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Skeletal muscle is generated by the successive incorporation of primary (embryonic), secondary (fetal), and tertiary (adult) fibers into muscle. Conditional excision of Pitx2 function by an MCK(Cre) driver resulted in animals with histological and ultrastructural defects in P30 muscles and fibers, respectively. Mutant muscle showed severe reduction in mitochondria and FoxO3-mediated mitophagy. Both oxidative and glycolytic energy metabolism were reduced. Conditional excision was limited to fetal muscle fibers after the G1–G0 transition and resulted in altered MHC, Rac1, MEF2a, and alpha-tubulin expression within these fibers. The onset of excision, monitored by a nuclear reporter gene, was observed as early as E16. Muscle at this stage was already severely malformed, but appeared to recover by P30 by the expansion of adjoining larger fibers. Our studies demonstrate that the homeodomain transcription factor Pitx2 has a postmitotic role in maintaining skeletal muscle integrity and energy homeostasis in fetal muscle fibers.

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-cell-targeted 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(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) 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 lineage-associated 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.

Late-stage Pitx2(+/LacZ) mouse embryos stained with x-gal appeared to have blue muscles, suggesting that Pitx2 expression specifically marks some phase of the myogenic progression or muscle anlagen formation. Detailed temporal and spatial analyses were undertaken to determine the extent and onset of Pitx2 expression in muscle. Pitx2 was specifically expressed in the vast majority of muscles of the head and trunk in late embryos and adults. Early Pitx2 expression in the cephalic mesoderm, first branchial arch and somatopleure preceded specification of head muscle. In contrast, Pitx2 expression appeared to follow muscle specification events in the trunk. However, Pitx2 expression was rapidly upregulated in these myogenic structures by E10.5. Upregulation correlated tightly with the apposition of a non-myogenic, Pitx2-expressing, cell cluster lateral to the dermomyotome. This cluster first appeared at the forelimb level at E10.25, gradually elongated in the posterior direction, appeared to aggregate from delaminated cells emanating from the ventrally located somatopleure, and was named the dorsal somatopleure. Immunohistochemistry on appendicular sections after E10.5 demonstrated that Pitx2 neatly marked the areas of muscle anlagen, that Pax3, Lbx1, and the muscle regulatory factors (MRFs) stained only subsets of Pitx2(+) cells within these areas, and that virtually all Pitx2(+) cells in these areas express at least one of these known myogenic markers. Taken together, the results demonstrate that, within muscle anlagen, Pitx2 marks the muscle lineage more completely that any of the known markers, and are consistent with a role for Pitx2 in muscle anlagen formation or maintenance.

The mouse homeodomain protein Hoxa-7, expressed under the control of an inducible promoter, was able to inducibly activate reporter genes containing multimerized Hoxa-7 binding sites in Saccharomyces cerevisiae . This tight regulation was exploited in an attempt to screen for HOXa-7 responsive elements. A reporter library consisting of a randomised 10bp element inserted into the minimal ga1 promoter was constructed. In a surprisingly small screen, 24 reporters were isolated which had all of the transactivation characteristics expected for a Hoxa-7 binding site insertion. However, further characterisation revealed that the selected elements lacked homeo-domain (HD) binding core motifs and were not bound by a purified Hoxa-7/β-galactosidase fusion protein capable of binding known sites. The minimal promoter context contains 16 HD core motifs in 410 bp. Careful re-examination of basal levels revealed a low residual response of the gal1 minimal promoter to Hoxa-7. The 11 characterised 10 bp inserts amplified Hoxa-7 responsiveness in a manner correlated to increases in basal reporter activity. Thus, a quantitative range of Hox-responsiveness was produced by slight sequence alterations that did not change HD binding sites of their relative spacing in the promoter. These data suggest how, without altering resident HD base contact zones, mouse promoters could be optimised by natural selection to give appropriate quantitative outputs in each anatomical region defined by an assortment of Hox proteins. The selected elements were pyrimidine rich on the sense strand, containing (T) n C motifs, strikingly similar to sequences which enhance Hoxa-7 binding activation from outside the HD contact zone. A search of defined sequence databases demonstrated that these elements were over-represented in promoters. Two elements altered the mobility shift patterns produced by cell extracts on minimal promoter fragments.

The murine developmental control gene product, Hoxa-7, was shown to function as a DNA-binding transactivator in Saccharomyces cerevisiae. The importance of the ATTA core, the preference for antp class flanking nucleotides, the importance of Asn-51 of the homeodomain (HD), and the synergism of multiple binding sites all reflect properties that have previously been described for HOM or Hox proteins in tissue culture systems. A comparison of contact positions among genes of paralog groups and classes of mammalian HDs points to a lack of diversity in positions that make base contact, suggesting that besides the combination of HD amino acid-base pair contacts, another means of recognizing differences between targets must exist if Hox genes select different targets. The HD of antennapedia is identical to the Hoxa-7 HD. The interaction of Hoxa-7 with the exact sequence used in the nuclear magnetic resonance three-dimensional structural analysis on the antennapedia HD was studied. Hoxa-7 binding and transactivation was influenced by sequences outside of the known base contact zone of this site. We conclude that Hoxa-7 protein has a second means to interact with DNA or/and that the sequences flanking the base contact zone influence HD interactions by distorting DNA within the contact zone (base or backbone). This result is discussed in terms of DNA flexure and two modes of transcription used in S. cerevisiae.

Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Deltatrx1 Deltatrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Deltatrx1 Deltatrx2 yeast. In contrast to wild-type cells, Deltatrx1 Deltatrx2 cells were unable to accumulate or maintain high levels of dNTPs when alpha-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Deltatrx1 Deltatrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.

The neural crest is a migratory cell population that gives rise to multiple cell types in the vertebrate embryo. The intrinsic determinants that segregate neural crest cells from multipotential dorsal progenitors within the neural tube are poorly defined. In this study, we show that the winged helix transcription factor Foxd3 is expressed in both premigratory and migratory neural crest cells. Foxd3 is genetically downstream of Pax3 and is not expressed in regions of Pax3 mutant mice that lack neural crest, implying that Foxd3 may regulate aspects of the neural crest differentiation program. We show that misexpression of Foxd3 in the chick neural tube promotes a neural crest-like phenotype and suppresses interneuron differentiation. Cells that ectopically express Foxd3 upregulate HNK1 and Cad7, delaminate and emigrate from the neural tube at multiple dorsoventral levels. Foxd3 does not induce Slug and RhoB, nor is its ability to promote a neural crest-like phenotype enhanced by co-expression of Slug. Together these results suggest Foxd3 can function independently of Slug and RhoB to promote the development of neural crest cells from neural tube progenitors.

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 thatOct-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.

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.

Thymidine kinase (TK) is representative of a class of enzymes involved in DNA precursor biosynthesis that declines as cells withdraw from the cell cycle. If TK activity is regulated exclusively by the availability of messenger RNA, changes in enzyme activity levels should not precede or excede changes in TK mRNA levels. This prediction was tested in several tissues during chicken embryogenesis and in differentiating muscle cells in culture. A sensitive method of determining absolute TK mRNA levels was developed. A synthetic complimentary RNA probe spanning an intron acceptor site in the chicken TK gene was hybridized with cellular RNA or synthetic colinear TK RNA of known concentration. After RNase digestion and gel electrophoresis, the intensity of the protected fragment was used to calculate absolute TK mRNA levels. As few as 0.02 molecules of TK mRNA per cell could be measured accurately. Depending on the tissue type, 8-day embryos contained between 3 and 12 TK mRNAs per cell. Proliferating mouse muscle cells transformed with the chicken TK gene contained between 30 and 150 TK mRNAs per cell. Both in vivo and in vitro , TK mRNA levels declined as cells withdrew from the cell cycle during differentiation. In vivo , the decline in TK activity never preceded or exceded observed changes in TK mRNA. However, in the cell culture system, TK activity consistently declined to a greater extent than TK mRNA. Thus, a translational or a post-translational mechanism must also be operative in controlling TK activity levels. Estimation of transcription rates in nuclei isolated from proliferating and differentiated muscle cell transformants indicated that the TK gene was transcriptionally repressed in postreplicative cells.

The molecular basis for replication-dependent expression of thymidine kinase (TK) activity (EC 2.7.1.21) was investigated in mouse skeletal muscle cells transformed with multiple copies of the chicken TK gene. When shifted to mitogen-depleted medium, proliferating myoblasts irreversibly withdraw from the cell cycle and commit to terminal differentiation. Early after commitment, postreplicative myocytes maintain nearly proliferative levels of TK mRNA but have greatly reduced levels of TK activity. Metabolic labeling studies with [35S]methionine indicated that the decrease in TK activity was associated with a 10-fold reduction in the rate of TK protein synthesis. Commitment had little effect on the stability or catalytic efficiency of TK protein. The decrease in TK synthetic rate in the continued presence of TK mRNA indicated that translation of TK mRNA was repressed in committed cells. The distribution of TK mRNA between ribonucleoprotein particles and polysomes was determined. In both proliferative cells and committed cells, TK mRNA levels were maximal in polysomes containing five to seven ribosomes. Thus, the synthesis of TK protein in nonreplicating muscle cells was inhibited by a translational mechanism that did not alter the average number of ribosomes engaged by TK mRNA.

Replication-dependent changes in levels of enzymes involved in DNA precursor biosynthesis are accompanied frequently by changes in levels of cognate mRNA. We tested the common assumption that changes in mRNA levels are responsible for growth-dependent expression of these enzymes using a line of mouse muscle cells that irreversibly withdraws from the cell cycle as part of its terminal differentiation program. Thymidine kinase (TK) mRNA, activity, and protein levels were quantitated in cells transformed with multiple copies of the chicken TK gene. The decline in TK mRNA (both whole cell and cytoplasmic) during myogenesis was poor (2-fold average) and variable (1.2 to 8-fold). In contrast, TK activity always was regulated efficiently (20-fold), even in cells which regulated TK mRNA very poorly. Thus, regulation of TK activity was independent of TK mRNA regulation as myoblasts withdrew from the cell cycle. A TK/beta-galactosidase fusion protein was used to derive an antibody against chicken TK. Immunoblot and immunoprecipitation analyses demonstrated TK protein levels, like TK activity levels, declined to a greater extent than TK mRNA levels. Thus, TK activity likely was regulated by a mechanism involving either decreased translation of TK mRNA or increased degradation of TK protein in committed muscle cells.

TK mRNA levels were determined in mouse L cells transformed with intron deletion mutations of the chicken TK gene. Whether normalized per cell, per integrated gene, or per internal control signal, intron deletion did not diminish the efficiency of TK mRNA formation in transformed L cells. The results demonstrated that introns are not required for efficient biogenesis of cellular mRNA in transformed mouse L cells.

The totipotent stem cells of the pregastrulation mouse embryo which give rise to all embryonic somatic tissues and germ cells express Oct-4. The expression is downregulated during gastrulation and is thereafter only maintained in the germline lineage. Oct-4/lacZ transgenes were used to determine how this pattern of expression was achieved, and resulted in the identification of two separate regulatory elements. The distal element drives Oct-4 expression in preimplantation embryos, in migratory and postmigratory primordial germ cells but is inactive in cells of the epiblast. In cell lines this element is specifically active in embryonic stem and embryonic germ cells. The proximal element directs the epiblast-specific expression pattern, including downregulation during gastrulation; in cell lines its activity is restricted to epiblast-derived cells. Thus, Oct-4 expression in the germline is regulated separately from epiblast expression. This provides the first marker for the identification of totipotent cells in the embryo, and suggests that expression of Oct-4 in the totipotent cycle is dependent on a set of factors unique to the germline.

In mammalian embryos, myogenic precursor cells emigrate from the ventral lip of the dermomyotome and colonize the limbs, tongue and diaphragm where they differentiate and form skeletal muscle. Previous studies have shown that Pax3, together with the c-Met receptor tyrosine kinase and its ligand Scatter Factor (SF) are necessary for the migration of hypaxial muscle precursors in mice. Lbx1 and Pax3 are co-expressed in all migrating hypaxial muscle precursors, raising the possibility that Lbx1 regulates their migration. To examine the function of Lbx1 in muscle development, we inactivated the Lbx1 gene by homologous recombination. Mice lacking Lbx1 exhibit an extensive loss of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests that Lbx1 is not required for the specification of particular limb muscles, and the muscle defects that occur in Lbx1−/− mice can be solely attributed to changes in muscle precursor migration. c-Met is expressed in Lbx1 mutant mice and limb muscle precursors delaminate from the ventral dermomyotome but fail to migrate laterally into the limb. Muscle precursors still migrate ventrally and give rise to tongue, diaphragm and some limb muscles, demonstrating Lbx1 is necessary for the lateral, but not ventral, migration of hypaxial muscle precursors. These results suggest that Lbx1 regulates responsiveness to a lateral migration signal which emanates from the developing limb.