Retinoic acid synthesis for the developing telencephalon.

The small lipid retinoic acid is known to promote neuronal differentiation in vitro and to act as a teratogen in the embryonic brain, but very little is known about the natural role of endogenously synthesized retinoic acid in forebrain development. Retinoic acid is synthesized mainly by three retinaldehyde dehydrogenases. We show here where the retinaldehyde dehydrogenases for the developing telencephalon are expressed and how their expression patterns change over developmental time. Retinoic acid diffusing from the retinaldehyde dehydrogenase sites is likely to influence the early telencephalon before the beginning of neurogenesis, as well as differentiation and radial migration of neurons into the cerebral cortex. Because of its diffusible character, retinoic acid represents a unique tool for the coordination of growth processes over an intermediate distance range in the developing telencephalon.     

Retinoic acid fails to reverse emphysema in adult mouse models

BACKGROUND: Previous work has shown that all-trans-retinoic acid reverses elastase induced emphysema in rats. Since there is currently no effective treatment for pulmonary emphysema, the effect of retinoic acid should be further investigated in other adult species. A study was undertaken using two murine models of emphysema to evaluate the effect of retinoic acid. METHODS: The models used were an elastase induced emphysema model for acute alveolar destruction and a tumour necrosis factor (TNF)-alpha transgenic mouse which exhibits chronic air space enlargement, loss of elastic recoil, increased lung volume, and pulmonary hypertension comparable to human pulmonary emphysema. All-trans-retinoic acid (2 mg/kg) was injected for 12 successive days after the establishment of emphysema. The effects of treatment were evaluated using physiological and morphometric analyses. RESULTS: In contrast to the rat, administration of all-trans-retinoic acid in these murine models did not improve the emphysema. Moreover, worsening of emphysema was observed in TNF-alpha transgenic mice treated with all-trans-retinoic acid. The level of keratinocyte chemoattractant (KC), a CXC chemokine, in bronchoalveolar lavage fluid was increased in TNF-alpha transgenic mice following retinoic acid treatment. These data raise the possibility that retinoic acid causes deterioration of emphysema by promoting inflammation in this model. CONCLUSIONS: In these models, retinoic acid did not show positive effects on emphysema. The effect of retinoic acid in the treatment of pulmonary emphysema remains controversial, and further studies are required to determine its physiological effects under a variety of experimental conditions.     

Retinoic acid induces Pdx1-positive endoderm in differentiating mouse embryonic stem cells

We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene (Pdx1). Analysis of chimeric embryos derived from blastocyst injection of Pdx1(GFP/w) ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1(+) pancreatic endoderm. RA was most effective at inducing Pdx1 expression when added to cultures at day 4 of ES differentiation, a period corresponding to the end of gastrulation in the embryo. RT-PCR analysis showed that Pdx1-positive cells from day 8 cultures expressed the early endoderm markers Ptf1a, Foxa2, Hnf4alpha, Hnf1beta, and Hnf6, consistent with the notion that they corresponded to the early pancreatic endoderm present in the embryonic day 9.5 mouse embryo. These results demonstrate the utility of Pdx1(GFP/w) ES cells as a tool for monitoring the effects of factors that influence pancreatic differentiation from ES cells.     

Layer-specific production of nitric oxide during cortical circuit formation in postnatal mouse brain

In the developing cerebral cortex, neuronal nitric oxide synthase (nNOS) is expressed abundantly, but temporarily. During the early postnatal stage, cortical neurons located in the multi-layered structure of the cortical plate start forming well-organized cortical circuits, but little is known about the molecular machinery for layer-specific circuit formation. To address the involvement of nitric oxide (NO), we utilized a new NO indicator (DAR-4M) and developed a protocol for the real-time imaging of NO produced in fresh cortical slices upon N-methyl-D-aspartic acid stimulation. At postnatal day 0 (P0), NO production was restricted to the deep layers (layers V and VI) of the somatosensory cortex where transient synapses are formed. At P10, the production of NO was expanded to layer IV where large numbers of thalamocortical axons form synapses. The pattern of NO production could correspond to active sites for synaptic formation. This study is the first clear demonstration of NO production in the postnatal mouse neocortex. The findings presented may reflect a function of NO in relation to the layer-specific development of neural circuits in the neocortex.     

Neurogenesis in the postnatal human epileptic brain

OBJECT: The normal adult human telencephalon does not reveal evidence of spontaneous neuronal migration and differentiation despite the robust germinal capacity of the subventricular zone (SVZ) astrocyte ribbon that contains neural stem cells. This might be because it is averse to accepting new neurons into an established neuronal network, probably representing an evolutionary acquisition to prevent the formation of anomalous neuronal circuits. Some forms of epilepsy, such as malformations of cortical development, are thought to be due to abnormal corticogenesis during the embryonic and early postnatal periods. The role of postnatal architectural reorganization and possibly postnatal neurogenesis in some forms of epilepsy in humans remains unknown. In this study the authors used resected specimens of epileptic brain to determine whether neurogenesis could occur in the diseased tissue. METHODS: The authors studied freshly resected brain tissue obtained in 47 patients who underwent neurosurgical procedures and four autopsies. Forty-four samples were harvested in patients who underwent resection for the treatment of pharmacoresistant epilepsy. RESULTS: Using organotypic brain slice preparations cultured with 5-bromodeoxyuridine (a marker for cell proliferation), immunohistochemistry, and cell trackers, the authors demonstrate the presence of spontaneous cell proliferation, migration, and neuronal differentiation in the adult human telencephalon that starts in the SVZ and progresses to the adjacent white matter and neocortex in human neocortical pathological structures associated with epilepsy. No cell migration or neuronal differentiation was found in the control group. CONCLUSIONS: The presence of spontaneous neurogenesis associated with some forms of human neocortical epilepsy may represent an erroneous and maladaptive mechanism for neuronal circuitry repair, or it may be an intrinsic part of the pathogenic process.     

Retinoic acid responsiveness of cells and tissues in developing fetal limbs evaluated in a RAREhsplacZ transgenic mouse model

Limb morphogenesis is a complex phenomenon in which retinoids play an important role. Abnormal maternal retinoid levels from high oral doses cause fetal malformations, including abnormalities of the musculoskeletal system. Our purpose was to identify the retinoid-responsive cells in bone and cartilage during limb development by using a transgenic line of mice containing a reporter gene insert consisting of a retinoic acid response element linked to an Escherichia coli β-galactosidase gene. Transgenic fetuses from day 11.5 after conception to birth (day 20) were analyzed histologically. Retinoid-responsive cells and tissues were first seen in the limb bud at 12.5 days in the webs between the forming digits. The webs stained maximally at 14.5 days, after which staining intensity subsided. Staining in the muscles was detectable at 13.5 days, at a stage coinciding with myoblast fusion. Specific regions of perichondrium and periosteum also stained at this Stage. Occasional staining was observed in individual chondroblasts in all chondrogenic regions, including hypertrophic chondroblasts and certain articular surfaces of developing joints. Staining of these tissues decreased in intensity in subsequent stages. Osteoclasts started to express β-galactosidase at 15.5 days and continued to stain into maturity. Our results indicate that specific subsets of cells respond to retinoids at specific stages in the course of normal limb development. In hypertrophic chondrocytes and cells in the webs and joints that display such a response, retinoid-induced effects may be linked to cell death that occurs in these regions. Staining in muscle, perichondrium, and periosteum may reflect retinoid-induced effects associated with cell differentiation and growth. These results suggest that retinoids play a role in a variety of tissues, including bone and cartilage, at specific stages during morphogenesis.     

Retinoic acid and glucocorticoids enhance the effect of 1,25-dihydroxyvitamin D3 on bone gamma-carboxyglutamic acid protein synthesis by rat osteosarcoma cells

Two 1,25-dihydroxyvitamin D3-controlled parameters in the osteoblastlike osteosarcoma cell line ROS 17/2, bone gamma-carboxyglutamic acid-containing protein (BGP) and collagen synthesis, were measured after pretreatments with either retinoic acid (RA), or triamcinolone acetate (TRM). RA and TRM both caused double the expected increase in BGP secretion at 16 hr after treatment with 1,25-dihydroxyvitamin D3. Triamcinolone acetate concentrations of 10(-8) and 10(-9) M or 10(-6) M retinoic acid were effective in enhancing the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion. Treatment with RA or TRM alone did not stimulate BGP secretion. RA alone had no effect on BGP secretion, while TRM inhibited BGP secretion. Collagen synthesis is inhibited by 1,25-dihydroxyvitamin D3. Neither retinoic acid nor triamcinolone acetate enhanced the 1,25-dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Retinoic acid by itself inhibited collagen synthesis but did not change the 1,25 dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Triamcinolone acetate by itself or together with 1,25-dihydroxyvitamin D3 increased collagen synthesis. We conclude that, although both triamcinolone acetate and retinoic acid increase the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion by ROS 17/2 cells, they have different effects on the regulation of collagen production. Thus, although both hormones increase the 1,25-dihydroxyvitamin D3 receptor concentration in these cells, their actions are not mediated solely by this mechanism.     

Effects of halothane on the synthesis of neurotransmitter amino acids in mouse brain

The effects of halothane on the synthesis of the three major neuroactive amino acids (θ–aminobutyric acid, aspartate and glutamate) and glutamine, which is closely related metabolically, were investigated in mouse brain using a labelled precursor ([¹³C]glucose) and a gas chromatography–mass spectrometry system. The ratios of newly synthesised amino acids were increased relative to baseline values when animals were exposed to 1% halothane, and decreased when they were exposed to 2% halothane. These findings suggest that halothane affects the synthesis of neurotransmitter amino acids in a concentration–dependent manner, without discrimination between excitatory and inhibitory amino acids.     

Endogenous GFAP-positive neural stem/progenitor cells in the postnatal mouse cortex are activated following traumatic brain injury

Interest in promoting regeneration of the injured nervous system has recently turned toward the use of endogenous stem cells. Elucidating cues involved in driving these precursor cells out of quiescence following injury, and the signals that drive them toward neuronal and glial lineages, will help to harness these cells for repair. Using a biomechanically validated in vitro organotypic stretch injury model, cortico-hippocampal slices from postnatal mice were cultured and a stretch injury equivalent to a severe traumatic brain injury (TBI) applied. In uninjured cortex, proliferative potential under in vitro conditions is virtually absent in older slices (equivalent postnatal day 15 compared to 8). However, following a severe stretch injury, this potential is restored in injured outer cortex. Using slices from mice expressing a fluorescent reporter on the human glial fibrillary acidic protein (GFAP) promoter, we show that GFAP+ cells account for the majority of proliferating neurospheres formed, and that these cells are likely to arise from the cortical parenchyma and not from the subventricular zone. Moreover, we provide evidence for a correlation between upregulation of sonic hedgehog signaling, a pathway known to regulate stem cell proliferation, and this restoration of regenerative potential following TBI. Our results indicate that a source of quiescent endogenous stem cells residing in the cortex and subcortical tissue proliferate in vitro following TBI. Moreover, these proliferating cells are multipotent and are derived mostly from GFAP-expressing cells. This raises the possibility of using this endogenous source of stem cells for repair following TBI.     

Retinoic acid binding protein in normal and neoplastic rat prostate

Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (³H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen FI rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5a reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.     

Retinoic acid affects the cell cycle and increases total protein content in epithelial cells

Retinoids and particularly retinoic acid (RA) have been incriminated in the adaptation to uninephrectomy and compensatory kidney growth in humans. However, there is no data assessing the effects of RA on renal cells. Since the bulk of the compensatory kidney growth is due to tubular cells, we studied the effects of RA, retinol and epidermal growth factor (EGF) on a rabbit kidney epithelial cell line RK13 in culture. RA significantly increased thymidine incorporation by 42 +/- 8% (P less than 0.01). This increase appeared as soon as three hours after adding RA and could still be observed after five days. Total protein content was also increased by RA by 37 +/- 4% (P less than 0.01). Flow cytometer analysis showed a significant decrease in the percentage of resting cells (G0-G1 phases) induced by RA (-9.4 +/- 2%; P less than 0.01). We observed similar results in growth factor free medium, and the RA induced changes were the same in confluent and non-confluent cells. Retinol did not modify thymidine incorporation or total protein content. EGF increased by 75% thymidine incorporation (P less than 0.01). In serum free conditions RA failed to have a synergistic effect with EGF. These data show that RA is able to induce modifications in kidney epithelial cells compatible with those observed in hypertrophy while retinol is not. These modifications are not due to other growth factor potentiation but to RA itself, and are independent of the contact-inhibition phenomenon.     

SET在不同发育阶段小鼠睾丸的表达及其功能

SET蛋白是一个多功能蛋白,在调节包括DNA复制、核小体装配、染色体修饰、DNA转录、细胞周期、细胞凋亡等细胞生物学过程中起重要作用。前期研究发现,SET在卵巢中调节雄激素合成。然而,SET在睾丸组织中的表达及其功能仍然不明确。在此,我们检测不同年龄段小鼠睾丸组织中SET表达,探讨其在精子发生及睾酮生成方面的潜在功能。48只不同年龄段的雄性小鼠（1周龄的ICR雄性小鼠作为幼年期组,4周龄小鼠作为性发育前组,12周龄小鼠作为性成熟期组,12月龄作为老年组）。免疫组织化学方法观察SET各年龄段小鼠的定位表达;qRT-PCR和Western Blot分别检测睾丸中SET mRNA和蛋白水平表达。SET表达定位于生精小管中的精原细胞、精母细胞,在青春期前及成熟期的单倍体和四倍体生殖细胞中高表达;成熟期及老年期的睾丸间质细胞中也有SET的表达;支持细胞中很少量表达。青春期前SET的mRNA与成熟期相比表达量最高（P〈0．05）,而SET蛋白在性成熟期小鼠睾丸中表达最高（P〈O．05）。SET主要表达于精原细胞和精母细胞,少量表达于支持细胞,表明SET可能与精子发生有关。SET还表达于睾丸间质细胞,则与睾酮生成有关。     

The developmental nephrome: systems biology in the developing kidney

PURPOSE OF REVIEW: A set of important genes and signaling pathways involved in kidney development is emerging from analyses of mutant mice, in-vitro models, and global gene expression patterns. Conversion of data into dynamic models or networks through the synthesis of information at multiple levels is crucial for a better understanding of kidney development. RECENT FINDINGS: Genetic and in-vitro evidence is beginning to provide a limited sense of the network topology in stages of kidney development. Intriguing data from other fields suggest how, with the aid of large-scale gene expression studies, these stages might be represented as dynamic attractor states. It is also suggested how branching morphogenesis of the epithelial ureteric bud may be sustained by an autocatalytic set of proteins whose interactions lead to repeated rounds of tip and stalk generation. Accumulating data in lower organisms suggest network topologies may be quite flexible, and the implications of these results for varieties of tubulogenesis and renal regeneration after acute injury are discussed. SUMMARY: Currently it may be feasible to build tentative dynamic multistage models of nephrogenesis that facilitate experimental thinking. As data accumulate, it may become possible to test their predictive value.     

Epidermal growth factor in the mouse kidney: developmental changes and intranephron localizations

The developmental changes in epidermal growth factor (EGF) have been studied in tissue homogenates, kidney slices, and microdissected nephron segments of the mouse. Immunoreactive EGF concentration per milligram of protein increased in the kidney by about 20-fold from 1 week to 3 weeks of life, reaching the highest levels between 5 and 7 weeks, and decreasing by 10 weeks of life. The time course of the changes was different from those of submaxillary and urinary EGF. Above 7 weeks of age, kidney EGF was higher in female than in male mice. Among various zones of the kidney (outer cortex, inner cortex, outer medulla I, II and papilla), the outer medulla I and II contained the highest quantities of EGF per gram of wet tissue. The highest EGF content per millimeter length was observed in the medullary and cortical thick ascending limbs of Henle's loop. The amounts exceeded by about 4.5-fold those found in glomeruli and in proximal convoluted and proximal straight tubules, and by about 3-fold those present in distal and collecting tubules. Unilateral nephrectomy resulted in no significant changes in EGF levels in the contralateral kidney. The results suggest that the ontogeny of kidney EGF is different from that of the EGF found in the submaxillary gland, and that there is nephron heterogeneity in EGF content.