12-day Embryos Research Articles

The origin of bmp16, a novel Bmp2/4relative, retained in teleost fish genomes

BackgroundWhole genome sequences have allowed us to have an overview of the evolution of gene repertoires. The target of the present study, the TGFβ superfamily, contains many genes involved in vertebrate development, and provides an ideal system to explore the relationships between evolution of gene repertoires and that of developmental programs.ResultsAs a result of a bioinformatic survey of sequenced vertebrate genomes, we identified an uncharacterized member of the TGFβ superfamily, designated bmp16, which is confined to teleost fish species. Our molecular phylogenetic study revealed a high affinity of bmp16 to the Bmp2/4 subfamily. Importantly, further analyses based on the maximum-likelihood method unambiguously ruled out the possibility that this teleost-specific gene is a product of teleost-specific genome duplication. This suggests that the absence of a bmp16 ortholog in tetrapods is due to a secondary loss. In situ hybridization showed embryonic expression of the zebrafish bmp16 in the developing swim bladder, heart, tail bud, and ectoderm of pectoral and median fin folds in pharyngula stages, as well as gut-associated expression in 5-day embryos.ConclusionComparisons of expression patterns revealed (1) the redundancy of bmp16 expression with its homologs in presumably plesiomorphic expression domains, such as the fin fold, heart, and tail bud, which might have permitted its loss in the tetrapod lineage, and (2) the loss of craniofacial expression and gain of swim bladder expression of bmp16 after the gene duplication between Bmp2, -4 and -16. Our findings highlight the importance of documenting secondary changes of gene repertoires and expression patterns in other gene families.

Dopamine D4 Receptors Regulate GABAA Receptor Trafficking via an Actin/Cofilin/Myosin-dependent Mechanism

The GABA(A) receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABA(A)R current is subject to the regulation of dopamine D(4) receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D(4) modulation of GABA(A)Rs. We found that the density of surface clusters of GABA(A)R beta2/3 subunits was reduced by D(4), suggesting that the D(4) reduction of GABA(A)R current is associated with a decrease in functional GABA(A)Rs at the plasma membrane. Moreover, the D(4) reduction of GABA(A)R current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D(4) regulates GABA(A)R trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser(3), provides the possible link between D(4) signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D(4) regulation of GABA(A)R trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D(4) reduction of GABA(A)R current. These results suggest that D(4) receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABA(A)R clusters to the surface.

Cytogenetics of preimplantation embryos sired by bulls heterozygous for the 1/29 translocation.

Eighty-eight embryos aged 1 to 15 days sired by bulls heterozygous for the 1/29 chromosome translocation and 28 embryos aged 7 to 15 days sired by a normal bull were chromosomally analysed. All donor females had normal karyotypes. Only 47.4% of the embryos could be karyotyped. Two embryos sired by a heterozygous bull were trisomic. One 2-celled embryo was a 2n/ln mosaic and one 1-celled egg was polyspermic. Both of these were collected from superovulated donors. The mean cell number and mitotic index for 7-day embryos sired by heterozygous bulls were 72 and 3.73%, respectively, and sired by normal bulls, 81 and 5.74%, respectively.

Histopathological and Ultrastructural Studies on the Effects of Electromagnetic Fields on the Liver of Preincubated White Leghorn Chicken Embryo

There are several reports indicating a linkage between exposures to 50–60 Hz electromagnetic fields and abnormalities in the early stages of embryonic development of chicken embryos. The present study was designed to demonstrate whether electromagnetic fields could be an environmental factor invoking histopathological and ultra-structural changes in livers of preincubated chicken embryos exposed to EMFs. Following other researchers and our previous results from different groups of Developmental Biology at the Animal Sciences, Faculty of Biological Sciences, Shahid-Beheshti University, effects of most effective intensities (1.33, 2.66, 5.52, and 7.32 mT) of electromagnetic fields (EMFs, 50 Hz ) on livers of pre-incubated white leghorn chicken embryos were investigated . 150 healthy, fresh, and fertilized eggs (55–65 gr) were divided into 6 groups of experimental(1–4, n = 30), control (n = 60), and sham (n = 50). Experimental eggs (inside coil) were exposed to 4 different intensities (1.33, 2.66, 5.52, and 7.32 mT). Sham groups were located inside same coil, with no exposure, for 24 h before incubation. Control, sham, and experimental groups (1–4) were then incubated in an incubator (38 ± 0.5°C, 60% humidity) for 17 days. At the end of this period, livers of experimental, sham, and control groups were processed for light and transmission electrom microscopes (TEM and SEM) studies. So, livers of 17-day old chicken embryos were removed by C-sections, fixed in formalin 10%, stained with H&E and reticulin, and studied under light microscope. Others were prepared for electron microscopes (TEM and SEM) investigations. Morphological observations indicated exencephalic embryos, embryos with asymmetrical faces, crossed beak, shorter upper beak, deformed hind limbs, gastroschesis, anophthalmia, and microphthalmia. H&E and reticulin stainings, TEMS, and SEMs studies indicated EMFs would create hepato-cytes with fibrotic bands, severe steatohepatitis, vacuolizations, swollen and extremely electron-dense mitochondria, reduced invisible cristae, crystalized mitochondria with degenerated cristae, myelin-like figures, macrophages engulfing adjacent cells, dentated nuclei, nuclei with irregular envelopes, degenerated hepatocytes, abnormal lipid accumulations, lipid droplets pushing hepatocytes' nuclei to the corner of the cells, abundant cellular infiltrations cellular infiltrations inside sinusoid and around central veins, disrupted reticulin plexus, and release of chromatin into cytosol,, with partially regular water layers. An elevated oxyradical generation and, subsequentely, cell membrane disruptions were the reasons for electromagnetic fields inducing cell damages.

New Aspects for the Treatment of Cardiac Diseases Based on the Diversity of Functional Controls on Cardiac Muscles: Diversity in the Excitation–Contraction Mechanisms of the Heart

The waveform of the myocardial action potential (AP) triggering contraction differs among the species, developmental stage, and pathological state. The species difference in heart rate, which inversely correlates with body size, originates in the ion-channel mechanisms responsible for diastolic depolarization of the sinoatrial node. In some cases, such as the chronically AV-blocked dog and 11- to 13-day chick embryo, the repolarization reserve is decreased making the heart useful for drug evaluation. The degree of dependence of contraction on sarcoplasmic reticulum (SR) function increases during development. The large SR dependence and short AP of the adult mouse and rat support their rapid contraction under high heart rate. The function of the Na+/Ca2+ exchanger is affected by AP waveform and ion concentrations; its major role is Ca2+ extrusion, but under pathological conditions such as ischemia-reperfusion, it allows Ca2+ influx and leads to myocardial injury, including loss of mitochondrial function. The role of mitochondria in ATP supply is less in the fetus where glycolysis plays a greater role. The pharmacological properties of the myocardium are affected by all of these factors and also by autonomic innervation and the hormonal status. Such comprehensive understanding is indispensable for the development of novel therapeutic strategies.

The Chromatin of Active Genes Is Not in a Permanently Open Conformation

Quantitative measurements of local chromatin accessibility to DNase I in 15-day chicken embryo erythrocyte nuclei have been performed using a range of nuclease concentrations and real-time TaqMan PCR to monitor the loss of short (∼80 bp) amplicons. At the β-globin locus, well-established DNase I hypersensitive sites stand out against a background in which actively transcribed gene sequences (e.g., β-adult and β-hatching) are no more sensitive than the nearby constitutive heterochromatin that has previously been shown to form the 30-nm fibre structure. Similar observations were made at the lysozyme locus containing the active Gas41 gene and also at the GAPDH locus. We conclude that active genes are not continuously held in an open ‘beads-on-a-string’ configuration, but adopt a 30-nm-type structure most of the time. This implies that the compact nucleosomal supercoil re-forms in the wake of the polymerase complex.

New Histopathological Experimental Model for Benign Prostatic Hyperplasia: Stromal Hyperplasia in Rats

Histological observations of clinical benign prostatic hyperplasia specimens show that benign prostatic hyperplasia tissue is mainly composed of stromal components, smooth muscle and fibrous tissue, so-called stromal hyperplasia. However, little is understood regarding the pathogenesis of this stromal hyperplasia due to no suitable stromal hyperplasia model to elucidate the pathology of benign prostatic hyperplasia. We created a novel model of benign prostatic hyperplasia accompanied by clinically relevant stromal hyperplasia. The urogenital sinus isolated from male rat 20-day embryos was implanted into pubertal male rat ventral prostates. Two to 8 weeks after the operation the implanted urogenital sinus was isolated, weighed and subjected to histochemical analysis. To distinguish between and characterize the epithelial and stromal components we stained for collagen, smooth muscle components, growth factors and proliferating cell nuclear antigen. In addition, to determine whether the implanted urogenital sinus had differentiated into functional prostate we stained for androgen receptor and dorsolateral prostatic secretory protein. Urogenital sinuses removed from male rat 20-day embryos initially weighed approximately 1 mg. After implantation into host rat ventral prostates they grew in time dependent fashion with no apparent change in the original ventral prostate weight in the host rat. Implanted urogenital sinus weight was more than 100 mg 3 weeks after implantation. Histological observation demonstrated that the ratio of stromal to total area was approximately 70%, which was much higher than that in age matched rat ventral prostates and in a testosterone induced epithelial hyperplasia model (approximately 20% and 15%, respectively). This predominantly stromal tissue composition was maintained up to 8 weeks after implantation. Proliferating cell nuclear antigen staining revealed that the ratio of proliferating cells in stroma was equal to or greater than that in epithelium. In this model the antiandrogen agent chlormadinone acetate (Wako Pure Chemicals Industries, Osaka, Japan) at a dose of 10 mg/kg prevented the increase in implanted urogenital sinus weight (19.1%) but its potency was less than that seen in the testosterone induced epithelial hyperplasia model, that is 93.4% at the 10 mg/kg dose. We have established a new experimental stromal hyperplasia model corresponding to clinical benign prostatic hyperplasia in terms of the composition of stromal components and functional differentiation of the prostate. Furthermore, the localization and time course of growth factor expression were also similar to those in men with benign prostatic hyperplasia.

Gene Expression Profiles of Neurotrophic Factors in Rat Cultured Spinal Cord Cells Under Cyclic Tensile Stress

An experimental study to investigate the in vitro gene expression of neurotrophic factors and receptors in cultured rat spinal cord cells subjected to cyclic mechanical stretch forces. We evaluated in vitro expression of neurotrophic factors and receptors in cultured rat spinal cord cells under cyclic tensile stress. Application of compressive mechanical stress to the spinal cord results in multiple changes making it difficult to examine the expression of neurotrophic factors and their receptors. There are no in vitro studies that investigated the biologic responses of cultured spinal cord cells to tensile stress. Spinal cord cells were isolated for culture from 15-day Sprague-Dawley rat embryos. We used the FX3000 Flexercell Strain Unit to induce mechanical stress. We analyzed the effects of mechanical stress on cell morphology, mRNA expression levels of various neurotrophic factors, and their immunoreactivities at 0, 2, 6, 12, 24, and 36 hours. Tensile stress for 6 hours resulted in reduction of spinal cord cells and loss of neurites. Cells that survived 24-hours stress showed swollen irregular-shaped soma, bleb formation, and fragmented neurites. The cell survival rate decreased, whereas lactate dehydrogenase release increased significantly at 6 hours. There were significant increases in mRNA expression levels of nerve growth factor, brain-derived neurotrophic factor, trkB, p75 neurotrophin receptor (p75), glial cell line-derived neurotrophic factor, and caspase-9 during the early period after application of tensile stress. Our results suggest survival of spinal cord neuronal cells under injurious tensile stress with increased synthesis and utilization of several neurotrophic factors, receptors, and expression of proteins related to cell apoptosis.

Primary chondrification foci in the wing basipodium of Struthio camelus with comments on interpretation of autopodial elements in Crocodilia and Aves

The present analysis consists of (1) description of the primary chondrification patterns and their transformation into ossified elements in the basipodium of Struthio camelus; (2) comparison of these with the conditions found in Alligator and Gallus; and (3) interpretation of the autopodial elements of Archaeopteryx. (1) The existence of five discrete metacarpal condensations in the 16-day embryo of Struthio argues for unique linear patterning process for each, and these are interpreted as digits 2,3,4 originating from metacarpal condensations 2,3,4. Nine chondrogenic foci appear in the Struthio carpus: radiale, centrale, intermedium, ulnare, pseudoulnare, pisiform, distal carpal 2+3, distal carpal 4, and distal carpal 5. It is evident that: (a) the avian "radiale" represents fused chondrogenic foci of the intermedium plus the radiale; (b) a neomorph carpal element, the pseudoulnare (probably avian autapomorphy), replaces the ulnare cartilage in Struthio; (c) the pseudoulnare in Struthio and Hinchliffe's element "X" are not identical to each other. (2) Spatio-temporal conditions of the autopodium are less constrained in the development of Struthio than they are in Gallus. This favors the ostrich model as the more appropriate for interpretation of the autopodial skeleton in the oldest birds and their ancestors. (3) An interpretation of the elements of the hand skeleton of Archaeopteryx is as follows: (a) digits 2,3,4; (b) distal carpal 2+3 (the semilunate); (c) distal carpal 4 (a missing element filling the gap between the semilunate and metacarpal IV); (d) the radiale+intermedium complex (the proximal carpal bone); (e) the pseudoulnare (the proximal carpal bone).

Comparison of mRNA expression of transcriptional factors and intercalated disk constituent proteins between in vivo and cultured cardiomyocytes

The weak contractile force exerted by engineered cardiac muscle is a big problem in cardiac muscle tissue engineering, even though the field has made great progress over the past decade. We believe that one major reason for the weak contractile force is that the expression of genes regulating cardiomyocyte differentiation and cardiac tissue syncytium may be different for in vivo and cultured cells. In the present study, we investigated the difference of mRNA expression under in vivo and culture conditions in order to seek a target for further gene transfer treatment in the process of cardiac tissue construction. To this end, mRNA expression of four major transcriptional factors (SRF, p300, Nkx2.5, and myocardin) and two intercalated disk constituent proteins (N-cadherin and connexin43) in rat cardiomyocytes was measured by means of ratiometric reverse-transcription polymerase chain reaction. Cardiomyocytes were harvested from the hearts of 18-day (about 3 days before birth) Wistar-rat embryos (embryonic cells), 12-day neonatal rat hearts (neonatal cells), or 14-day successive dish culture of the embryonic cells harvested from 18-day embryos (cultured cells). The results indicated that, except for SRF, the mRNAs had a lower expression tendency in cultured cells than in embryonic and in neonatal cells; in particular, the mRNA expression of myocardin, N-cadherin, and connexin43 of cultured cardiomyocytes was significantly lower than that of neonatal cells. Therefore, myocardin is a candidate for forced gene up-expression during the construction of engineered cardiac tissue; in addition, a plausible reason for the weak contractile force of engineered cardiac tissue is the weak constitution of intercalated disk, because it was elucidated that mRNA expression of proteins related to intercalated disk were lower in culture.

Cardiac myofibrillogenesis inside intact embryonic hearts

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 μm filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.

Systolic and diastolic ventricular function in zebrafish embryos: Influence of norepenephrine, MS-222 and temperature

BackgroundZebrafish are increasingly used to study the influences of gene mutation and manipulation on cardiac development, structure and function. In this study, a video edge detection system was used to characterise, continuously, cardiac ventricle function in 2–5 days old zebrafish embryos embedded in 0.6% agar and examined under light microscopy at room temperature (22°C). Using video edge detection software (IonOptix Inc), the motion of a small region of the cardiac ventricle wall was converted to a continuous chart trace allowing analysis of wall motion amplitude (WMA) and myocardial wall velocity during systole (MWVs) and diastole (MWVd).ResultsCardiac wall motion characteristics changed progressively from day 2 to 5 (WMA, 2-days, 17.6 ± 4.4 μm vs 5-days, 24.6 ± 4.7 μm, p < 0.01). MWVd was more rapid than MWVs at all developmental time points. Embryonic hearts were also assessed after increasing concentrations of norepenephrine (NE) and the anaesthetic agent MS222 (tricaine) were added to the bathing water. In response to NE, WMA increased significantly more in 4 day embryos compared with 2 day embryos (change in WMA,13.6 ± 8.2 μm vs 4.0 ± 8.8 μm, p = 0.01, respectively) while the decrease in WMA in response to MS222 was similar in both 2 and 4-day embryos. Heart rate, MWVs and MWVd were significantly higher at 28°C compared with 22°C. No differences in cardiac function were observed between AB and Golden strains.ConclusionVideo edge detection appears sufficiently sensitive to detect subtle changes in diastolic and systolic cardiac function during development and changes resulting from pharmacological and environmental interventions. Such measurements could be valuable in assessment of altered cardiac function after genetic manipulation.

Ontogeny of melatonin, Per2 and E4bp4 light responsiveness in the chicken embryonic pineal gland

The chicken pineal gland possesses the capacity to generate circadian oscillations, is able to synchronize to external light:dark cycles and can generate an hormonal output — melatonin. We examined the light responses of the chicken pineal gland and its effects on melatonin and Per2, Bmal1 and E4bp4 expression in 19-day old embryos and hatchlings during the dark phase, subjective light phase and in constant darkness. Expression of Per2 and E4bp4 were rhythmic under light:dark conditions, but the rhythms of E4bp4 and Bmal1 mRNA did not persist in constant darkness in 19-day old embryos. Per2 mRNA expression persisted in constant darkness, but with a reduced amplitude. Per2 expression was inducible by light only during the subjective day. Melatonin release was inhibited by light only at end of the dark phase and during the subjective light phase in embryos. Our data demonstrate that the embryonic avian pineal pacemaker is light sensitive and can generate rhythmic output, however the effects of light were diminished in chick embryos in compared to hatchlings.

Analysis of differentiation of stem/progenitor cells in tissue culture of neocortical primordium of mouse brain

Differentiation of neural stem/progenitor cells from neocortical primordium of the brain from 14-day mouse embryos was studied by immunohistochemical methods during their culturing. Non-differentiated cells expressing nestin and vimentin persisted in freely floating neurospheres throughout the experiment. Glioblasts, neuroblasts, and differentiated neurons were found in neurospheres cultured in differentiating medium. However, neurons disappeared with increasing the number of passages, the formation of neuroblasts was terminated, and only astrocytes and nestin-positive cells were seen in the culture. It was found that cells of mouse embryonic neocortex lose the capacity for spontaneous multipotent differentiation during culturing.

Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.

Soluble HLA-G is absent from human embryo cultures: A reassessment of sHLA-G detection methods

In recent years, the number of patients receiving in vitro fertilization (IVF) has been increasing, though the rate of successful implantations has remained at 10–20%. A major goal of this procedure is to afford the ability to select embryos with the most potential for implantation and development. Previous studies claimed to have detected soluble HLA-G (sHLA-G) protein in culture supernatant from 2 to 3-day embryos using ELISA methods, and concluded that sHLA-G protein levels were associated with successful implantation. This result, if substantiated could provide an important tool for IVF. In this study, we have re-examined these experiments by attempting to detect sHLA-G in the medium from 2 to 3-day embryos (84 samples) and 4 to 6-day embryos (25 samples) in which a part of blastocyst has started to differentiate into trophoblasts. Using a highly specific and sensitive ELISA, no sHLA-G protein was detectable in any sample, despite the fact that 27 of the 109 samples were from successfully implanted embryos. These results indicate that 2–6-day embryos do not secrete sHLA-G detectable by ELISA, and therefore that sHLA-G in culture medium is not a useful for successful implantation at this stage of development.

NeuroD1: developmental expression and regulated genes in the rodent pineal gland

NeuroD1/BETA2, a member of the bHLH transcription factor family, is known to influence the fate of specific neuronal, endocrine and retinal cells. We report here that NeuroD1 mRNA is highly abundant in the developing and adult rat pineal gland. Pineal expression begins in the 17-day embryo at which time it is also detectable in other brain regions. Expression in the pineal gland increases during the embryonic period and is maintained thereafter at levels equivalent to those found in the cerebellum and retina. In contrast, NeuroD1 mRNA decreases markedly in non-cerebellar brain regions during development. Pineal NeuroD1 levels are similar during the day and night, and do not appear to be influenced by sympathetic neural input. Gene expression analysis of the pineal glands from neonatal NeuroD1 knockout mice identifies 127 transcripts that are down-regulated (>twofold, p < 0.05) and 16 that are up-regulated (>twofold, p < 0.05). According to quantitative RT-PCR, the most dramatically down-regulated gene is kinesin family member 5C ( approximately 100-fold) and the most dramatically up-regulated gene is glutamic acid decarboxylase 1 ( approximately fourfold). Other impacted transcripts encode proteins involved in differentiation, development, signal transduction and trafficking. These findings represent the first step toward elucidating the role of NeuroD1 in the rodent pinealocyte.

Development of multidrug resistance type I Cmdr1 expression in chicken embryonic gonads

A primary role of P-glycoprotein (P-gp), encoded by the multidrug resistance type I gene, is to protect against naturally occurring xenotoxics. Recently, the preferential expression of chicken multidrug resistance type I (Cmdr1) was identified in the embryonic gonads during the early periods of development. Here we investigated the expression of Cmdr1 and P-gp in the gonads during embryogenesis, and compared to that in the ovarian follicles of domestic hens (Gallus gallus). As revealed by immunohistochemistry, P-gp was highly expressed in theca cells of mature follicles, whereas the expression was low in immature follicles. Immunohistochemical analysis showed that expression of Cmdr1-type P-gp was very low in embryonic gonads. Cmdr1 mRNA was undetectable in the gonads of 5-day embryos (E5) by RT-PCR, whereas Cmdr1 mRNA was significantly detectable in the developing gonads at E9 and E21. In the testicular tissues, germ cells were distributed along developing seminiferous cords as identified by a specific marker gene, whereas Cmdr1-type P-gp positive cells were observed evenly on testicular tissues. Collectively, it is concluded that Cmdr1 expression is initiated in the chicken ovary and testis after sexual differentiation, but expression of Cmdr1-type P-gp is very low through embryogenesis.

Protein Inhibitor of Activated STAT1 Interacts with and Up-regulates Activities of the Pro-proliferative Transcription Factor Krüppel-like Factor 5

Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates proliferation of various cell types, including fibroblasts, smooth muscle cells, and intestinal epithelial cells. To identify proteins that interact with KLF5, we performed a yeast two-hybrid screen of a 17-day mouse embryo cDNA library with KLF5 as bait. The screen revealed 21 preys clustered in four groups as follows: proteins mediating gene expression, metabolism, trafficking, and signaling. Among them was protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) ligase that regulates transcription factors through SUMOylation or physical interaction. Association between PIAS1 and KLF5 was verified by co-immunoprecipitation. Structural determination showed that the acidic domain of PIAS1 bound to both the amino- and carboxyl-terminal regions of KLF5 and that this interaction was inhibited by the amino terminus of PIAS1. Indirect immunofluorescence demonstrated that PIAS1 and KLF5 co-localized to the nucleus. Furthermore, the PIAS1-KLF5 complex was co-localized with the TATA-binding protein and was enriched in RNA polymerase II foci. Transient transfection of COS-7 cells by PIAS1 and KLF5 significantly increased the steady-state protein levels of each other. Luciferase reporter and chromatin immunoprecipitation assays showed that PIAS1 significantly activated the promoters of KLF5 and PIAS1 and synergistically increased the transcriptional activity of KLF5 in activating the cyclin D1 and Cdc2 promoters. Importantly, PIAS1 increased the ability of KLF5 to enhance cell proliferation in transfected cells. These results indicate that PIAS1 is a functional partner of KLF5 and enhances the ability of KLF5 to promote proliferation.

Hydrogen peroxide mimics the responses to oxygen in chicken ductus arteriosus

Oxygen-induced vasoconstriction is an initial trigger for ductus arteriosus (DA) closure. The aim of this study was to investigate the developmental responses to O2 in chicken DA. Experiments were performed in the pulmonary and the aortic sides of the DA isolated from embryos incubated for 15 or 20 days. In 20-day embryos, the increase in O2 tension from 0 to 21% caused a contraction in rings obtained from the pulmonary side while a relaxant response was elicited in those obtained from the aortic side. In contrast, no changes were found following 21% O2 exposure in either the pulmonary or the aortic sides of the 15-day DA. The contraction to O2 was markedly prevented by the NADPH oxidase inhibitor apocynin, the mitochondrial electron transport chain inhibitor rotenone and by polyethylenglycol-catalase. Furthermore, addition of t-butyl-hydroperoxide mimicked the responses induced by 21% O2 (no effect in 15-day and contraction and relaxation in pulmonary and aortic sides of the 20-day DA, respectively). Our results are in favor of H2O2 as a possible mediator in the contractile responses to O2 in the DA. Supported by AGL2004-06685/ALI and SAF2005-03770