Premature expression of the winged helix transcription factor HFH-11B in regenerating mouse liver accelerates hepatocyte entry into S phase.

Two-thirds partial hepatectomy (PH) induces differentiated cells in the liver remnant to proliferate and regenerate to its original size. The proliferation-specific HNF-3/fork head homolog-11B protein (HFH-11B; also known as Trident and Win) is a family member of the winged helix/fork head transcription factors and in regenerating liver its expression is reactivated prior to hepatocyte entry into DNA replication (S phase). To examine whether HFH-11B regulates hepatocyte proliferation during liver regeneration, we used the -3-kb transthyretin (TTR) promoter to create transgenic mice that displayed ectopic hepatocyte expression of HFH-11B. Liver regeneration studies with the TTR-HFH-11B mice demonstrate that its premature expression resulted in an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This liver regeneration phenotype is associated with protracted expression of cyclin D1 and C/EBPbeta, which are involved in stimulating DNA replication and premature expression of M phase promoting cyclin B1 and cdc2. Consistent with the early hepatocyte entry into S phase, regenerating transgenic livers exhibited earlier expression of DNA repair genes (XRCC1, mHR21spA, and mHR23B). Furthermore, in nonregenerating transgenic livers, ectopic HFH-11B expression did not elicit abnormal hepatocyte proliferation, a finding consistent with the retention of the HFH-11B transgene protein in the cytoplasm. We found that nuclear translocation of the HFH-11B transgene protein requires mitogenic signalling induced by PH and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than in wild type.

Elevated levels of hepatocyte nuclear factor 3beta in mouse hepatocytes influence expression of genes involved in bile acid and glucose homeostasis.

The winged helix transcription factor, hepatocyte nuclear factor-3beta (HNF-3beta), mediates the hepatocyte-specific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3beta in regulating these genes remains unknown because homozygous null HNF3beta mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3beta, we created transgenic mice in which the -3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3beta protein. Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3beta on the hepatic expression of the endogenous mouse HNF-3alpha,-3beta, -3gamma, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3beta transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.

Formation of undifferentiated mesenteric tumors in transgenic mice expressing human neurotropic polymavirus early protein.

The human polyomavirus, JCV, is the established etiologic agent of the human demyelinating disease, progressive multifocal leukoencephalopathy (PML) seen in immunosuppressed individuals. In PML patients, the viral early protein, which is produced exclusively in glial cells is responsible for initiation of the viral lytic cycle. The JCV early protein, T-antigen, has greater than 70% homology to the well characterized SV40 early protein which has established oncogenic properties. To investigate the role of JCV T-antigen in tumorigenesis, transgenic mice containing the viral early genome were produced. Of the four positive transgenic animals, one developed severe neurological abnormalities and succumbed to death at 3 weeks of age. Another animal died with no visible gross pathology and the cause of death was not determined. The remaining two founders developed massive, undifferentiated, solid mesenteric tumors with no obvious neurological symptoms. Results from histologic analysis demonstrated the presence of highly cellular, poorly differentiated neoplastic cells in the tumor tissue. Electron microscopic evaluation of the tumor revealed the presence of a small blue cell-like tumor of epithelial/neuroectodermal origin. Results from RNA analysis by non-quantitative and highly sensitive RT-PCR indicated the presence of the JCV early transcript in various tissues, including kidney, liver, spleen, heart, lung, and brain, as well as in the tumors. However, analysis of the viral early protein by Western blot and immunohistochemistry indicated high level production of JCV early protein in the tumor tissue, but not in any other tissues. These observations present the first evidence for the development of inheritable neuroectodermal tumors induced by the human polyomavirus, JCV, early protein in a whole animal system.

Modifications in protein binding to upstream sequences of the sea urchin cytoplasmic actin gene CyIIa in comparison to its linked neighbors, CyI and CyIIb.

The sequences corresponding to regions upstream of the ATG and transcription start site of the CyIIa cytoplasmic actin gene of the sea urchin Strongylocentrotus purpuratus were determined and compared to the genomically linked CyI and CyIIb actin genes. Sites of protein-DNA interaction in the CyIIa upstream sequences were identified by DNase I footprinting. The similarity between CyIIa and CyI (and CyIIb) upstream sequences was limited and included a consensus octamer sequence, serum response element (SRE) and some short sequences within the proximal promoter region. The octamer sequence was found to bind protein. A single DNase I hypersensitive site was detected within the SRE and to two flanking nucleotides, but otherwise, the SRE did not appear to be protected. This is in contrast to strong protein binding to the CyIIb SRE. A region in the CyIIa gene with limited identity to the functionally significant protein binding site D in CyI also did not bind protein. Four additional sites of protein-DNA interaction were identified in CyIIa upstream sequences. One of these is similar to a protein binding site previously located in the CyI upstream sequences, whereas the others appear to be unique. These data indicate that the CyIIa upstream sequences differ extensively from those of CyI. The pattern of CyIIa expression is likely a consequence of these alternations in DNA sequence and protein-DNA interactions.

Protein tyrosine kinase 6 regulates mammary gland tumorigenesis in mouse models.

Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the majority of human breast tumors and breast cancer cell lines, but its expression has not been reported in normal mammary gland. To study functions of PTK6 in vivo, we generated and characterized several transgenic mouse lines with expression of human PTK6 under control of the mouse mammary tumor virus (MMTV) long terminal repeat. Ectopic active PTK6 was detected in luminal epithelial cells of mature transgenic mammary glands. Lines expressing the MMTV-PTK6 transgene exhibited more than a two-fold increase in mammary gland tumor formation compared with nontransgenic control animals. PTK6 activates signal transducer and activator of transcription 3 (STAT3), and active STAT3 was detected in PTK6-positive mammary gland epithelial cells. Endogenous mouse PTK6 was not detected in the normal mouse mammary gland, but it was induced in mouse mammary gland tumors of different origin, including spontaneous tumors that developed in control mice, and tumors that formed in PTK6, H-Ras, ERBB2 and PyMT transgenic models. MMTV-PTK6 and MMTV-ERBB2 transgenic mice were crossed to explore crosstalk between PTK6 and ERBB2 signaling in vivo. We found no significant increase in tumor incidence, size or metastasis in ERBB2/PTK6 double transgenic mice. Although we detected increased proliferation in ERBB2/PTK6 double transgenic tumors, an increase in apoptosis was also observed. MMTV-PTK6 clearly promotes mammary gland tumorigenesis in vivo, but its impact may be underrepresented in our transgenic models because of induction of endogenous PTK6 expression.

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo.

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.

Regulation of histone synthesis during early Urechis caupo (echiura) development.

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [3H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [3H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.

In vivo competition identifies positive cis-regulatory elements required for lineage-specific gene expression in the sea urchin embryo.

Several cis-regulatory elements within the 5' regulatory region of the lineage-specific CyIIIa actin gene have been identified by in vivo competition. Sea urchin eggs were coinjected with a fusion construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by the CyIIIa regulatory domain, together with molar excesses of various DNA subfragments that are derived from this region. Each subfragment studied includes one or several known sites where highly specific interactions occur in vitro with nuclear DNA-binding proteins. Coinjection of excess molecules of some of these subregions results in a decrease in the activity of the CyIIIa-CAT fusion gene, as a function of the molar subfragment: CyIIIa-CAT ratio. This result implies that these sites complete with cis sequences linked to the CAT reporter gene for limited factors that positively regulate CyIIIa transcription in the embryo, and demonstrates the functional importance of a number of the DNA-protein interactions that have been observed in vitro.

Spatially deranged though temporally correct expression of Strongylocentrotus purpuratus actin gene fusion in transgenic embryos of a different sea urchin family.

We report the unexpected observation that cis-regulatory sequences of a Strongylocentrotus purpuratus actin gene, which direct a particular, lineage-specific pattern of embryonic expression, confer a completely different spatial pattern of expression when introduced into embryos of another sea urchin species. We utilized a fusion gene construct in which the bacterial chloramphenicol acetyl transferase (CAT) reporter gene is driven by CyIIIa actin regulatory sequences. We previously showed that the regulatory region that is included suffices to promote the accumulation of CAT mRNA in transgenic S. purpuratus embryos, on the same developmental schedule and in the same embryonic region, the aboral ectoderm, in which the CyIIIa actin gene is normally expressed (Flytzanis et al. 1987; Hough-Evans et al. 1987). When injected into zygotes of Lytechinus variegatus, which belongs to a different echinoid family, the expected temporal pattern of expression of CAT enzyme was observed. Thus, in both S. purpuratus and L. variegatus embryos, expression is activated at the early blastula stage, although this stage is attained several hours sooner in L. variegatus embryo cultures. Similar kinetics of CAT enzyme accumulation were obtained whether the gene was introduced directly into the L. variegatus zygote nucleus or into the cytoplasm. However, when examined by in situ hybridization, the transgenic L. variegatus embryos were found to display a totally new pattern of CAT mRNA accumulation. Copious CAT transcripts were detected not only in aboral ectoderm cells, but also in skeletogenic mesenchyme cells, gut cells, and oral ectoderm, all cell types that in the transgenic S. purpuratus controls are invariably devoid of detectable CAT transcripts.

Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA.

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs.

A fusion gene construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of CyIIIa actin gene regulatory sequences was injected into unfertilized eggs of the urchin Strongylocentrotus purpuratus, and early pluteus stage embryos that developed from these eggs were fixed and sectioned for analysis by in situ hybridization. A [3H]RNA antisense probe for CAT mRNA was hybridized to 5-micron embryo sections. Autoradiographic signal denoting the presence of CAT mRNA was detected only over aboral ectoderm cells, in which the CyIIIa gene is normally expressed, and not over any recognizable regions of gut or oral ectoderm included in the same sections.

A regulatory domain that directs lineage-specific expression of a skeletal matrix protein gene in the sea urchin embryo.

DNA sequences derived from the 5' region of a gene coding for the 50-kD skeletal matrix protein (SM50) of sea urchin embryo spicules were linked to the CAT reporter gene and injected into unfertilized eggs. CAT mRNA and enzyme were synthesized from these fusion constructs in embryos derived from these eggs, and in situ hybridization with a CAT antisense RNA probe demonstrated that expression is confined to skeletogenic mesenchyme cells. A mean of 5.5 of the 32-blastula-stage skeletogenic mesenchyme cells displayed CAT mRNA (range 1-15), a result consistent with earlier measurements indicating that incorporation of the exogenous injected DNA probably occurs in a single blastomere during early cleavage. In vitro mutagenesis and deletion experiments showed that CAT enzyme activity in the transgenic embryos is enhanced 34-fold by decreasing the number of SM50 amino acids at the amino-terminus of the fusion protein from 43 to 4. cis-regulatory sequences that are sufficient to promote lineage-specific spatial expression in the embryo are located between -440 and +120 with respect to the transcriptional initiation site.

Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene.

Previous studies have located some twenty distinct sites within the 2.3 kb 5' regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development.

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo.

The CyIIIa.CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene.

Territorial expression of three different trans-genes in early sea urchin embryos detected by a whole-mount fluorescence procedure.

We have developed a new procedure for detection of the protein product of chloramphenicol acetyltransferase (CAT) reporter genes in whole mounted sea urchin embryos. The position of a commercially available anti-CAT antibody is visualized by video or confocal microscopy, and thus the spatial domains of exogenous reporter gene expression can be determined with regard to the intact three-dimensional structures of the embryo. We show that in pluteus stage embryos CAT protein expression patterns for SM50 . CAT or CyIIIa . CAT reporter genes are similar to those previously obtained by in situ hybridizations with radioactive probes. Taking advantage of the superior resolution of cellular CAT expression patterns using the antibody visualization method, we found for the first time that, in addition to the expression in aboral ectoderm, some cells in the ciliated band of the pluteus express CyIIIa . CAT. The expression of a new fusion construct, CyIIa . CAT, was also examined. As expected from the localization of endogenous CyIIa mRNA, CAT protein was expressed under control of the CyIIa promoter in gut and skeletogenic mesenchyme cells.

Maternal-fetal interactions affect growth of human immunodeficiency virus type 1 transgenic mice.

Infants vertically infected with human immunodeficiency virus type 1 (HIV-1) often manifest profoundly deficient growth with failure to thrive. The pathologic mechanisms that produce growth failure associated with pediatric HIV infection are not clear. Transgenic mice homozygous for a gag/pol deletion mutant of the infectious provirus pNL4-3 have been found to manifest a similar growth failure pattern. To explore the influence of HIV-1 on fetal growth and maternal-fetal interactions, we examined intrauterine growth of transgenic and nontransgenic mice and evaluated the consequence of embryo transfer into normal and heterozygous transgenic mothers. Mice homozygous for the HIV transgene had normal intrauterine and birth weights but uniformly displayed severe growth retardation postnatally. Transgene expression was prominent in transgenic fetuses and their placentas and in uteri of transgenic mothers, as determined by Northern analysis. Although embryo transfer did not affect intrauterine growth, the pregnancy rate in transgenic mothers was markedly lower than in nontransgenic controls. In both fetal and neonatal tissues, transgene expression was significantly greater in homozygous animals when compared with heterozygotes, but the difference was magnified postnatally. These results suggest that HIV gene expression affected both mother and neonate. In the mother, expression of the HIV-1 transgene reduced postfertilization pregnancy rate. Once the animal was pregnant, however, the effects of transgene expression on the homozygous fetus were overcome in utero, possibly by the contribution of maternal factors or by inhibition of HIV-1 gene expression by a fetal or maternal factor(s). In the neonate, HIV-1 transgene expression increased dramatically in homozygotes and was associated with profound growth failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth failure and AIDS-like cachexia syndrome in HIV-1 transgenic mice.

The mechanisms which predispose to growth failure in infants and children infected with immunodeficiency virus type-1 (HIV-1) are not fully understood. The contributions of viral replication and CD4+ T cell depletion to growth failure in an HIV-1 transgenic mouse model were investigated. Mice homozygous for the transgene, a gag-pol deletion mutant of the HIV-1 provirus pNL4-3, exhibited marked cachexia, growth retardation, lymphoproliferation with a reduction in the percentage of CD4+ T cells but an increase in the absolute number of splenic CD4+ and CD8+ T cells, thymic hypoplasia, and early death. Despite the absence of T cells, athymic nude mice, homozygous for the HIV transgene, displayed comparable growth failure. The results indicate that AIDS-like cachexia may be produced by expression of viral envelope or accessory genes, need not be accompanied by absolute depletion of CD4+ T cells, and may occur independent of T cell function.