Overexpression of Lis1 in different stages of spermatogenesis does not result in an aberrant phenotype.

Previous studies showed that in the mouse mutant Lis1(GT/GT) gene trap integration in intron 2 of Lis1 gene leads to male infertility in homozygous Lis1(GT/GT) mice. We further analyzed this line and could confirm the suggested downregulation of a testis-specific Lis1 transcript in mutant animals in a quantitative manner. Moreover, we analyzed the gene trap mutation on different genetic backgrounds in incipient congenic animals and could exclude a genetic background effect. To gain further insights into the role and requirement of LIS1 in spermatogenesis, 3 transgenic lines were generated, that overexpress Lis1 under control of the testis-specific promoters hEF-1α, which is exclusively active in spermatogonial cells, PGK2, which is active in pachytene spermatocytes and following stages of spermatogenesis, and Tnp2 which is active in round spermatids and following stages of spermatogenesis, respectively. All 3 transgenic lines remained fertile and testis sections displayed no abnormalities. To overcome the infertility of Lis1(GT/GT) males, these transgenic Lis1-overexpressing animals were mated with Lis1(GT/GT) mice to generate 'rescued' Lis1(GT/GT)/Lis1(Tpos) males. 'Rescued' animals from all transgenic lines remained infertile, thus overexpression of Lis1 in different stages of spermatogenesis could not rescue the infertility phenotype of homozygous gene trap males.

Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C.

Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.

Transcription of the muscle regulatory gene Myf4 is regulated by serum components, peptide growth factors and signaling pathways involving G proteins.

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell-surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle-specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.

The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development.

The spatial and temporal expression pattern of the muscle regulatory gene Myf-6 (MRF4/herculin) has been investigated by in situ hybridization during embryonic and fetal mouse development. Here, we report that the Myf-6 gene shows a biphasic pattern of expression. Myf-6 transcripts are first detected in the most rostral somites of the mouse embryo at 9 d of gestation and accumulate progressively in myotomal cells along the rostro-caudal axis. This expression is transient and Myf-6 mRNA can no longer be detected in myotomal cells after day 12 post coitum (p.c.). In contrast to other muscle determination genes (MyoD1, myogenin, Myf-5), Myf-6 mRNA is not detected in limb buds or visceral arches and skeletal muscle of the mouse embryo (day 8-15 p.c.). In fetal mice, Myf-6 transcripts appear at day 16 p.c. in all skeletal muscles, and the gene continues to be expressed at a high level after birth. These results suggest that early Myf-6 expression may be restricted to a population of myogenic cells that does not contribute to the embryonic muscle masses in limb buds and visceral arches. The reappearance of Myf-6 mRNA in fetal skeletal muscle coincides approximately with secondary muscle fiber formation and the onset of innervation.

Muscle differentiation: more complexity to the network of myogenic regulators.

Recent genetic and biochemical approaches have advanced our understanding of control mechanisms underlying myogenesis in vertebrate organisms. In particular, systematic combinations of targeted gene disruptions in mice have revealed unique and overlapping functions of members of the MyoD family of transcription factors within the regulatory network that establishes skeletal muscle cell lineages. Moreover, Pax3 has been identified as a key regulator of myogenesis which seems to act genetically upstream of MyoD. In addition, novel genes have been discovered that modulate myogenesis and the activity of myogenic basic helix-loop-helix (bHLH) proteins in positive or negative ways. The molecular mechanisms of these interactions and cooperativity are being elucidated, most notably between the myogenic bHLH factors and MEF2 transcription factors.

The homeobox gene NKX3.2 is a target of left-right signalling and is expressed on opposite sides in chick and mouse embryos.

Vertebrate internal organs display invariant left-right (L-R) asymmetry. A signalling cascade that sets up L-R asymmetry has recently been identified (reviewed in [1]). On the right side of Hensen's node, activin represses Sonic hedgehog (Shh) expression and induces expression of the genes for the activin receptor (ActRIIa) and fibroblast growth factor-8 (FGF8) [2] [3]. On the left side, Shh induces nodal expression in lateral plate mesoderm (LPM); nodal in turn upregulates left-sided expression of the bicoid-like homeobox gene Pitx2 [4] [5] [6]. Here, we found that the homeobox gene NKX3.2 is asymmetrically expressed in the anterior left LPM and in head mesoderm in the chick embryo. Misexpression of the normally left-sided signals Nodal, Lefty2 and Shh on the right side, or ectopic application of retinoic acid (RA), resulted in upregulation of NKX3.2 contralateral to its normal expression in left LPM. Ectopic application of FGF8 on the left side blocked NKX3.2 expression, whereas the FGF receptor-1 (FGFR-1) antagonist SU5402, implanted on the right side, resulted in bilateral NKX3.2 expression in the LPM, suggesting that FGF8 is an important negative determinant of asymmetric NKX3.2 expression. NKX3.2 expression was also found to be asymmetric in the mouse LPM but, unlike in the chick, it was expressed in the right LPM. In the inversion of embryonic turning (inv) mouse mutant, which has aberrant L-R development, NKX3.2 was expressed predominantly on the left side. Thus, NKX3.2 transcripts accumulate on opposite sides of mouse and chick embryos although, in both the mouse and chick, NKX3.2 expression is controlled by the L-R signalling pathways.

A novel E1A domain mediates skeletal-muscle-specific enhancer repression independently of pRB and p300 binding.

The adenovirus E1A oncoprotein completely blocks muscle differentiation and specifically inhibits the transactivating function of myogenic basic helix-loop-helix (bHLH) transcription factors. This inhibition is dependent on the conserved region CR1 of E1A, which also constitutes part of the binding sites for the pocket proteins pRB, p107, and p130 and the transcriptional coactivators p300 and CBP. Here we report a detailed mutational analysis of E1A and the identification of a muscle inhibition motif within CR1. This motif encompasses amino acids 38 to 62 and inhibits Myf-5- or MyoD-mediated activation of myogenin and the muscle creatine kinase gene. Overexpression of this E1A region also inhibits the conversion of 10T1/2 fibroblasts to the myogenic lineage. The sequence motif EPDNEE (amino acids 55 to 60) within CR1 appears to be particularly important, because point mutations of this sequence diminish the E1A inhibitory activity. Interactions of E1A with pRB and with p300 do not seem to be necessary for the muscle-specific enhancer repression, because E1A mutants which lack these interactions still inhibit Myf-5- and MyoD-mediated transactivation. Moreover, overexpression of p300 fails to overcome muscle-specific inhibition by wild-type E1A and mutant E1A protein which lacks pRB binding. Since we have no evidence for direct E1A interaction with bHLH proteins, we propose that E1A may target a necessary cofactor of the muscle-specific bHLH transcription complex.

Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

Initiation of protein synthesis in bacillus subtilis in the presence of trimethoprim or aminopterin.

Initiation of protein synthesis has been studied in the presence of the tetrahydrofolic acid analogues trimethoprim or aminopterin in Bacillus subtilis. This bacterium can grow in the presence of the inhibitors, when the medium is supplemented with the low molecular weight products of tetrahydrofolate-dependent pathways. In an attempt to show whether formylation of initiator tRNA is a prerequisite for the iniation of protein synthesis in procaryotic cells, the amount of N-formylmethionine in tRNA and in protein has been determined. The level of formylation of methionyl-tRNA was found to be 70% in control cells and approximately 2% in inhibitor-treated cells. The content of formyl groups in protein has also been found to be drastically reduced. Trimethoprim or aminopterin did not alter the amount of tRNAMet nor the degree of aminoacylation of tRNAMet in vivo. These results indicate that in B. subtilis inititation of protein synthesis is possible without prior formylation of initiator tRNA.

Purification and characterization of tRNAMet-f, tRNAPhe and tRNATyr2 from Baccillus subtilis.

Three tRNAs specific for methionine, phenylalanine and tyrosine were isolated from the total tRNA of Bacillus subtilis by chromatographic procedures using BD-cellulose and reversed-phase (5) chromatography. The acceptor activities of the purified tRNAs are 1160, 1260 and 1320 pmoles per A260nm unit for tRNAMetf, tRNAPhe and tRNATyr2 respectively. In tRNAMetf and tRNAPhe ribothymidine, pseudouridine and dihydrouridine are present, in addition, in tRNAPhe 7-methyguanosine and a 2'-O-methylated nucleoside were found. The modified nucleosides of tRNATyr2 are ribothymidine, pseudouridine, dihydrouridine, 4-thiouridine and 1-methyladenosine. The results suggest the presence of 2-methylthio-N6(delta 2-isopentenyl)adenosine in tRNAPhe and tRNATyr2. The thermal denaturation profiles of the three tRAN species are presented.

Nkx2-9 is a novel homeobox transcription factor which demarcates ventral domains in the developing mouse CNS.

Nkx homeobox transcription factors are expressed in diverse embryonic cells and presumably control cell-type specification and morphogenetic events. Nkx2-9 is a novel family member of NK2 genes which lacks the conserved TN-domain found in all hitherto known murine Nkx2 genes. The prominent expression of Nkx2-9 in ventral brain and neural tube structures defines a subset of neuronal cells along the entire neuraxis. During embryonic development, Nkx2-9-expressing cells shift from the presumptive floor plate into a more dorsolateral position of the neuroectoderm and later become limited to the ventricular zone. Nkx2-9 expression overlaps with that of Nkx2-2 but is generally broader. While initially Nkx2-9 is expressed in close proximity to sonic hedgehog, its expression domain clearly segregates from sonic hedgehog at later developmental stages. The dynamic expression pattern of Nkx2-9 in ventral domains of the CNS is consistent with a possible role in the specification of a distinct subset of neurons.

Chicken NKx2-8, a novel homeobox gene expressed during early heart and foregut development.

cNkx2-8 represents a novel member of the NK2-family transcription factors. The gene contains three highly conserved regions, the TN-, NK2-, and homeodomains which are diagnostic for this group of proteins. cNkx2-8 is expressed during chick embryogenesis in ventral foregut endoderm, myocardial mesoderm, epithelium of the branchial arches and the dorsal mesocardium. While cNkx2-8 expression partially overlaps with other NK genes, such as Nkx2-5 and Nkx2-3, its onset and aspects of its expression domains are specific. Thus, structural data and the expression profile suggest that cNkx2-8 constitutes a new homeobox protein which may cooperate with its known relatives in defining an antero-ventral field including the developing heart and pharyngeal endoderm.

BMP-2 induces ectopic expression of cardiac lineage markers and interferes with somite formation in chicken embryos.

In Drosophila induction of the homeobox gene tinman and subsequent heart formation are dependent on dpp signaling from overlying ectoderm. In order to define vertebrate heart-inducing signals we screened for dpp-homologues expressed in HH stage 4 chicken embryos. The majority of transcripts were found to be BMP-2 among several other members of the BMP family. From embryonic HH stage 4 onwards cardiogenic mesoderm appeared to be in close contact to BMP-2 expressing cells which initially were present in lateral mesoderm and subsequently after headfold formation in the pharyngeal endoderm. In order to assess the role of BMP-2 for heart formation, gastrulating chick embryos in New culture were implanted with BMP-2 producing cells. BMP-2 implantation resulted in ectopic cardiac mesoderm specification. BMP-2 was able to induce Nkx2-5 expression ectopically within the anterior head domain, while GATA-4 was also induced more caudally. Cardiogenic induction by BMP-2, however remained incomplete, since neither Nkx2-8 nor the cardiac-restricted structural gene VMHC-1 became ectopically induced. BMP-2 expressing cells implanted adjacent to paraxial mesoderm resulted in impaired somite formation and blocked the expression of marker genes, such as paraxis, Pax-3, and the forkhead gene cFKH-1. These results suggest that BMP-2 is part of the complex of cardiogenic signals and is involved in the patterning of early mesoderm similar to the role of dpp in Drosophila.

Chick NKx-2.3 represents a novel family member of vertebrate homologues to the Drosophila homeobox gene tinman: differential expression of cNKx-2.3 and cNKx-2.5 during heart and gut development.

NKx homeodomain proteins are members of a growing family of vertebrate transcription factors with strong homology to the NK genes in Drosophila. Here, we describe the cloning of cNKx-2.3 and cNKx-2.5 cDNAs and their expression during chick development. Both genes are expressed in the developing heart with distinct but overlapping spatio-temporal patterns. While cNKx-2.5 is activated in early precardiac mesoderm and continues to be uniformly expressed throughout the mature heart, expression of NKx-2.3 starts later in differentiated myocardial cells with regional differences compared to NKx-2.5. Additionally, both genes are expressed in adjacent domains of the developing mid- and hindgut mesoderm as well as in branchial arches. The highly conserved structure of cNKx-2.5 and its similar expression to mouse and Xenopus NKx-2.5 genes and to the Drosophila gene tinman argue that it constitutes the chick homologue of these genes. Different temporal and spatial activity of cNKx-2.3 in heart and gut as well as in a regionally restricted expression domain in the neural tube suggest that cNKx-2.3 is a member of the NK-2 gene family which may be involved in specifying mesodermally and ectodermally derived cell types in the embryo.

BMP2 is required for early heart development during a distinct time period.

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.

Distinct temporal expression of mouse Nkx-5.1 and Nkx-5.2 homeobox genes during brain and ear development.

The mouse Nkx-5.1 and Nkx-5.2 genes have been identified by sequence homology to Drosophila NK genes within the homeobox domain. Here, we report the isolation of the Nkx-5.2 cDNA and a detailed comparative analysis of the spatio-temporal expression patterns for Nkx-5.1 and Nkx-5.2 genes. Nkx-5.2 transcripts are first detected in E13.5 embryos where they colocalize with Nkx-5.1 mRNA in the developing central nervous system and the inner ear. However, the onset of Nkx-5.1 transcription begins much earlier in 10 somite stage embryos (E8.5) in the otic placode and the branchial region. Nkx-5.1 expression in the ear persists until birth, whereas in branchial arches it is transient between E8.5 to E11.5. Transcript distribution appears regionalized in the otic vesicle concentrating at the anterior and posterior margin and later at the dorsal side of the otocyst. These domains are distinct from regions expressing Pax-2 and sek, two other early markers for otic development. From E11.5 to birth several Nkx-5.1 expression domains appear in the brain between the ventral diencephalon and the myelencephalon. The same expression domains also exist for Nkx-5.2 beginning at E13.5. The regionally restricted expression pattern of both Nkx-5 genes during mouse development suggests their involvement in cell type specification of neuronal cells.

Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review.

The role of the four myogenic regulating genes Myf-5, myogenin, MyoD, and MRF4 (herculin, Myf-6) during mouse embryogenesis has been investigated by targeted gene inactivation. Null mutations for the MyoD gene generate no skeletal muscle phenotype due to a compensatory activation of the Myf-5 gene. Mice carrying a homozygous Myf-5 mutation exert considerably delayed myotome formation with unexpected consequences. While skeletal myogenesis in these mutant mice resumes normally at the onset of MyoD expression, a skeletal defect of the ribs persists. Apparently, Myf-5 and MyoD individually are not absolutely essential for skeletal muscle development, most likely because they have overlapping or redundant functions. In fact, double mutants lacking both, MyoD and Myf-5, fail to develop skeletal musculature and the muscle forming regions seem to be devoid of myoblasts. Homozygous inactivation of the myogenin gene leads to drastically reduced myofiber formation. These mice accumulate apparently normal numbers of myoblasts which are arrested in their terminal differentiation program. Myf-6 null mutant mice exhibit drastically reduced expression of Myf-5 for reasons presently unknown. The phenotype is very similar to Myf-5 mutants with an additional reduction of deep back muscles and minor alterations in sarcomeric protein isoforms. Based on the phenotypes obtained from these various gene "knock-out" mice, we now begin to understand the regulatory network and the homostatic relationship of genes which are critically involved in myogenesis of vertebrates.

Alkali myosin light chains in man are encoded by a multigene family that includes the adult skeletal muscle, the embryonic or atrial, and nonsarcomeric isoforms.

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC1 and MLC3; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLCemb); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC1, MLC3 and MLCemb, and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC1 and MLC3 is located on human chromosome 2.

Treatment of human peripheral lymphocytes with concanavalin A activates expression of glutathione reductase.

A cDNA clone corresponding to mRNA present only in proliferating cells was isolated and its nucleotide sequence determined. This cDNA is 723 nucleotides long and encodes a portion of the human glutathione reductase mRNA corresponding to the amino acids 77-318 of the mature protein. Expression of glutathione reductase mRNA was undetectable in resting human T-lymphocytes and was induced shortly after cells had been triggered to proliferate by the treatment with concanavalin A. This result suggests that synthesis of glutathione reductase is generally activated in replicating cells which may indicate that this enzyme plays a functional role during cell proliferation.