A minimal c-fes cassette directs myeloid-specific expression in transgenic mice.

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) human c-fes genomic fragment was previously shown to contain cis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR, c-fes transgenes were analyzed in mice. Myeloid-cell-specific, deoxyribonuclease-I-hypersensitive sites localized to the 3' boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446-base-pair promoter, integration-site-independent, copy-number-dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo.

GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient (GATA6(-/-)) ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6(-/-) ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, alpha-fetoprotein (AFP), and HNF3beta. Homozygous GATA6(-/-) mice died between embryonic day (E) 6.5 and E7. 5 and exhibited a specific defect in endoderm differentiation including severely down-regulated expression of GATA4 and absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activated the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development, GATA6(-/-)-C57BL/6 chimeric mice were generated. lacZ-tagged GATA6(-/-) ES cells contributed to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium.

Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.

gamma-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine gamma-sarcoglycan gene was disrupted using homologous recombination. Mice lacking gamma-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma-, and delta-sarcoglycan function as a unit. Importantly, mice lacking gamma-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, beta-dystroglycan and laminin were left intact, implying that the dystrophin-dystroglycan-laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking gamma-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking gamma-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.

GATA4 transcription factor is required for ventral morphogenesis and heart tube formation.

Previous studies have suggested that the GATA4 transcription factor plays an important role in regulating mammalian cardiac development. In the studies described in this report we have used gene targeting to produce GATA4-deficient mice. Homozygous GATA4-deficient (GATA4-/-) mice died between 8.5 and 10.5 days post coitum (dpc). GATA4-/- embryos displayed severe defects in both rostral-to-caudal and lateral-to-ventral folding, which were reflected in a generalized disruption of the ventral body pattern. This resulted in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube. Analysis of cardiac development in the GATA4-/- mice demonstrated that these embryos developed splanchnic mesoderm, which differentiated into primitive cardiac myocytes that expressed contractile proteins. However, consistent with the observed defect in ventral morphogenesis, these GATA4-/- procardiomyocytes failed to migrate to the ventral midline to form a linear heart tube and instead formed aberrant cardiac structures in the anterior and dorsolateral regions of the embryo. The defect in ventral migration of the GATA4-/- procardiomyocytes was not cell intrinsic because GATA4-/- cardiac myocytes and endocardial cells populated the hearts of GATA4-/- -C57BL/6 chimeric mice. Taken together, these results demonstrated that GATA4 is not essential for the specification of the cardiac cell lineages. However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis.

Prostaglandin secretion by guinea pig tracheal epithelial cells caused by eosinophil major basic protein.

We examined the effect of eosinophil major basic protein (MBP) on prostaglandin (PG) secretion from guinea pig tracheal epithelial (GPTE) cells. Primary cultures of GPTE cells were incubated with 10(-6) M MBP for up to 6 h and then stimulated with 10(-6) M bradykinin (BK). PGE2, 6-ketoprostaglandin F1 alpha (PGF1 alpha), PGF2 alpha, and thromboxane B2 (TxB2) concentrations in media were determined by enzyme-linked immunoabsorbent assay (EIA). Incubation with MBP for 6 h caused secretion of both PGE2 (17,614 +/- 4,416 vs. 1,426 +/- 555 pg/10(6) cells at baseline, P < 0.001, n = 7) and PGF2 alpha (20,303 +/- 5,724 vs. 3,790 +/- 1.075 pg/10(6) cells at baseline, P < 0.002, n = 7). Secretion of PGE2 and PGF2 alpha stimulated by MBP required at least 2 h. Incubation with MBP for 6 h also augmented the subsequent response to BK: PGE2 secretion was 29,215 +/- 6,853 vs. 3,445 +/- 1,041 pg/10(6) cells for BK alone (P < 0.0001), and PGF2 alpha secretion was 25,407 +/- 6,237 vs. 5,213 +/- 1,535 pg/10(6) cells for BK alone (P < 0.0001). MBP did not change 6-keto-PGF1 alpha and TxB2 secretion. Incubation of GPTE cells from seven animals with polylysine, a protein with mass and ion charge similar to MBP, for 2 h, both caused secretion of PGE2 (8,579 +/- 3,244 vs. 788 +/- 419 pg/10(6) cells at baseline, P < 0.01) and augmented the response to BK (12,732 +/- 4,788 vs. 1,653 +/- 680 pg/10(6) cells after BK alone, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of guinea pig tracheal epithelial cells induced by calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) is contained within and secreted by nerves and neuroepithelial bodies in the airway epithelium. To determine whether CGRP is mitogenic for airway epithelial cells, tracheal epithelial cells isolated from 26 guinea pigs were grown in primary culture for 2 days. Subconfluent cells were exposed to 10(-13) to 10(-9) M CGRP for 4 h and then returned to CGRP-free medium. Proliferation was quantified by direct cell count and by measurement of fractional labeling with the thymidine analog, bromodeoxyuridine (BrdU). CGRP exposure increased both cell number (53,980 +/- 9,870 cells after 10(-9) M CGRP versus 33,910 +/- 5,150 cells after control, P < 0.05) and fractional BrdU labeling (12.9 +/- 2.2% after 10(-11) M CGRP versus 3.9 +/- 0.9%, control; P < 0.01, n = 9) at 24 h after exposure. The mitogenic effect of CGRP persisted at least 3 days after exposure. CGRP-induced proliferation was attenuated by co-incubation with the CGRP receptor antagonist, hCGRP-(8-37). These data demonstrate that CGRP causes proliferation of guinea pig tracheal epithelial cells in primary culture through stimulation of a specific receptor, and suggest a role for this neuropeptide in regulating airway epithelial cell growth.

Topographic distribution of prostaglandin secretion caused by bradykinin in canine tracheal epithelial cells.

Inflammatory mediators promote the synthesis and secretion of prostaglandin (PG) mediators in airway epithelial cells. In this study, we examined the topographic and kinetic profile of PG secretion in canine tracheal epithelial cells harvested from the tracheal posterior membrane (PM) and those obtained from the immediately anterior cartilage-associated membrane (CM). Primary cultures of tracheal epithelial cells obtained from 23 disease-free dogs were grown to confluence in serum-enriched medium. Cells then were incubated in serum-free medium for 1 h and stimulated with 10(-7) to 10(-5) M bradykinin. Baseline secretion of PGE2 was similar to both PM and CM cells; however, PM cells secreted greater concentrations in both PGI2 (measured as 6-keto-PGF1 alpha) (1,269 +/- 160 versus 775 +/- 91 pg/10(6) cells, P less than 0.01) and PGF2 alpha (436 +/- 54 versus 234 +/- 45 pg/10(6) cells, P less than 0.002) compared with CM cells. Bradykinin (BK) stimulation caused substantial secretion in less than or equal to 20 min of PGE2 and 6-keto-PGF1 alpha from PM but not CM cells: after stimulation with 10(-6) M BK, 6-keto-PGF1 alpha secretion was 348 +/- 74% in PM cells versus 157 +/- 18% of baseline secretion in CM cells (P less than 0.005); PGE2 secretion was 310 +/- 53% in PM cells versus 163 +/- 15% of baseline secretion in CM cells (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)