Association and relay neurons that are generated in the dorsal spinal cord play essential roles in transducing somatosensory information. During development, these two major neuronal classes are delineated by the expression of the homeodomain transcription factor Lbx1. Lbx1 is expressed in and required for the correct specification of three early dorsal interneuron populations and late-born neurons that form the substantia gelatinosa. In mice lacking Lbx1, cells types that arise in the ventral alar plate acquire more dorsal identities. This results in the loss of dorsal horn association interneurons, excess production of commissural neurons, and disrupted sensory afferent innervation of the dorsal horn. Lbx1, therefore, plays a critical role in the development of sensory pathways in the spinal cord that relay pain and touch.

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.

The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo-and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.[Key Words: POU; Oct; Sox; osteopontin; preimplantation embryo] Received March 9, 1998; revised version accepted April 14, 1998.The fertilized oocyte undergoes cleavage until a uniform cluster of cells, the morula, is formed. The first apparent differentiation occurs as the inner cell mass (ICM) separates from the trophectoderm during blastocoel formation (Gardner 1983). Trophectoderm refers to the epithelial cell layer that encloses the ICM and blastocoel. Subsequently, cells dissociate from the ICM and cover its blastocoelic surface to form the hypoblast (also called primitive endoderm). These cells do not form a welldefined polarized epithelium, eventually loose cell contacts, and contain an extensive rough endoplasmatic reticulum, which is often swollen with secretory material (Nadijcka and Hillman 1974). The hypoblast differentiates into the parietal and visceral endoderms (Gardner 1983). Parietal endoderm cells form from hypoblast precursors that migrate and adhere to the thin basal lamina on the inner surface of the trophectoderm. In contrast, visceral endoderm cells do not migrate and consists of a columnar epithelial layer surrounding the late ICM or early epiblast (also called primitive endoderm). Relatively little is known about the molecular signals guiding ce...

Interneurons in the ventral spinal cord are essential for coordinated locomotion in vertebrates. During embryogenesis, the V0 and V1 classes of ventral interneurons are defined by expression of the homeodomain transcription factors Evx1/2 and En1, respectively. In this study, we show that Evx1 V0 interneurons are locally projecting intersegmental commissural neurons. In Evx1 mutant embryos, the majority of V0 interneurons fail to extend commissural axons. Instead, they adopt an En1-like ipsilateral axonal projection and ectopically express En1, indicating that V0 interneurons are transfated to a V1 identity. Conversely, misexpression of Evx1 represses En1, suggesting that Evx1 may suppress the V1 interneuron differentiation program. Our findings demonstrate that Evx1 is a postmitotic determinant of V0 interneuron identity and reveal a critical postmitotic phase for neuronal determination in the developing spinal cord.

Pax3 RNA is expressed in neural crest when Schwann cell (SC) precursors migrate to the PNS. Pax3 RNA and SC markers were monitored in sciatic nerves of mice during development and nerve repair. An inverse correlation was observed between expression of Pax3 RNA and myelin basic protein (MBP). Inverse correlation was also observed in SC primary cultures. Treating cultures with forskolin, an adenylate cyclase agonist, repressed Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and elevated MBP. Subsequent microinjection with Pax3 expression vector elevated Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and repressed MBP. Thus, Pax3 is likely involved in the differentiation pathway to myelinating SCs. Pax3 repressed a 1.3 kb MBP promoter fragment in cotransfection assays, suggesting that it represses MBP transcription.

Pitx2 expression is observed during all states of the myogenic progression in embryonic muscle anlagen and persists in adult muscle. Pitx2 mutant mice form all but a few muscle anlagen. Loss or degeneration in muscle anlagen could generally be attributed to the loss of a muscle attachment site induced by some other aspect of the Pitx2 phenotype. Muscles derived from the first branchial arch were absent, whereas muscles derived from the second branchial arch were merely distorted in Pitx2 mutants at midgestation. Pitx2 was expressed well before, and was required for, initiation of the myogenic progression in the first, but not second, branchial arch mesoderm. Pitx2 was also required for expression of premyoblast specification markers Tbx1, Tcf21, and Msc in the first, but not second, branchial arch. First, but not second, arch mesoderm of Pitx2 mutants failed to enlarge after embryonic day 9.5, well before the onset of the myogenic progression. Thus, Pitx2 contributes to specification of first, but not second, arch mesoderm. The jaw of Pitx2 mutants was vestigial by midgestation, but significant size reductions were observed as early as embryonic day 10.5. The diminutive first branchial arch of mutants could not be explained by loss of mesoderm alone, suggesting that Pitx2 contributes to the earliest specification of jaw itself.homeobox gene ͉ muscle development

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-celltargeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4 1 CD8 1 CD3 lo double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4 1 CD8 À and CD4 À CD8 1 single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineageassociated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.Key words: Mouse . T-cell differentiation . Transcription factor Supporting Information available online Introduction T-cell differentiation is a complex and dynamic process that leads to the production of functionally distinct populations within the thymus -gd and ab T-cell subsets, the latter of which include helper CD4 1 T cells, cytotoxic CD8 1 T cells, Treg cells, and NKT cells. Hematopoietic progenitor cells enter the thymus as CD4 À CD8 À double-negative (DN) cells and proceed through successive steps of maturation. DN thymocytes are further divided into at least four developmental stages based on the differential expression of CD44 and CD25: CD44 1 CD25 À (DN1), CD44 1 CD25 1 (DN2), CD44 À CD25 1 (DN3), and CD44 À CD25 À Ã Joint senior authors; additional correspondence, Dr. Mark Leid Eur. J. Immunol. 2010. 40: 2143-2154 DOI 10.1002 HIGHLIGHTS 2143Frontline (DN4). gd T cells differentiate from DN3 thymocytes, following rearrangement of the b, g, and d TCR chains. ab T cells develop from DN4 thymocytes that further differentiate into CD4 1 CD8 1 double-positive (DP) CD3 lo abTCR lo thymocytes. Positive selection events between the TCR expressed by DP cells and MHC molecules expressed by thymic stromal cells lead to the appearance of mature CD4 1 and CD8 1 single-positive (SP) CD3 hi /TCR hi thymocytes, and NKT cells, all presumably resulting from large-scale changes in gene expression programs.Transcription factors essential for the ab T-cell developmental programs have been identified [1][2][3]. In particular, Zbtb7b (also known as ThPok) is required for CD4 1 T-cell differentiation [4,5]. Zbtb7b is not expressed in DP thymocytes, but is activated downstream of TCR signaling by TOX [...

Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60؇C. The enzyme was highly tolerant to denaturing agents, proteases, and repeated freezing and thawing. The molecular weight of the produced levan depended on temperature, salinity, and sucrose concentration. The enzyme had levan-degrading activity and did not accept raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20 amino acids. By comparison of all levansucrases which have been sequenced to date, the enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product levan prompted a new assessment of the enzyme's role in pathogenesis. Depending on formation conditions, levan solutions exclude other polymer solutions. This behavior supports the presumption that the levansucrase is important in the early phase of infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.