG modules of ϳ200 amino acid residues, designated LG1 through LG5. By analogy with the identification of the Arg-Gly-Asp (RGD) cell-adhesive motif in fibronectin, many attempts have been made to identify specific sequences mimicking the integrin binding activity of laminins. However, neither recombinant fragments of the G domain nor synthetic peptides modeled after the sequences in the G domain ...

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as ␣-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their ␣ chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (␣5␤1␥1) for ␣ 3 ␤ 1 and ␣ 6 ␤ 1 integrins and ␣-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4 -5 modules did not compromise the binding of laminin-10 to ␣ 3 ␤ 1 and ␣ 6 ␤ 1 integrins but completely abrogated its binding to ␣-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to ␣-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the ␣-dystroglycan binding by the LG4 module.

Polydom affects remodeling of lymphatic vessels in both mouse and zebrafish. Polydom deposited around lymphatic vessels seems to ensure Foxc2 upregulation in lymphatic endothelial cells, possibly via the Ang-2 and Tie1/Tie2 receptor system.

Background: Polydom/SVEP1 is a putative extracellular matrix protein of unknown function. Results: Polydom/SVEP1 is a potent ligand for integrin ␣9␤1 and colocalizes with the integrin in vivo. Conclusion: Polydom/SVEP1 is a hitherto unknown high affinity ligand for integrin ␣9␤1. Significance: The identification of this high affinity ligand offers important clues toward better understanding of the consequences of integrin ␣9␤1-mediated cell-extracellular matrix interactions.

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.