Retinoic acid signaling is required for proper morphogenesis of mammary gland.

Retinoic acid (RA), a bioactive chemical compound synthesized from dietary derived vitamin A, has been successfully used as a chemopreventive and chemotherapeutic agent through the regulation of cell proliferation, differentiation, and apoptosis acting via the retinoic acid receptors. Despite two decades of research on the function of retinoic acid, the physiological role of RA in mammary gland development is still not well characterized. In this report, we demonstrate that RA is required for proper morphogenesis of mouse mammary gland in a novel transgenic mouse model system. It was found that inhibition of RA signaling in vivo leads to excessive mammary ductal morphogenesis through upregulation of cyclin D1 and MMP-3 expression. Furthermore, we show that the transgene-induced excessive branching morphogenesis could be reversed by treatment with RA, demonstrating the direct physiological effect of RA signaling in vivo. In addition, we demonstrate that excessive branching morphogenesis in the transgenic mammary gland are cell-autonomous and do not require stromal signals within the transgenic mammary gland. Finally, we provide evidence suggesting that retinoic acid signaling is required for appropriate mammary gland differentiation. Collectively, our data indicate for the first time that retinoic acid signaling is required to maintain the homeostasis of mammary gland morphogenesis.     

Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification.

Because tissues from all three germ layers contribute to the pharyngeal arches, it is not surprising that all major signaling pathways are involved in their development. We focus on the role of retinoic acid (RA) signaling because it has been recognized for quite some time that alterations in this pathway lead to craniofacial malformations. Several studies exist that describe phenotypes observed upon RA perturbations in pharyngeal arch development; however, these studies did not address whether RA plays multiple roles at distinct time points during development. Here, we report the resulting phenotypes in the hindbrain, the neural crest-derived tissues, and the pharyngeal endoderm when RA synthesis is disrupted during zebrafish gastrulation and pharyngeal arch morphogenesis. Our results demonstrate that RA is required for the post-gastrulation morphogenesis and segmentation of endodermal pouches, and that loss of RA does not affect the length of the pharyngeal ectoderm or medial endoderm along the anterior-posterior axis. We also provide evidence that RA is not required for the specification of pharyngeal pouch endoderm and that the pharyngeal endoderm consists of at least two different cell populations, of which the pouch endoderm is sensitive to RA and the more medial pharyngeal endoderm is not. These results demonstrate that the developmental processes underlying pharyngeal arch defects differ depending on when RA signaling is disturbed during development.     

Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development.

Studies on nonmammalian vertebrate embryos have indicated that retinoic acid (RA) is required for pancreas development. We have analyzed mouse embryos carrying a null mutation of the gene encoding retinaldehyde dehydrogenase 2 (Raldh2), which controls RA synthesis. Raldh2-/- embryos specifically lack expression of Pdx1 (a homeobox gene required for pancreas development) and Prox1 in dorsal endodermal but not ventral endodermal pancreatic precursor tissues. Ventral endodermal expression of Hex is not affected in Raldh2-/- embryos, indicating that liver specification is not dependent upon RA. Also, expression of Foxa2 across the dorsoventral axis of the endoderm is not affected in Raldh2-/- embryos, indicating that a lack of RA does not cause a general defect in foregut endoderm development. Comparison of wild-type and Raldh2-/- embryos carrying an RA-reporter transgene demonstrates that RA activity is normally present throughout the endoderm except in the ventral-most region but is totally missing in endoderm of Raldh2-/- embryos. Thus, Raldh2 expressed in adjacent splanchnic lateral plate mesoderm provides an RA signal to dorsal endoderm. Dorsal Pdx1 expression is rescued in Raldh2-/- embryos by low-dose maternal administration of RA, which preferentially restores RA-reporter expression in the dorsal endoderm. Our findings demonstrate a specific role for RA in mouse embryos as a mesodermally synthesized signal needed for dorsal endodermal expression of Pdx1 during development of the dorsal pancreatic lineage.     

MHC-restricted T cell receptor signaling is required for alpha beta TCR replacement of the pre T cell receptor

A developmental block is imposed on CD25(+)CD44(-) thymocytes at the beta-selection checkpoint in the absence of the pre T cell receptor (preTCR) alpha-chain, pTalpha. Early surface expression of a transgenic alphabeta TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4(+)CD8(+) double-positive stage. We wanted to analyze whether a restricting MHC element is required for alphabeta TCR-expressing double-negative (DN) thymocytes to overcome the developmental block in pTalpha-deficient animals. We used the HY-I knock-in model that endows thymocytes with alphabeta TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pTalpha-deficient background, this HY-I TCR transgene 'rescued' CD25(+)CD44(-) thymocytes from apoptosis and enabled progression to later differentiation stages. On a non-selecting MHC background, however, pTalpha-deficient HY-I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY-TCR-mediated rescue of pTalpha-deficient thymocytes.     

Retinoic acid receptor beta 2 mRNA is elevated by retinoic acid in vivo in susceptible regions of mid-gestation mouse embryos.

Many of the biological effects of retinoic acid are mediated by its nuclear receptors (RAR-alpha, RAR-beta, and RAR-gamma), and each of these three receptors exist in multiple isoforms. As a first step to identify if any of the receptor isoforms are involved in dysmorphogenesis which is induced in mouse embryos after treatment with retinoic acid (RA), we examined the levels of mRNA of several isoforms of each RAR in the limb buds and other embryonic regions of normal and RA-treated embryos. Within 3 to 6 hr after treatment of mice on day 11 of gestation with RA, RAR-beta 2 mRNA levels in the whole embryo increased 7-fold while both RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated only 2-fold. Since RA treatment of day 11 embryos especially produces limb defects in virtually every embryo, we next examined individual embryonic regions separately. Limb buds showed the highest elevations in RAR-beta 2 mRNA levels (12-fold) compared to a moderate elevation in the head/craniofacial region (8-fold) and a small elevation in the remainder of the body (4-fold). In contrast, RAR-alpha 2 and RAR-gamma 1 mRNA levels were elevated in all these tissues to a similar extent, which amounted to only about a 2-fold increase. Retinol, the precursor of RA in the embryo, was also capable of elevating RAR-beta 2 mRNA levels in the limb bud, but the increase was delayed, apparently indicating that metabolic conversion of retinol to RA preceded the effect on mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrin alpha5 is required for somite rotation and boundary formation in Xenopus.

The morphogenesis of somites in Xenopus laevis is characterized by a complex process of cell turning that requires coordinated regulation of cell shape, adhesion, and motility. The integrin alpha5 subunit has been implicated in the formation of somite boundaries in organisms utilizing epithelialization to create morphologically distinct somites, but its function has not been examined in Xenopus. We used a splice-blocking morpholino to knock down expression of integrin alpha5 during somite formation. Loss of integrin alpha5 delayed somite turning and accumulation of integrin beta1 at somite boundaries, and disrupted the fibronectin matrix surrounding developing somites. Irregular somite boundaries with a sparse and discontinuous fibronectin matrix formed upon eventual completion of somite turning. Recovery of somite morphology was improved, but still incomplete in far posterior somites. These data demonstrate that the role of integrin alpha5 in somite boundary formation is conserved in a species using a unique mechanism of somitogenesis.     

Signaling via interleukin-4 receptor alpha chain is required for successful vaccination against schistosomiasis in BALB/c mice

Although protective immunity in C57BL/6 mice induced by a single dose of the radiation-attenuated schistosome vaccine is believed to be mediated by Th1-type immune responses, we here report that in BALB/c mice protection can also depend upon signaling via the interleukin-4 (IL-4) receptor which conventionally governs the development of Th2-type immune responses. We show that in BALB/c mice deficient for the IL-4 receptor alpha chain (IL-4Ralpha(-/-)), which are unresponsive to IL-4 and IL-13, vaccine-induced protection is abrogated compared with that in wild-type (WT) mice. In vaccinated IL-4Ralpha(-/-) mice, IL-12p40 production by cells from the skin exposure site was elevated, although gamma interferon (IFN-gamma) production in draining lymphoid tissues was similar in WT and IL-4Ralpha(-/-) mice. Nevertheless, the effector response in IL-4Ralpha(-/-) mice was Th1 biased with elevated IFN-gamma in the lungs and higher immunoglobulin G2a (IgG2a) and IgG2b titers but negligible quantities of Th2-associated IgG1 and IgE. Interestingly, levels of IL-4 were equivalent in WT and IL-4Ralpha(-/-) mice, indicating that Th2 responses were not dependent upon signaling by IL-4 or IL-13. No differences in the phenotype and composition of the pulmonary effector mechanism that might explain the failure to induce protection in IL-4Ralpha(-/-) mice were detected. However, passive transfer of partial protection to naive IL-4Ralpha(-/-) mice, using serum from vaccinated WT mice, indicates that Th2-associated antibodies such as IgG1 have a role in parasite elimination in BALB/c strain mice and that signaling via IL-4R can be an important factor in the generation of protection.     

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

TrkB receptor signaling is required for establishment of GABAergic synapses in the cerebellum

Neurotrophins are essential to the normal development and maintenance of the nervous system. Neurotrophin signaling is mediated by Trk family tyrosine kinases such as TrkA, TrkB and TrkC, as well as by the pan-neurotrophin receptor p75NTR. Here we have deleted the trkB gene in cerebellar precursors by Wnt1-driven Cre--mediated recombination to study the function of the TrkB in the cerebellum. Despite the absence of TrkB, the mature cerebellum of mutant mice appears similar to that of wild type, with all types of cell present in normal numbers and positions. Granule and Purkinje cell dendrites appear normal and the former have typical numbers of excitatory synapses. By contrast, inhibitory interneurons are strongly affected: although present in normal numbers, they express reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations. Thus, TrkB is essential to the development of GABAergic neurons and regulates synapse formation in addition to its role in the development of axon terminals.     

A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila.

We recently identified from the Drosophila genome database a large family of G protein-coupled receptor genes, the Gr genes, and predicted that they encode taste receptors on the basis of their structure and specificity of expression. The expression of Gr genes in gustatory neurons has subsequently been confirmed and 56 family members have been reported. Here we provide functional evidence that one Gr gene, Gr5a, encodes a taste receptor required for response to the sugar trehalose. In two different mutants that carry deletions in Gr5a, electrophysiological and behavioral responses to trehalose were diminished but the response to sucrose was unaffected. Transgenic rescue experiments showed that Gr5a confers response to trehalose. The results correlate a particular taste ligand with a Gr receptor and indicate a role for G protein-mediated signaling in the transduction of sweet taste in Drosophila.     

Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia

While erythropoietin (Epo) and its receptor (EpoR) have been widely investigated in brain, the expression and function of the soluble Epo receptor (sEpoR) remain unknown. Here we demonstrate that sEpoR, a negative regulator of Epo's binding to the EpoR, is present in the mouse brain and is down-regulated by 62% after exposure to normobaric chronic hypoxia (10% O₂ for 3 days). Furthermore, while normoxic minute ventilation increased by 58% in control mice following hypoxic acclimatization, sEpoR infusion in brain during the hypoxic challenge efficiently reduced brain Epo concentration and abolished the ventilatory acclimatization to hypoxia (VAH). These observations imply that hypoxic downregulation of sEpoR is required for adequate ventilatory acclimatization to hypoxia, thereby underlying the function of Epo as a key factor regulating oxygen delivery not only by its classical activity on red blood cell production, but also by regulating ventilation.     

Tropomyosin is required for the cell fusion process during conjugation in fission yeast

BACKGROUND: Tropomyosin is an actin-binding protein, which is thought to stabilize actin filaments and influence many aspects of F-actin. In fission yeast, the cdc8 gene encodes tropomyosin, and the gene product Cdc8p is known to be essential for the formation of the F-actin contractile ring and hence for cytokinesis in the mitotic cell cycle. RESULTS: We isolated fission yeast mutants that were defective in cell fusion during conjugation. One of them turned out to carry a point mutation in cdc8. We found that the original temperature-sensitive cdc8 mutant frequently failed to undergo cell fusion when mated at a semi-permissive temperature. Additional cdc8 mutants isolated by targeted mutagenesis also showed defects in both cell fusion and cytokinesis. A decrease in the amount of intracellular Cdc8p also affected both, but cell growth was more severely blocked than cell fusion in this case. Immunostaining revealed that Cdc8p was localized as a spot at the cell-to-cell attachment site during conjugation, without overlapping with F-actin patches. CONCLUSIONS: Tropomyosin Cdc8p is indispensable for cell fusion during conjugation in fission yeast. However, cell fusion appears to require fewer tropomyosin molecules than cytokinesis. We speculate that tropomyosin may organize a small F-actin-containing organelle at the cell-to-cell contact site in each mating cell, which plays a key role in cell fusion.     

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.     

Vascular endothelial growth factor receptor signaling is required for cardiac valve formation in zebrafish.

Vascular endothelial growth factor-receptors (VEGF-Rs) are pivotal regulators of vascular development, but a specific role for these receptors in the formation of heart valves has not been identified. We took advantage of small molecule inhibitors of VEGF-R signaling and showed that blocking VEGF-R signaling with receptor selective tyrosine kinase inhibitors, PTK 787 and AAC 787, from 17-21 hr post-fertilization (hpf) in zebrafish embryos resulted in a functional and structural defect in cardiac valve development. Regurgitation of blood between the two chambers of the heart, as well as a loss of cell-restricted expression of the valve differentiation markers notch 1b and bone morphogenetic protein-4 (bmp-4), was readily apparent in treated embryos. In addition, microangiography revealed a loss of a definitive atrioventricular constriction in treated embryos. Taken together, these data demonstrate a novel function for VEGF-Rs in the endocardial endothelium of the developing cardiac valve.     

Retinoic acid receptor beta2 promotes functional regeneration of sensory axons in the spinal cord

The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor beta2 (RARbeta2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARbeta2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARbeta2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARbeta2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARbeta2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.     

A frameshift mutation in the mouse {alpha}1 glycine receptor gene (Gira1) results in progressive neurological symptoms and juvenile death

The neurologic mutant mouse, oscillator, Is characterized bya fine motor tremor and muscle spasms that begin at 2 weeksof age and progressively worsen, resulting in death by 3 weeksof age. We report the localization of the oscillator mutationto the central region of mouse Chr 11, and demonstrate its allelismwith spasmodic, a recessive viable neurological mutation whichdisplays excessive startle. Oscillator Is caused by a microdeletionIn the gene coding for the     

动脉内皮损伤后平滑肌细胞维甲酸受体的表达及维甲酸的作用

目的 研究动脉内皮损伤后ＶＳＭＣ中ＲＡＲ表达的规律及其与内膜增生的关系,方法 放射配基法测量用维甲酸与未用药两组内皮损伤后不同时期的受体最大结合容量和平衡解离常数抽时用形态学方法测量血管内膜面积。结果 动脉内皮损伤后早期ＶＳＭＣ中ＲＡＲ数量显著低于正常（Ｐ〈０．０１）,于第４周恢复正常,同时血管膜面积增加,用维甲酸后,ＲＡＲ显著增加,于术后２周即接近正常,血管内膜面积显著少于未用药组,结论 动脉损     

Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice

Here we describe a family of GPI-anchored cell surface proteins that function as ligands for the mouse activating NKG2D receptor. These molecules are encoded by the retinoic acid early inducible (RAE-1) and H60 minor histocompatibility antigen genes on mouse chromosome 10 and show weak homology with MHC class I. Expression of the NKG2D ligands is low or absent on normal, adult tissues; however, they are constitutively expressed on some tumors and upregulated by retinoic acid. Ectopic expression of RAE-1 and H60 confers target susceptibility to NK cell attack. These studies identify a family of ligands for the activating NKG2D receptor on NK and T cells, which may play an important role in innate and adaptive immunity.