Explants In Organ Culture Research Articles

Glucocorticoids Maintain the Extracellular Matrix of Differentiated Mammary Tissue During Explant and Whole Organ Culture

Glucocorticoids Maintain the Extracellular Matrix of Differentiated Mammary Tissue During Explant and Whole Organ Culture

Gene expression signatures in BALB3T3 fibroblasts exposed to cobalt micro/nano-particles and cobalt ions

Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation.ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota.IL17A- and IL22-producing, natural cytotoxicity receptor–negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor–positive cells, in a dose-dependent manner.IL6 contributes to activation of colonic natural cytotoxicity receptor–negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor–positive cells.

Mouse Embryonic Stem Cell–Derived Embryoid Bodies Generate Progenitors That Integrate Long Term into Renal Proximal Tubules In Vivo

The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP+ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP+ cells into embryonic kidney explants in organ culture, beta-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP+ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.

Leukemia Inhibitor Factor (LIF) Inhibits HIV-1 Replication Via Restriction of Stat 3 Activation

Leukemia inhibitor factor (LIF) has been shown to potently inhibit HIV-1 replication in vitro and in human organ explant cultures. Furthermore, LIF activates the Jak/Stat signaling pathway with which many viruses, including HIV-1, interfere. We used CXCR4 and the LIF signaling receptor (gp130)-expressing cMAGI cells transfected with CD4, CCR5, and HIV-LTR-beta-galactosidase as a model system to investigate the potential involvement of Stat proteins in the anti-HIV-1 effect of LIF. Pretreatment with recombinant human (rh)LIF resulted in a significantly reduced uptake of HIV-1(BaL) , HIV-1(LAI), and SIVmac251 viral particles without affecting uptake of murine leukemia retroviral particles. HIV-1(BaL), HIV-1(LAI), as well as rhLIF selectively induced phosphorylation of Stat 3 but not Stat 1 or Stat 5. However, treatment of cMAGI cells with rhLIF prior to HIV-1 infection downregulated the HIV-1-mediated Stat 3 phosphorylation. In addition, peripheral blood mononuclear cells (PBMCs) transfected with Stat 3 siRNA prior to HIV-1(LAI) or HIV-1(BaL) infection produced significantly less HIV-1 p24 antigen as compared to nontransfected HIV-1(LAI) and HIV-1(BaL)-infected PBMCs. Thus, the Jak/Stat signaling pathway is important for the HIV-1 replication life cycle and rhLIF excerts its anti-HIV-1 activity by disrupting this signaling cascade.

Reduced endothelin converting enzyme‐1 and endothelin‐3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease–like distal intestinal aganglionosis

Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.

Cigarette Smoke Causes Small Airway Remodeling by Direct Growth Factor Induction and Release

Small airway remodeling (SAR) is one of the anatomic causes of airflow obstruction in patients with chronic obstructive pulmonary disease. The pathogenesis of SAR is poorly understood, but there are two general theories: (1) SAR is caused by smoke-induced inflammatory cells that damage the airway and evoke a repair process and (2) SAR is caused by direct induction of growth factors by cigarette smoke. To investigate this process, we briefly exposed rat tracheal explants, a model system free of exogenous inflammatory cells, to whole cigarette smoke, and then maintained the explants in organ culture for varying periods. With explants cultured in air, smoke induced up-regulation of both transforming growth factor (TGF)-β1 and procollagen gene expression at 24 h after initial exposure. Increased procollagen gene expression was prevented in a dose–response fashion by the TGF-β antagonist fetuin, indicating that TGF-β was driving fibrosis. Collected supernatant from explants exposed to smoke and cultured in fluid medium showed increased release of TGF-β1, and this was abolished by the oxidant scavenger tetramethylthiourea. Gene expression of connective tissue growth factor (CTGF) was sharply increased 2 h after smoke exposure. Using pure recombinant proteins, smoke-conditioned medium oxidized TGF-β latency-associated peptide and also caused release of active TGF-β1 from latent TGF-β1 . We conclude that cigarette smoke directly induces release of preformed TGF-β1 by oxidizing the latency-associated peptide in rat tracheal explants, and that release must be extremely rapid, since gene expression of CTGF, the downstream fibrogenic driver of TGF-β effects on collagen synthesis, is elevated very shortly after smoke exposure. Although tracheal explants are only an approximate model of small airways, these findings suggest that smoke-induced SAR may reflect direct induction of growth factor release and signaling and does not require smoke-evoked inflammatory cells.

A tissue explant system for assessing tendon overuse injury

Tendon overuse injuries are common athletic and occupational problems. When studying mechanisms that cause these injuries, inherent complexities associated with controlling in vivo loading necessitates alternative approaches such as in vitro organ culture. Current devices for loading explants in organ culture, whether custom-built or commercial, have various deficiencies in their loading capability, control mechanism and strain assessment. To overcome these shortcomings, an advanced tissue loading device with video strain analysis capabilities was developed for investigating overuse injuries and its performance/calibration were evaluated. Two tests were used to assess the ability of the system to create and monitor mechanical changes with overuse. Overuse loading significantly increased strains and decreased strength, showing the ability of this system to create and monitor tissue damage. Furthermore, the device design allows for its use in a standard incubator. Coupled with custom loading and data collection programs, this system is suitable for long-term overuse injury studies.

ORGAN-CULTURED AIRWAY EXPLANTS: A NEW MODEL OF AIRWAY HYPERRESPONSIVENESS

Respiratory pathology research is limited by the number of appropriate multicellular models suitable for studying mechanical properties and signaling pathways that are involved in airway responsiveness. In this study, the electrophysiological and pharmacomechanical properties of organ-cultured explants derived from normal guinea pig bronchi and trachea were investigated. The explants maintained their basic histological phenotype but became hyperreactive to excitatory (muscarinic, histaminergic, serotinergic, and thromboxane receptor agonists, 60 mM KCl) and inhibitory (norepinephrine, isoproterenol) stimuli within the first 3 days in culture, with or without serum in the culture medium. Indomethacin pretreatment did not modify the spasmogen responses of the explant. The onset of this intrinsic overreactivity was highly dependent on the initial presence of epithelium, took 3 days to reach its maximum, and lasted over several days (days 3 to 7). Removal of Ca2+ from the bathing solution initially normalized the inotropic responses of the cultured versus freshly isolated airway tissues. However, the responses to repetitive carbachol challenges in the absence of Ca2+ displayed a slower inactivation in the cultured explants compared to fresh tissues. Smooth muscle resting membrane potential and potassium-induced depolarizations were unaffected by organ culture. Immunohistochemical analyses revealed the presence of apoptotic bodies in the submucosa and epithelial layers, but none in the smooth muscle layer of cultured airways. These functional and histological findings may prove useful in understanding signaling processes involved in tissue hyperresponsiveness related to asthma.

Expression of C-type lectin receptors by subsets of dendritic cells in human skin.

C-type lectins are cell surface receptors that recognize carbohydrate structures which are often part of microbial pathogens. Several of these molecules are expressed on dendritic cells and are involved in antigen uptake. Expression of C-type lectins on dendritic cells of the human skin, i.e. Langerhans cells of the epidermis and dermal dendritic cells, has been incompletely studied to date. We therefore investigated C-type lectins in situ and on dendritic cells obtained by migration from skin explants by immunofluorescence and flow cytometry. Emphasis was laid on expression patterns of DEC-205/CD205 and BDCA-2, a marker for plasmacytoid dendritic cells. Langerhans cells in situ expressed low levels of DEC-205. Expression was upregulated upon maturation in skin explant organ culture. Most dermal dendritic cells were found to be positive for DEC-205 and DC-SIGN/CD209. Few BDCA-2-expressing cells were found in most skin samples. They were located in small groups in the dermis close beneath the basement membrane. The vast majority of all types of dendritic cells in normal human skin was of immature phenotype, i.e. did not express DC-LAMP/CD208. It is concluded that normal appearing human skin harbors different subsets of dendritic cells including few scattered BDCA-2-expressing cells, presumably plasmacytoid dendritic cells, expressing variable sets of C-type lectin receptors. This may critically contribute to the capacity of the skin immune system to flexibly respond to the world of microbial pathogens.

Racial disparity in membrane response to infectious stimuli: a possible explanation for observed differences in the incidence of prematurity: Community Award Paper

Objective This study compares the immune responsiveness of amniochorionic membranes (AC) derived from African American (AA) and white (C) women to an infectious stimulus ex vivo. Study design AC derived from AA and C women were placed in an organ explant culture for 48 hours and then stimulated with endotoxin. Enzyme-linked immunosorbent assay measured the concentration of matrix metalloproteinase 9 (MMP9), tumor necrosis factor-α (TNF-α), and soluble TNF receptors (sTNFR1and sTNFR2) in culture media from stimulated and unstimulated AC. Results The C group produced 8-fold more TNF-α after stimulation than did the AA group. Both soluble receptor (R1 and R2) production increased in the C group and decreased in the AA group after stimulation. Although the C group–derived membranes produced more MMP9 at rest, a 6-fold increase in MMP9 concentration was seen in the AA group–derived membranes after stimulation. No change in MMP9 concentration was seen after stimulation of the C group–derived membranes. Conclusion Although the C group produced more TNF, they also produce higher sTNFRs, which may serve a protective role. The increased MMP9 release by the AA group may be suggestive of the greater risk of premature rupture of membranes in the AA group.

Delivery of genes to the eye using lentiviral vectors.

The primary aim of gene transfer into the retinal cells has been to investigate the developmental mechanisms of the retinal cells or to reverse retinal diseases. Retroviruses have been used to investigate the differentiation of retinal cells, to study the embryonic retina in vivo or explant organ culture, and to trace the fate of the cells that were dividing at the time of gene transfer. Using adenovirus, Bennett et al. showed the possibility of using gene therapy to correct degenerative diseases of the central nervous system (CNS) (6). However, owing to the short duration of the gene expression, adenovirus is not suitable for correcting chronic diseases. Currently, lentivirus (7-9) and adenoassociated virus vectors (10-14) are being used for studying and correcting gene therapy of retinal degenerative diseases. Using an HIV vector carrying the green fluorescent protein (GFP) gene expressed from the cytomegalovirus (CMV) promoter, we showed that efficient and long-lasting gene expression could be obtained in the retina (7,8). Moreover, gene expression was restricted to the photoreceptor cells and was more efficient with the rhodopsin promoter. Similar results were reported using adeno-associated virus (AAV) vector. Using a lentivirus vector carrying the phosphodiesterase beta subunit (PDEbeta) gene, the mutation of which causes retinal degeneration called retinitis pigmentosa in rd mice, photoreceptor cells were rescued from degeneration in rd mice for at least 6 mo by PDEbeta transduction using HIV-based lentivirus vector (9).

遺伝性内耳障害モデルにおける前庭形態異常と遺伝子導入の試み―ｓｈａｋｅｒ‐２マウス，ｗａｌｔｚｉｎｇ ｇｕｉｎｅａ Ｐｉｇの検討から―

We reviewed the hair cell pathology of genetic hearing loss and circling behavior and discussed possibility of gene therapy in human genetic inner ear disease through genetic inner ear disease animal models. First, we focused on the cytocauds containing pathological actin filaments in the vestibular type 1 hair cell of waltzing guinea pigs (WGP) and in auditory and vestibular hair cells of shaker-2 mice. The cytocaud, which seems like to be an actin filaments ladder, penetrates the basement membrane and runs along nerve fiber. One possible explanation is that hair cells detach from the basement membrane during development but do not in WGP and shaker-2 mice. However, the molecular signals that are required for detachment are unknown.Furthermore, we tested the possibility of gene transudation in shaker-2 mice, the model of DFNB3, i.e. human deafness, in order to treat the hair cell pathology including cytocaud. Vestibular sensory epithelia of explant organ culture obtained from mouse pups at P1, P3, and P5 were transfected by adenovirus vector containing the bacterial lacZ in vitro and processed to detect the reporter gene expression. Vestibular hair cells can be transduced at all stages in shaker-2 mice. Our data demonstrate that differentiating hair cells in mouse explants can be virally transduced, suggesting that wild-type genes can be inserted into these cells to correct the phenotype. We need further investigations of gene therapy using animal in vivo data to discuss the possibility of clinical application for the genetic inner ear diseases.

ALA and ALA hexyl ester induction of porphyrins after their systemic administration to tumour bearing mice.

The use of synthetic lipophilic molecules derived from 5-aminolevulinic acid (ALA) is currently under investigation to enhance cellular ALA penetration. In this work we studied the effect of systemic administration to mice of the hexyl ester of ALA (He-ALA) on porphyrin tissue synthesis as compared to ALA. In most normal tissues as well as in tumour, He-ALA induced less porphyrin synthesis than ALA after its systemic administration either intravenous or intraperitoneal, although explant organ cultures exposed to either ALA or He-ALA revealed equally active esterases. The only tissue that accumulated higher porphyrin levels from He-ALA (seven times more than ALA) was the brain, and this correlated well with a rapid increase in ALA/He-ALA content in brain after administration of He-ALA. This may be ascribed to a differential permeability to lipophilic substances controlled by the blood–brain barrier, a feature which could be further exploited to treat brain tumours.British Journal of Cancer (2002) 87, 790–795. doi:10.1038/sj.bjc.6600559 www.bjcancer.com© 2002 Cancer Research UK

Using rats as a research model for the study of endometriosis.

Although there are disadvantages of extrapolating data across species, the rat model may be used to study events involved in the pathogenesis and pathophysiologies of endometriosis or novel therapeutic approaches for this disorder that are not accessible in humans. Rat endometriotic tissues are similar to human lesions in vivo, and rat endometriotic tissues and cells perform in a similar manner as human endometriotic tissues and cells in organ explant culture and isolated cell culture. The rat model permits studies of mechanisms and regulators in a controlled manner free from confounding influences such as individual patient variation and environmental influences. The primary method used for induction of endometriosis in rats has been autotransplantation of uterine squares (implants) into the peritoneal cavity. Beyond mere growth of endometrium in ectopic locations, rats with endometriosis display similar symptoms, including a reduction in fertility and fecundity, and the endometriotic implants react similarly to therapeutics as those of humans with the disease. Similar alterations in gene expression and protein production have been observed in endometriotic tissues from rats and humans that may, in part, be causative agents involved in the pathogenesis or pathophysiologies of endometriosis.

Apolipoprotein B48 glycosylation in abetalipoproteinemia and Anderson's disease

Background & Aims: Abetalipoproteinemia and Anderson's disease are hereditary lipid malabsorption syndromes. In abetalipoproteinemia, lipoprotein assembly is defective because of mutations in the microsomal triglyceride transfer protein. Here, we evaluated the intracellular transport of apolipoprotein B48 to localize the defect in Anderson's disease. Methods: Asparagine-linked oligosaccharide processing of apolipoprotein B48 in normal and affected individuals was determined by the endoglycosidase H and F sensitivities of the protein after metabolic labeling of intestinal explants in organ culture. Cell ultrastructure was evaluated with electron microscopy. Results: In Anderson's disease as in normal individuals, there was a time-dependent transformation of high mannose endoglycosidase H–sensitive oligosaccharides, of endoplasmic reticulum origin, to complex endoglycosidase H–resistant oligosaccharides, added in the Golgi network. In contrast, despite the translocation of apolipoprotein B48 into the endoplasmic reticulum in patients with abetalipoproteinemia and in biopsies treated with Brefeldin A, which blocks anterograde transport between the endoplasmic reticulum and the Golgi network, there was no transformation of endoglycosidase H–sensitive oligosaccharides. Conclusions: In abetalipoproteinemia and Anderson's disease, apolipoprotein B48 is completely translocated into the endoplasmic reticulum, but only in Anderson's disease is the protein transported to the Golgi apparatus. This suggests that Anderson's disease is caused by a post-Golgi cargo-specific secretion defect.GASTROENTEROLOGY 2001;121:1101-1108

Intraembryonic, but Not Yolk Sac Hematopoietic Precursors, Isolated before Circulation, Provide Long-Term Multilineage Reconstitution

The relative contribution of yolk sac and intraembryonic precursors to hematopoiesis has been a matter of long-standing controversy. As reconstitution activity has so far only been found in embryonic tissues after the onset of circulation, the origin of reconstituting cells could not be formally established. Here, we separated yolk sac and intraembryonic splanchnopleura prior to circulation and maintained the explants in organ culture before transfer. Precursors derived from the intraembryonic site generated multilineage hematopoietic progeny in adult mice for more than 6 months. Yolk sac cells only provided myeloid short-term reconstitution. The results reveal a differential hematopoietic capacity of precirculation embryonic tissues in vivo, and indicate that the only cells capable of adult long-term hematopoiesis are of intraembryonic origin.

Interleukin-10 inhibition of gelatinases in fetal membranes: therapeutic implications in preterm premature rupture of membranes

OBJECTIVE: To examine the effect of interleukin-10 on production and regulation of gelatinases by amniochorion in an in vitro model of infection. METHODS: We placed amniochorionic membranes collected from eight women who had elective repeat cesareans at term in an organ explant culture system. After 48 hours in culture, the membranes were stimulated with lipopolysaccharide (50 ng/mL), and some were costimulated with interleukin-10 (500 ng/mL). Tissue and media samples were collected after 24-hour stimulation. Quantitative polymerase chain reactions and enzyme-linked immunosorbent assays were used to evaluate matrix metalloproteinase 2 and matrix metalloproteinase 9 messenger RNA and proteins, respectively. RESULTS: Lipopolysaccharide stimulation induced 55.14 transcripts of matrix metalloproteinase 9, compared with 0.83 in control tissues ( P < .001). Costimulation with interleukin-10 and lipopolysaccharide significantly reduced matrix metalloproteinase 9 messenger RNA levels to 10 transcripts ( P < .001). Lipopolysaccharide stimulation produced 29.25 ng/mL of immunoreactive matrix metalloproteinase 9, which was reduced to 6.3 ng/mL ( P adj = .016) after costimulation with interleukin-10. Although not significant, matrix metalloproteinase 2 messenger RNA levels were higher in lipopolysaccharide-stimulated tissues (4.37 × 10 6 transcripts) compared with control (2.8 × 10 5 transcripts; P adj = .08), with a significant decrease in matrix metalloproteinase 2 messenger RNA levels in interleukin-10- costimulated tissues (2.9 × 10 6; P adj = .007). Interleukin-10 costimulation resulted in a significant decrease in matrix metalloproteinase 2 protein production (203.1 [lipopolysaccharide] and 149.75 [with interleukin-10]; P adj < .001). CONCLUSION: Interleukin-10 eliminated lipopolysaccharide induction of matrix metalloproteinase 2 and 9 in amniochorion.

NSAID gastropathy: prevention by celery seed extracts in disease-stressed rats

Previous studies showed that some anti-inflammatory celery seed extracts (CSEs) were not gastrotoxic, in contrast to many OTC NSAIDs, when dosed to arthritic rats. The present investigation was designed to quantify the potential activity of CSEs against NSAID injury in rats with severe acute or chronic inflammation and to define the possible relationship of this to effects on mucosal prostaglandin production. Oral doses of alcoholic (A-CSE) (150-300 mg/kg) and supercritical fluid (S-CSE)(20-50 mg/kg) extracts of seeds of wild celery Apium graveolens from north India (Beagle Int. Nerang, Qld.) profoundly suppressed gastric injury elicited in disease-stressed female rats (Wistar, DA) with (a) chronic arthritic inflammation or (b) severe acute inflammation (from oleyl alcohol, 0.1 ml in tail base), fasted overnight and then dosed either (i) orally with ibuprofen (50 mg/kg), sodium naproxen (27.5 mg/kg), ketoprofen (5 mg/kg) or acidic ethanol (150 mg/kg); or (ii) parenterally with piroxicam (5 mg/kg) or nabumetone (100 mg/kg). By contrast several conventional gastroprotectants, e.g. sucralfate, cimetidine, bismuth salts, all given orally were ineffective in preventing gastric injury from parenteral piroxicam. Gastroprotection by CSEs was not over-ridden by co-dosing with isotonic HCl. Most other celery seed 'oils' were ineffective in these assays. A-CSE was found to have marked inhibitory effects on PGE2 production by porcine gastric (fundic) mucosal explants in organ culture. Quercetin and mycrecetin which are reported components of some CSEs also inhibited PGE2 production in concentrations of 10 μM, whereas limonene, another reported component of CSEs had little effect at the same concentration. These results suggest that gastroprotective effects of CSEs are probably mediated through non-prostaglandin mechanisms.

Pulmonary Hypoplasia in Mice Lacking Tumor Necrosis Factor-α Converting Enzyme Indicates an Indispensable Role for Cell Surface Protein Shedding during Embryonic Lung Branching Morphogenesis

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor-α converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-α (TNF-α). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a “sheddase” during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-α or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development.

CFU-S11 activity does not localize solely with the aorta in the aorta-gonad-mesonephros region

AbstractThe aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site in the midgestation mouse conceptus and first contains colony-forming units–spleen day 11 (CFU-S11) at embryonic day 10 (E10). Because CFU-S11 activity is present in the AGM region before the onset of hematopoietic stem cell (HSC) activity, CFU-S11 activity in the complex developing vascular and urogenital regions of the AGM was localized. From E10 onward, CFU-S11 activity is associated with the aortic vasculature, and is found also in the urogenital ridges (UGRs). Together with data obtained from organ explant cultures, in which up to a 16-fold increase in CFU-S11 activity was observed, it was determined that CFU-S11 can be increased autonomously both in vascular sites and in UGRs. Furthermore, CFU-S11 activity is present in vitelline and umbilical vessels. This, together with the presence of CFU-S11 in the UGRs 2 days before HSC activity, suggests both temporally and spatially distinct emergent sources of CFU-S11.