Analysis of Growth Factor Signaling in Embryos (Methods in Signal Transduction Series)

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  1. Methods in Signal Transduction Series - Routledge
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Analysis of Growth Factor Signaling in Embryos Methods in Signal Transduction Series

These two databases also provide bioinformatics tools online to combine specific biochemical information on a certain organism and facilitate the interpretation of biological meanings for experimental data. By using a combined approach of Microarray-Bioinformatic technologies, a potential metabolic mechanism contributing to colorectal cancer CRC has been demonstrated [] Several environmental factors may be involved in a series of points along the genetic pathway to CRC. These include genes associated with bile acid metabolism, glycolysis metabolism and fatty acid metabolism pathways, supporting a hypothesis that some metabolic alternations observed in colon carcinoma may occur in the development of CRC.

Cellular models are instrumental in dissecting a complex pathological process into simpler molecular events. Parkinson's disease PD is multifactorial and clinically heterogeneous; the aetiology of the sporadic and most common form is still unclear and only a few molecular mechanisms have been clarified so far in the neurodegenerative cascade.

In such a multifaceted picture, it is particularly important to identify experimental models that simplify the study of the different networks of proteins and genes involved. Cellular models that reproduce some of the features of the neurons that degenerate in PD have contributed to many advances in our comprehension of the pathogenic flow of the disease. In particular, the pivotal biochemical pathways i. The central role of a-synuclein has generated many models aiming to elucidate its contribution to the dysregulation of various cellular processes.

Classical cellular models appear to be the correct choice for preliminary studies on the molecular action of new drugs or potential toxins and for understanding the role of single genetic factors. Moreover, the availability of novel cellular systems, such as cybrids or induced pluripotent stem cells, offers the chance to exploit the advantages of an in vitro investigation, although mirroring more closely the cell population being affected. Synaptic degeneration and death of nerve cells are defining features of Alzheimer's disease AD , the most prevalent age-related neurodegenerative disorders.

In AD, neurons in the hippocampus and basal forebrain brain regions that subserve learning and memory functions are selectively vulnerable. Studies of postmortem brain tissue from AD people have provided evidence for increased levels of oxidative stress, mitochondrial dysfunction and impaired glucose uptake in vulnerable neuronal populations. Studies of animal and cell culture models of AD suggest that increased levels of oxidative stress membrane lipid peroxidation , in particular may disrupt neuronal energy metabolism and ion homeostasis , by impairing the function of membrane ion-motive ATPases , glucose and glutamate transporters.

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Such oxidative and metabolic compromise may thereby render neurons vulnerable to excitotoxicity and apoptosis. Recent studies suggest that AD can manifest systemic alterations in energy metabolism e. Emerging evidence that dietary restriction can forestall the development of AD is consistent with a major "metabolic" component to these disorders, and provides optimism that these devastating brain disorders of aging may be largely preventable.

From Wikipedia, the free encyclopedia. Gomperts; Peter E. Tatham; Ijsbrand M. Kramer Signal transduction Pbk. Amsterdam [u. Berg; John L. Tymoczko; Lubert Stryer Biochemistry 6. New York: Freeman. In Prasanna, V. LNCS Springer-Verlag, pp. Nature Reviews Genetics.

J Neurosci Nurs 35 2 : —8. New York, N. Journal of Biomedicine and Biotechnology. Current Opinion in Cell Biology. Journal of Clinical Investigation. March Cancer Letters. Cold Spring Harbor Perspectives in Biology. Schiff; Willis C. Maddrey; Michael F. Sorrell, eds. Schiff's diseases of the liver 11th ed. Ross, Wojciech 23 April Biochemical Journal. European Journal of Cell Biology. International Journal of Biological Sciences. Arias; Harvey J. Alter Chichester, UK: Wiley-Blackwell.

DNA and Cell Biology. Signaling pathways in liver diseases 2. Berlin: Springer.

Vascular Pharmacology. Life Sciences. BioMed Research International. Proceedings of the Japan Academy, Series B. Journal of Clinical Oncology. Therapeutic Advances in Medical Oncology. Berlin [u. Nature Reviews Neuroscience. Trends in Neurosciences. Expert Reviews in Molecular Medicine. Proceedings of the National Academy of Sciences.

Methods in Signal Transduction Series - Routledge

The Journal of Neuroscience. December Annals of the New York Academy of Sciences. The Journal of Cell Biology. June April Nature Reviews. Nature Reviews Molecular Cell Biology. Fundamentos de Imunologia. Cellular and molecular immunology. Abbas, Andrew H. Lichtman, Shiv Pillai; illustrations by David L. Baker, Alexandra 7th ed. Encyclopedia of life sciences. Hoboken, NJ [u. Retrieved 8 January Journal of Immunology. Nature Immunology. Japanese Dental Science Review. Database Oxford. Frontiers in Endocrinology. Cancer Research. Human Reproduction.

The slides were examined with a Zeiss Epifluorescence photomicroscope equipped with a high pressure mercury burner HBO and tungsten lamp 12 V, 60 W. Gel electrophoresis. Embryonic lungs were collected on d 13 to 17 of gestation E13 to E17, Theiler stages 21 to 25 and lungs from litters were pooled.

Two independent samples were evaluated by Western analysis. Receptors were activated by incubation with nM dexamethasone at room temperature for 1 h. Protein concentrations ranged from 5. The molecular weight of proteins was calculated using known molecular weight markers Bio-Rad, Richmond, CA. Proteins were electrophoretically transferred to Immobilon filters Millipore, Bedford, MA and immunostained with anti-rat GR or anti-hsp hsp70 and hsp90 antibodies essentially as previously described This extensive blocking was necessary because the secondary goat anti-mouse antibody used with MAb nonspecifically binds to endogenous Ig in the mouse lungs.

After further washing in TBST, the blots were developed with 0. The reaction was stopped with the addition of 0. Blue immunostained bands were photographed. Two control and CORT-treated litters were treated as described above and embryonic lungs were collected on d 3 E15 , d 4 E16 , and d E17 postinjection. Lungs were fixed in alcohol:acetic acid fixative, processed, and immunostained with anti-rat SP-A antibodies as previously described Control sections were incubated in the absence of the primary antibodies; the controls were routinely negative data not shown.

Three control and CORT-treated lungs per day were evaluated. Gametchu and R. This MAb recognizes epitopes near the DNA-binding domain 31 ; its specificity, cross-reactivity with mouse GR, and recognition of both cytoplasmic and nuclear receptors have been determined previously 31 — Victoria, Canada ; their specificities have been determined by StressGen Biotechnologies.

MAb hsp90 also cross-reacts with rodent GR. MAb hsp70 has been shown to recognize both the constitutive hsp73 and inducible hsp72 proteins. The cross-reactivity of MAb hsp70 and hsp90 with mouse proteins has previously been demonstrated A polyclonal antibody against rat SP-A was generously provided by Dr. Whitsett; its specificity and cross-reactivity to mouse SP-A has previously been demonstrated 25 , 35 — A mouse lungs.

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Frozen organs, usually pooled from different litters, were processed as described above for Western blotting. DNA binding by GR was evaluated with the electrophoretic mobility-shift assay 38 , Dennis Sakai. When used, unlabeled competing binding sites were added at to fold molar excess 10 ng. All incubation mixes were matched for contributions from added extract. GR partially purified from adult rat liver provided by Dr. Dennis Sakai was also used as a standard. A minimum of two independent samples were evaluated per day. Glucocorticoid treatment of mice was performed as described above and RNA was isolated from lungs collected 3 d E15 after hormone injection.

Two to four independent samples each of control and treated animals were prepared with 7 to 11 lungs from 1 litter. Total cellular RNA was extracted with acidic guanidine isothiocyanate Purified RNA was quantified by UV spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Steady-state levels of specific transcripts were determined by Northern analysis. To facilitate accurate determination of transcript sizes, RNA size markers 0. Ratios were arcsin transformed, means were calculated, and differences between mean ratios were compared by a t test.

Stephen Yen. A subclone for rat SP-A was obtained from Dr. John Shannon 46 ; its recognition of mouse SP-A transcripts has been previously determined. Analysis of GR in the developing mouse lung. We initially conducted immunolocalization studies in embryonic and fetal B A mouse lungs to determine the cell-specific spatial distribution of the GR. In the early pseudoglandular stage E , when the respiratory epithelium branches into tubular structures lined by columnar epithelium and is embedded within densely packed undifferentiated mesenchyme, the receptor is diffusely distributed throughout the pulmonary mesenchyme, being localized to the membrane and the cytoplasm, but absent from nuclei Fig.

The absence of the GR from nuclei indicates that the receptors have not yet been activated in these early embryonic lungs. As development progresses, respiratory epithelia undergo extensive branching, with the mesenchymal cells adjacent to the epithelia becoming concentrically condensed and aligned around it By the late canalicular stage of development E16 to E17 , the number of receptors immunolocalized in the mesenchyme appears to increase with development Fig. The GR is primarily detected in the nuclei of the aligned mesenchyme adjacent to bronchial and air sac epithelia Fig.

GR immunolocalization in embryonic and fetal mouse lungs. A Early pseudoglandular stage E The GR, absent from the nuclei, is localized in the plasma membranes and the cytoplasm of the undifferentiated mesenchyme m and branching epithelia e. B The canalicular stage E By the canalicular stage of development seen in the E17 mouse lung, GR is immunolocalized in the aligned mesenchymal cells double arrows adjacent to the differentiating bronchial and air sac epithelia.

Because developmental differences in the GR have previously been shown 48 — 50 , we investigated possible qualitative and quantitative differences in embryonic pulmonary GR with progressive gestational age. Western analysis demonstrates that the major kD receptor is similar in form and relative mobility on all gestational days evaluated E13 to E17 Fig. To verify that the complexes involved DNA sequence-specific interaction with GR, E17 extracts were also incubated in the presence of excess unlabeled GRE or nonspecific oligodeoxyribonucleotides.

These results indicate that the embryonic GR, as well as the fetal GR, can be transformed and translocated into the nucleus i. Western analysis of E13 to E17 mouse lungs shows that the relative mobility of the GR protein is similar with progressive development. GRE binding by embryonic E13 to E17 pulmonary GR demonstrates that the receptor is functional on all gestational days evaluated. A Electrophoretic mobility-shift assays were performed with a 32 P-labeled oligodeoxyribonucleotide containing a high-affinity GRE from the rat tyrosine aminotransferase gene and BSA - or extracts of d 13 to 17 mouse lungs.

Hsp and progressive mouse lung development.

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They have been shown to be involved in ligand-receptor binding and GR transformation from the non-DNA-binding cytoplasmic form into the DNA-binding nuclear activated receptor 26 , 51 — However, little is known about embryonic pulmonary hsp90 and hsp Therefore, to further characterize the embryonic CORT-GR signal transduction pathway, we evaluated hsp90 and hsp70 in embryonic E13 to E17 mouse lungs by Western analysis.

Hsp90 and hsp70 proteins are present on all gestational days analyzed Fig. Western blot analysis of hsp90 A and hsp70 B in E13 to E17 mouse lungs shows that the proteins are qualitatively similar with progressive days of gestation. Note the cross-reactivity of anti-hsp90 with the kD GR. Our previous observation that administration of exogenous CORT to pregnant mice on d 12 of gestation results in accelerated lung maturation in vivo 10 , as well as the demonstration that CORT induces precocious SP-A protein expression when embryonic mouse lungs were cultured under chemically-defined conditions 9 , suggest that CORT-stimulation of lung maturation and SP-A expression may be mediated by the embryonic GR.

However, the direct determination that the embryonic GR is functional in vivo has not previously been reported. Quantitation by PhosphorImager analysis of four independent samples was conducted, the data were arcsin transformed, and the means tested for differences. Further, to determine whether in vivo CORT treatment of embryonic lungs also modulates SP-A protein expression, lungs were collected 3 d E15 , 4 d E16 , and 5 d E17 postinjection and evaluated by indirect immunofluorescence. Although SP-A is not normally detected in B A mouse lungs until d With increasing days of gestation d 16 and 17 , the distribution and intensity of immunostain are markedly greater in CORT-exposed lungs compared with controls compare Figs.

These results indicate that CORT induces precocious epithelial histodifferentiation. A and B E C and D E E and F E As normal development continues, SP-A is immunolocalized in E17 E lungs within the presumptive air sac spaces and in the epithelia surrounding them; CORT effects an increase in the distribution and relative amount of immunodetectable SP-A F compared with controls E.

Vertical bars represent 1 SEM. Pulmonary morphogenesis, mouse or human, is characterized by an ordered and precise schedule of cellular spatiotemporal events e. Understanding the gestational endocrinology regulating these events is critical to clarifying the pathogenesis of neonatal disease RDS and developing effective preventive and treatment strategies. The present in vivo mouse study, and others, indicate that the CORT-GR signal transduction pathway is operative during the embryonic period of lung ontogeny and that it is likely to be important to normal organogenesis: 1 GR is present from the early pseudoglandular stage EE13 in mesenchymal cytoplasm and translocates to the nucleus with progressive development 73 present study.

The mouse lung begins development on embryonic d 11 E11 as an epithelial evagination from the posterior pharyngeal region into undifferentiated mesenchyme, this epithelium bifurcating to form the lung primordium 47 , Given that lung morphogenesis is the result of a complex series of epithelial-mesenchymal interactions for review, see Ref. The GR, initially diffusely localized throughout the densely-packed embryonic pulmonary mesenchyme, becomes nonrandomly distributed in the fetal mesenchyme, with a higher concentration detected in the mesenchyme concentrically arranged adjacent to the differentiating bronchial and air sac epithelia.

We highlight the functions of the vascular endothelial growth factor VEGF and fibroblast growth factor FGF pathways in regulating these processes, with an emphasis on the migration and differentiation of primary mesenchyme cells PMCs and the formation of the embryonic skeleton. We compare the functions of growth factors in early sea urchin development with the roles that these and other growth factors play in early mesoderm morphogenesis in Drosophila and vertebrates.

Skeletal morphogenesis has been studied intensively, and several reviews discuss this process in detail Ettensohn, ; Killian and Wilt, ; Lyons et al. In brief, the PMCs are progeny of the large micromeres, four blastomeres that arise at the vegetal pole as a consequence of the fourth and fifth cleavage divisions, which are unequal in the vegetal region of the embryo Fig.

Methods in Signal Transduction Series

After a brief period of quiescence, the cells begin to migrate away from the vegetal pole, adhering to the wall of the blastocoel and translocating along a thin basal lamina that covers the basal surfaces of the epithelial cells that form the outer wall of the embryo Fig. The PMCs migrate by means of filopodia, which engage in continuous cycles of extension and retraction Malinda et al. Soon after the subequatorial ring forms, a single chain of PMCs migrates longitudinally from each VLC toward the animal pole of the embryo, with its advance led by numerous filopodia Fig.

PMCs secrete the calcified embryonic endoskeleton, which forms from two triradiate spicule rudiments, one of which is deposited within each VLC late in gastrulation Fig. The spicules are deposited within the PMC filopodial cables; therefore, the spatial arrangement of the PMCs and the resultant positioning of the cables serves directly as a template for the embryonic endoskeleton.

Both the branching pattern and fine structure of the endoskeleton are highly consistent within individuals of a species but vary among different species of sea urchin. Several additional skeletal elements arise during later larval development Smith et al. The skeleton serves as the primary determinant of the shape of the larva and plays an important role in its orientation, swimming, and feeding see Ettensohn, and references therein.

In particular, pigment cells and blastocoelar cells, two cell populations that function in immune surveillance Li et al. The guidance cues that direct these movements have not been identified. In contrast, considerable progress has been made in dissecting the mechanisms of PMC guidance, as discussed below.

The complex and highly reproducible pattern of PMC migration during sea urchin gastrulation originally suggested that external cues from the ectoderm play an important role in directing PMC movements. Gustafson and Wolpert , Okazaki et al. More significantly, perturbation of ectodermal patterning with NiCl 2 was shown to alter PMC migration and skeletogenesis Armstrong et al.

A series of cell transplantation and cell marking studies showed that PMC guidance cues arise progressively during development and that PMCs are specifically competent to respond to these cues Ettensohn and Malinda, ; Ettensohn and McClay, ; Malinda and Ettensohn, ; Peterson and McClay, Additional evidence that PMCs respond to signals from the ectoderm came from studies showing that localized photoablation of ectoderm cells blocked the deposition of skeletal material by neighboring PMCs Ettensohn and Malinda, and from the finding that several mRNAs encoding biomineralization—related proteins are expressed selectively in the VLCs, where skeletal growth is initiated presumably in response to ectodermal cues Harkey et al.

Although these various studies clearly showed that local ectodermal cues regulate PMC migration, gene expression, and skeletogenesis, the molecular nature of the cues remained unknown until relatively recently. Growth factor signaling pathways have been linked to a wide variety of essential processes during embryonic development. Most growth factor receptors are receptor tyrosine kinases RTKs , a family of transmembrane glycoproteins that function in transmitting extracellular signals into the cell reviewed in Hubbard and Till, Various RTKs differ mostly in the architecture of their extracellular domains, and in the absence of a ligand usually exist as monomers.

The binding of a ligand to a specific receptor triggers the dimerization and autophosphorylation of the receptor, leading to the sequential phosphorylation and activation of downstream effectors of RTK signaling. In a comprehensive survey of RTK signaling genes in the genome of the purple sea urchin, Strongylocentrotus purpuratus , Lapraz et al. The P. Overexpression of vegf3 leads to an increased number of PMCs and the formation of supernumerary skeletal rods. Similarly, knockdown of vegf3 in L. PMC migration is dependent on the dynamic motility of filopodia Malinda et al.

Thus, inhibition of VEGF signaling in vivo does not prevent PMCs from spreading on the basal lamina or moving away from the vegetal plate, and the velocity of PMC migration in an in vitro motilility assay is not affected by axitinib. In other model systems, VEGF functions as a chemoattractant and a regulator of filopodial dynamics Gerhardt, A variety of studies suggest that VEGF also acts as chemoattractant for developing neurons Chauvet et al.

A homolog of vegf3 has also been identified in the sea urchin Hemicentrotus pulcherrimus. Similar to previous observations, Hp vegf expression is restricted to two domains of ectoderm overlying the VLCs, and the overexpression of this ligand leads to the formation of supernumerary skeletal rods Fujita et al.

Interestingly, overexpression of the H. These data point to a possible role for heparan sulfate proteoglycans in mediating VEGF signaling during skeletogenesis. The expression pattern of vegf3 has been examined in embryos of two other groups of echinoderms: sea stars Patiria pectinifera and brittle stars Amphipholis kochii Morino et al. In contrast, vegf3 is not expressed in sea star embryos, which lack an endoskeleton, though this gene is expressed at late larval stages in epithelial cells that overlie the primordia of the adult skeleton Morino et al.

These comparative studies point to an important, conserved role of VEGF signaling in echinoderm skeletogenesis and suggest that evolutionary modifications in VEGF expression played a role in the appearance of new patterns of skeletogenesis within the phylum. Recently, the pattern of expression of a second VEGF ligand, vegf2 , was described in the sea urchin Strongylocentrotus intermedius Kipryushina et al.

Unlike vegf3 , which is expressed specifically in the ectoderm, vegf2 is ubiquitously expressed during early embryonic development but at later stages is expressed selectively by PMCs, particularly in the scheitel region at the posterior apex of the larva.


The function of vegf2 is unknown, though its expression pattern raises the possibility this ligand might also play a role in skeletogenesis. VEGF signaling is one of several signaling pathways that control these regional patterns of gene expression and skeletal rod growth within the PMC network. It has long been known that the formation of spicules by isolated micromeres requires horse serum McCarthy and Spiegel, ; Okazaki, ; Page and Benson, , and VEGF may be the active factor in serum.

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These effects are consistent with patterns of skeletal growth in vivo ; vegf3 mRNA is expressed at high levels during gastrulation when the triradiate spicules are synthesized, and at lower levels at the prism and pluteus stages, when additional skeletal rods e. The local expression of several biomineralization genes by PMCs at the tips of the ventral rods is also dependent on VEGF signaling Sun and Ettensohn, unpublished observations.

Because skeletal rods elongate by the addition of new biomineral at their tips Decker and Lennarz, ; Ettensohn and Malinda, , it seems likely that VEGF regulates skeletal growth by locally stimulating the expression of biomineralization proteins by PMCs at the tips of the rods. Though VEGF signaling plays an essential role in PMC gene expression and skeletal growth ventrally, other unidentified signaling pathways must operate on the dorsal side of the embryo. FGFs are a conserved family of polypeptide growth factors ubiquitous among vertebrates and invertebrates Ornitz and Itoh, FGF signaling has been shown to play a significant role in mesoderm formation in the sea urchin, as in other metazoans.

The role of FGF has been studied in two sea urchin species, P.