help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Servoss, S. J.
Right arrow Articles by Hill, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Servoss, S. J.
Right arrow Articles by Hill, J. M.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 142, No. 8 3348-3353
Copyright © 2001 by The Endocrine Society

ARTICLES

IGF-I as a Mediator of VIP/Activity-Dependent Neurotrophic Factor-Stimulated Embryonic Growth

Stephen J. Servoss, Susan J. Lee, Gretchen Gibney, Illana Gozes, Douglas E. Brenneman and Joanna M. Hill

Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University (I.G.), Tel Aviv, Israel 69978

Address all correspondence and requests for reprints to: Joanna M. Hill, Ph.D., Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 5A38, 9000 Rockville Pike, Bethesda, Maryland 20892-4480. E-mail: jh139h{at}nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I and the IGF-I receptor are necessary for normal embryonic growth. VIP is an important regulator of early postimplantation growth and acts indirectly through the release of other factors, including activity-dependent neurotrophic factor. The relationship of IGF-I growth regulation to VIP/activity-dependent neurotrophic factor-stimulated growth was examined with whole cultured embryonic d 9.5 mouse embryos. Somite numbers and DNA and protein contents were measured in embryos treated with IGF-I, anti-IGF-I, VIP, activity-dependent neurotrophic factor, and anti-activity-dependent neurotrophic factor-14 (antiserum to an activity-dependent neurotrophic factor agonist). IGF-I mRNA content was measured after incubation with and without VIP for 30 and 60 min using competitive RT-PCR. IGF-I induced a significant, dose-dependent increase in growth as measured by somite number, DNA levels, and protein content. Furthermore, anti-IGF-I inhibited embryonic growth and also prevented exogenous IGF-mediated growth. Both VIP- and activity-dependent neurotrophic factor-stimulated growth were blocked by anti-IGF-I, whereas anti-activity-dependent neurotrophic factor-14 had no detectable effect on IGF-I-induced growth. Treatment with VIP resulted in a 2-fold increase in embryonic IGF-I mRNA. These data suggest that IGF-I is a downstream mediator of VIP and activity-dependent neurotrophic factor in a regulatory pathway coordinating embryonic growth and that VIP may function as a regulator of IGF-I gene expression in the embryo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EARLY STUDIES demonstrating that IGF-I mRNA increases approximately 9-fold between embryonic d (E) 11 and E13 in the rat (1) suggested an important role for IGF-I during early postimplantation embryogenesis. This developmental period is characterized by organogenesis, including neural tube closure, neurogenesis, and somite formation and corresponds to E9–11 in the mouse and approximately d 22–32 in human gestation (2). Subsequent work in the mouse showed that both IGF-I and the IGF-I receptor were necessary for normal embryonic growth (3, 4, 5). Overexpression of IGF-I was shown to enhance embryonic growth (6), and newborn mice lacking a functional IGF-I gene or functional IGF-I receptor gene weighed 60% and 45%, respectively, of wild-type littermates (3, 4, 5). By E13.5 of gestation, the weights of IGF-I gene knockout mice were significantly less than those of wild-type littermates, and significantly slower weight gain was apparent as early as E11 in the IGF-I receptor knockout (3).

The neuropeptide VIP has been shown to be an important regulator of embryonic growth (7, 8, 9, 10, 11, 12, 13, 14, 15), and a surge of VIP in the rat maternal serum accompanied by a peak of VIP in the embryo and extraembryonic membranes (10) coincide with the 9-fold increase in IGF-I transcript reported in the embryonic rat (1). VIP treatment of whole E9.5 mice in culture resulted in dose-related growth (7, 11, 13, 15) with an increased rate of cell division through shortening of G1 and S phases of the cell cycle (12). VIP treatment induced significant increases in somite number, cross-sectional area, and DNA and protein contents (7, 13, 15), with the morphological proportions of the embryos remaining normal. Blockage of VIP action through the treatment of pregnant mice with a VIP antagonist (neurotensin 6–11 VIP 7–28) (16, 17) from E9–11 resulted in significant inhibition of embryonic growth (8), as did treatment of whole cultured E9.5 embryos with an antibody to VIP (15), illustrating that VIP was necessary to maintain the normal growth rate. As blockage of VIP action in pregnant mice after E11 did not retard growth (8), the period of VIP regulation of early postimplantation embryonic growth apparently ended by E11 in the mouse.

VIP is known to be a secretagogue for a variety of survival-promoting substances (18, 19, 20, 21), and many VIP actions are thought to occur through the VIP-induced release of activity-dependent neurotrophic factor (ADNF), a femtomolar-acting protein isolated from conditioned medium of cerebral cortical astrocytes (20). In cell culture studies both VIP and ADNF exhibited protective actions against a number of toxic substances, including N-methyl-D-aspartate, tetrodotoxin, ß-amyloid peptide, and gp120, the envelope protein of human immunodeficiency virus (11, 20, 22). Detailed structure/activity studies of peptide sequences related to ADNF revealed peptides exhibiting neuroprotective potencies comparable to that of the parent protein (23). Antiserum to ADNF-14 (a 14-amino acid ADNF agonist) was developed and has been shown to neutralize the survival-promoting activity of both ADNF peptides and intact ADNF in vitro (20) and to detect a 60-kDa form of ADNF in Western analyses of conditioned medium from VIP-stimulated astrocyte cultures (24, 25).

ADNF has been found to stimulate embryonic growth, and treatment of whole E9.5 cultured mouse embryos with as little as 10-13 M ADNF induced an increase in growth with a potency a million-fold greater than that of VIP (15). Antiserum to ADNF protein inhibited embryonic growth with respect to controls and also blocked VIP-induced growth. However, anti-VIP did not inhibit ADNF-induced growth, indicating that ADNF is a downstream effector of VIP action (15). These data suggest that both VIP and ADNF serve as potent growth factors during embryonic development, that their presence on E9.5, a critical time point in multisystem organogenesis, is necessary for normal development, and that VIP action occurs at least in part through ADNF.

Because of the importance of both VIP and IGF-I in embryonic growth and the observation that peaks of activity for these two factors coincide during the early postimplantation period of development, the present study was designed to evaluate the relation of IGF-I regulation of embryonic growth to the growth stimulatory properties of VIP/ADNF during the this period of development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Whole embryo culture
E9.5 NIH Swiss (Harlan Sprague Dawley, Inc., Indianapolis, IN) mouse embryos were cultured as previously described (7, 11, 13, 15, 26, 27). Briefly, embryos were explanted with their yolk sac and ectoplacental cone intact. Pairs of embryos, with 16–18 somites, were incubated in 1 ml adult male Sprague Dawley rat serum (Harlan Sprague Dawley, Inc.) containing 13 mM glucose in polypropylene tubes placed on a rotating agitator (30 rpm) for 4 or 6 h at 37 C in a gas phase of 5% CO2 and 95% air. Treatments were added in a volume of 10 µl. The serum of control embryos received 10 µl PBS. The presence of a vaginal plug at the end of a 4-h mating period indicated the beginning of d 0 of pregnancy. Embryos were either untreated (control; n = 44) or treated with IGF-I (Bachem, King of Prussia, PA) at concentrations of 10-13 M (n = 4), 10-11 M (n = 6), 10-9 M (n = 10), and 10-7 M (n = 28); nerve growth factor (R & D Systems, Minneapolis, MN) at concentrations of 10-11 M (n = 6), 10-9 M (n = 6), and 10-7 M (n = 9); VIP (Peninsula Laboratories, Inc., Belmont, CA) at 10-7 M, a concentration based on previous studies (7, 11, 13, 15) (n = 10); ADNF (extracted and purified as previously described) (20) at 10-13 M, a concentration based on previous studies (13) (n = 22); anti-IGF-I (Upstate Biotechnology, Inc., Lake Placid, NY) at concentrations of 50 µg/ml (n = 4), 5 µg/ml (n = 36), 0.5 µg/ml (n = 12), and 0.05 µg/ml (n = 10); anti-ADNF-14, 25 µl/ml antiserum (n = 8); cotreatments of 10-9 M IGF-I and anti-IGF-I (5 µg/ml; n = 6); 10-7 M VIP and anti-IGF-I (5 µg/ml; n = 11); 10-13 M ADNF and anti-IGF-I (5 µg/ml; n = 11); 10-9 M IGF-I and anti-ADNF-14 (25 µl/ml; n = 14); 10-7 M VIP and 10-7 M IGF-I (n = 6); 10-9 M VIP and 10-9 M IGF-I (n = 6); 10-13 M ADNF and 10-7 M IGF-I (n = 6); and 10-15 M ADNF and 10-9 M IGF-I (n = 6). Anti-ADNF-14 was prepared in rabbits using a keyhole limpet hemocyanin conjugate of the ADNF agonist peptide VLGGGSALLRSIPA (20, 24, 25). A Western analysis of conditioned medium from VIP-stimulated astrocyte cultures detected a single band at 60 kDa (24) using a 1:1000 dilution of the antiserum. Specificity of anti-ADNF-14 was shown, in that a 1:500 dilution did not detect 5 µg of the closely homologous protein 60-kDa heat shock protein in a dot blot, and therefore was considered specific for ADNF (24, 25). The experiments testing the effective dose range of anti-IGF-I were performed for 6 h; however, in all other experiments 4-h incubations were performed. VIP and IGF-I were dissolved with 0.02% acetic acid; all other test materials were dissolved in PBS. All defined concentrations were attained by serial dilution in PBS. To test the potential impact of serum on experimental treatments, embryos were incubated with anti-IGF-I antiserum in the presence (n = 12) and absence of VIP (n = 12) in serum-free incubation medium consisting of HEPES-buffered saline (122 mM NaCl, 3.3 mM KCl, 1.2 mM CaCl2, 0.4 mM MgSO4, 1.2 mM KH2PO4, 13 mM glucose, and 25 mM HEPES, pH 7.3, containing 20 µg/ml leupeptin). The culture medium was not changed throughout the incubation period. After incubation, the embryos were visualized under a dissecting microscope, and the number of somites, an indicator of growth and development (28), was counted. Embryos were frozen on dry ice and stored at -70 C for protein and DNA analysis. For each treatment group, data were obtained from a minimum of two experiments. To quantitate protein and DNA, frozen embryos (n = 6–22) were thawed and homogenized in PBS. Total protein was assayed by the method of Bradford (29), using a protein assay reagent (Bio-Rad Laboratories, Inc. Melville, NY), and total DNA was measured using the method of Burton (30), as modified by Munro (31).

The data were analyzed using ANOVA, with repeated measures where appropriate, followed by contrasts between controls and all other treatment groups with Fisher’s protected least significant difference (StatView, Abacus Concepts, Inc., Berkeley, CA). All data are presented as the mean ± 1 SE.

The experimental protocol for this study followed the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and was approved by the animal care and use committee of the NICHD.

RT-PCR
RT-PCR was performed on whole cultured E9.5 mouse embryos treated for 30 or 60 min with or without 10-7 M VIP as described above. After incubation, embryos were washed three times in PBS, immersed in 1 ml Stat-60 (Tel-Test B, Friendswood, TX), and dissolved by sonication for isolation of total RNA by the acid guanidium/phenol chloroform method (32). As previously described, 20% of each RNA sample was reverse transcribed with random hexamer primers (33). DNA sequences for cyclophilin (M60456) and IGF-Iß (X04482) were obtained from GenBank. Primer sets were designed using Oligo software (National Biosciences, Plymouth, MN) and were synthesized by Bio-Synthesis, Inc. (Louisville, TX). The following primer sequences were used: IGF-Iß/5, 5'-CTGGTGGATGCTCTTCAGTTC-3'; IGF-Iß/3, 5'-CAGCTTCGTTTTCTTGTTTGTC-3'; cyclophilin/5, 5'-ATGGCACAGGAGGAAAGAGCA-3'; and cyclophilin/3, 5'-TTGCCGGAGTCGACAATGAT-3'. Competitive cDNA mimics containing the precise IGF-I and cyclophilin sequences with an internal deletion were constructed by PCR amplification of E9.5 mouse embryo cDNA, isolated as described above, using a 5'-primer homologous to two regions of the above gene sequences, separated by 79 and 72 bp for IGF-I and cyclophilin, respectively, along with the appropriate 3'-primers (34). 5'-Deletion primers, designed and synthesized as described above, were: IGF-Iß/5, 5'-CTGGTGGATGCTCTTCAGTTCTTGTGGATGAGTGTTGC-3'; and cyclophilin/5, 5'-ATGGCACAGGAGGAAAGAGCAATGCAGGCAAAGACACC-3'.

Products obtained from the mimic synthesis reactions were washed three times with cold ethanol and precipitated in sodium acetate. Once redissolved, mimic reaction products were separated on a 2% agarose gel. Bands of appropriate mol wt were collected on NA 45 DEAE membrane (Schleicher & Schuell, Keene, NH) and redissolved for use in competitive PCR.

PCR was carried out with 5 µl embryo cDNA and 3 µl of six known dilutions of purified cDNA mimic using the primers described above (19). Reactions were carried out in Taq DNA polymerase buffer (50 mM KCl and 10 mM Tris-HCl, pH 8.3) containing 200 µM NTP, 2 mM MgCl2, 5 µM of each primer, and 2.5 U Taq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT). To prevent evaporation, samples were overlaid with 50 µl mineral oil. PCR amplification was carried out in a Perkin-Elmer Corp. (Norwalk, CT) N801 programmable thermal controller, with amplification programs consisting of a 1-min denaturing step at 95 C, a 1-min annealing step at 61 or 65 C (for IGF-I and cyclophilin, respectively), and a 1-min synthesis step at 72 C. Products were separated through a 4–20% acrylamide minigel (Novex, San Diego, CA) and stained in 1 µg/ml ethidium bromide (Life Technologies, Inc., Gaithersburg, MD). Gel Marker-1 (Research Genetics, Inc., Huntsville, AL) was used as an electrophoresis marker for determination of product size. A trace amount of [32P]deoxy-CTP was added to the PCR buffer for quantitation of band intensity. After drying on a gel drier (model 583, Bio-Rad Laboratories, Inc., Richmond, CA), gels were exposed to a storage phosphor screen (Molecular Dynamics, Inc., Sunnyvale, CA) for 18–24 h. Relative band intensities were analyzed using ImageQuant software (Molecular Dynamics, Inc.). Data are reported relative to cyclophilin concentrations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Whole embryo culture
Incubation of E9.5 mouse embryos in vitro with IGF-I resulted in a significant dose-dependent increase in growth, with maximum growth occurring at the maximum dose tested (10-7 M; Figs. 1Go and 2AGo). Although control embryos cultured for 4 h grew 2.4 somites, data consistent with previous experiments (7, 11, 13, 15), embryos treated with 10-7 M IGF-I grew a mean of 4.6 somites. DNA and protein contents were also significantly greater in the IGF-treated groups (Table 1Go) with 10-7 M IGF-I-treated embryos having 213% of the DNA and 180% of the protein of control embryos. Treatment with the combinations of VIP and IGF-I or ADNF and IGF-I showed no additive effect on embryonic growth. A comparable dose range of treatment with nerve growth factor demonstrated no significant effect on embryonic growth, as measured by somite numbers after a 4-h incubation (Table 2Go).



View larger version (102K):
[in this window]
[in a new window]
  		Figure 1. Photomicrograph of control, IGF-I-treated, and anti-IGF-I-treated E9.5 mouse embryos after 4-h whole embryo culture. The IGF-I-treated embryo received 10-7 M IGF-I. IGF-I treatment stimulated the coordinated growth of both embryo brain and body. Anti-IGF-I inhibited embryonic growth. Bar, 0.5 mm.

 


View larger version (29K):
[in this window]
[in a new window]
  		Figure 2. Measures of the stimulation and inhibition of embryonic growth by IGF-I, VIP, ADNF, anti-IGF-I antibody, and anti-ADNF-14 antibody. A, Mean somites grown ± SEM after culture of E9.5 mouse embryos. Embryos were cultured with IGF-I for 4 h at concentrations ranging from 10-13–10-7 M. IGF-I treatment resulted in a significant and dose-related increase in somites (P < 0.0001, by ANOVA). Embryos were cultured with anti-IGF-I for 6 h at dilutions from 0.05–50 µg/ml. Anti-IGF-I treatment resulted in significant retardation of embryonic growth (P < 0.0001, by ANOVA). ***, Group differs from controls, P < 0.001; **, P < 0.01. B, Mean somites grown ± SEM in 4-h incubation of E9.5 mouse embryos. Treated embryos were cultured with VIP (10-7 M) or ADNF (10-13 M) with and without 5 µg/ml anti-IGF-I. Anti-IGF-I- and anti-IGF-I- plus ADNF-treated embryos grew significantly fewer somites than control embryos. In cotreated embryos, anti-IGF-I inhibited both VIP- and ADNF-induced increases in somite numbers (P < 0.0001, by ANOVA). ***, Group differs from controls, P < 0.001; **, P < 0.01. C, Mean somites grown ± SEM in 4-h incubation of E9.5 mouse embryos. Treated embryos were cultured with anti-IGF-I (5 µg/ml) or anti-ADNF-14 (25 µl/ml) with and without IGF-I (10-9 M). Anti-IGF-I, anti-ADNF-14, and IGF-I- and anti-IGF-I-treated embryos grew significantly fewer somites than IGF-I-treated embryos. Cotreatment of anti-ADNF-14 with IGF-I did not result in fewer somites grown than IGF-I treatment alone. In addition, anti-ADNF-14-treated embryos grew significantly fewer somites than controls (P < 0.0001, by ANOVA). ***, Group differs from IGF-I, P < 0.001. D, IGF-Iß transcripts relative to cyclophilin transcripts 30 min and 1 h after treatment with and without 10-7 M VIP. VIP doubled the amount of IGF-Iß mRNA in embryos 30 min and 1 h after treatment (P < 0.0001, by ANOVA). ***, Group differs from control, P < 0.001.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Protein and DNA contents of treated and control embryos after 4-h incubation

 

View this table:
[in this window]
[in a new window]
  		Table 2. Somites grown in 4 h in dose range of nerve growth factor

 
Embryos treated with a dose range of neutralizing anti-IGF-I antibody (anti-IGF-I) were incubated for 6 h to provide maximum separation between treated and control embryo growth rates and showed a significant and concentration-dependent inhibition of growth compared with control embryos. The maximum inhibition occurred at a concentration of 5 µg/ml antiserum (Figs. 1AGo and 2Go), and this concentration was used for all cotreatment experiments. Anti-IGF-I-treated (5 µg/ml) embryos grew a mean of 1.6 somites, whereas 6-h control embryos grew a mean of 4.0 somites. DNA and protein contents were also significantly reduced in the 6-h anti-IGF-I-treated embryos (55% and 51% of 6-h control value, respectively; data not shown). A 4-h incubation with anti-IGF-I (5 µg/ml) produced significant inhibition of somite growth (Fig. 2BGo and Table 1Go), but the change in size was not sufficient to be reflected in significantly less DNA and protein content. All cotreatment experiments were 4-h incubations, and anti-IGF-I was shown to inhibit the growth stimulation induced by VIP and ADNF treatment (Fig. 2BGo and Table 1Go) as well as growth stimulation by IGF-I (Fig. 2CGo and Table 1Go). Although anti-ADNF-14 antiserum significantly inhibited embryonic growth (Fig. 2CGo and Table 1Go), it did not inhibit IGF-I-induced growth (Fig. 2CGo and Table 1Go). In serum-free incubation medium, anti-IGF-I inhibited embryonic growth in both the presence and absence of VIP.

RT-PCR
As determined by RT-PCR, incubation of whole mouse embryos in vitro in the presence of VIP (10-7 M) for 30 and 60 min resulted in a significant increase in IGF-Iß transcript levels to a mean of 194% and 191% of time-matched control values, respectively (Fig. 2DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate that 1) IGF-I is a potent stimulator of embryonic growth of the E9.5 mouse embryo, as measured by increases in somite number and DNA and protein contents; 2) blockage of endogenous IGF-I action with an IGF-I antibody not only inhibits embryonic growth, but also the growth stimulation brought about by exogenously administered IGF-I, VIP, and ADNF; 3) blockage of ADNF does not inhibit IGF-I-induced growth, suggesting that IGF-I action occurs downstream from ADNF and that IGF-I at least in part accounts for the action of ADNF; and 4) VIP treatment increases IGF-I transcripts in the embryo.

These data confirm and extend previous studies linking the action of IGF-I to embryonic growth. In IGF-I receptor and IGF-I knockout mice growth restriction, as measured by weight gain, was apparent on E11 and E13.5, respectively (3). The deficits in growth detected at these ages presumably reflect deficits in IGF-I action occurring earlier in development.

Previous studies indicated that ADNF action accounted at least in part for the role of VIP in growth regulation, as 1) VIP was known to induce the release of ADNF (20); and 2) blockage of ADNF inhibited VIP-stimulated growth, but blockage of VIP did not inhibit ADNF-stimulated growth (15). The current study suggests that IGF-I stimulation of growth accounts at least in part for VIP/ADNF-induced growth during this embryonic period. Blockage of embryonic growth with the use of the ADNF-14 antibody did not inhibit IGF-I growth stimulation; however, anti-IGF-I antibody inhibited growth stimulation by IGF-I, VIP, and ADNF. Furthermore, VIP stimulation was shown to increase transcripts for IGF-I in the embryo. These data indicate that IGF-I may be a downstream effector of VIP/ADNF growth regulation. They further indicate that VIP may be a signal regulating IGF-I gene expression during embryogenesis. Although additional IGF-I isoforms may be expressed, the current data clearly show that VIP increased IGF-Iß transcripts in the early postimplantation mouse embryo. VIP is both present and necessary for normal embryonic development during the early postimplantation period of embryogenesis; however, the mRNA for VIP is not detectable in the embryo until after this period, suggesting that extraembryonic VIP is acting on the embryo (10, 14). In the mouse, VIP transcripts have been identified in the decidua, and VIP has been localized to {gamma}{delta} lymphocytes in the extraembryonic decidua (14). IGF-I is regulated by GH; however, as GH appears only later in fetal development (37, 38), the embryonic signal is not known. It may be particularly significant that the surge of VIP in rat maternal serum occurs during E11–13 in the rat (10), the time period overlapping the 9-fold increase in message for IGF-I in the rat embryo (1). This combined with the demonstration that VIP increased IGF-I mRNA in the embryo indicate that VIP may regulate embryonic IGF-I during this developmental period. The lack of an additive effect on embryonic growth after combined treatments with VIP and IGF-I or ADNF and IGF-I is also consistent with the hypothesis that VIP, ADNF, and IGF-I function in a related pathway and that with each of these treatments, similar mechanisms operate to regulate embryonic growth and development.

As discussed earlier, VIP treatment of embryos resulted in an increased rate of cell division through shortening of G1 and S phases of the cell cycle (12) providing a mechanism through which embryonic development can occur. As IGF-I gene expression is stimulated by VIP and is apparently an effector of VIP actions, the potent mitogenic properties of IGF-I (39, 40, 41) suggest that this factor may be an active mediator of the cell cycle stimulation observed after VIP treatment.

IGF-I belongs to the insulin family of factors, which also includes IGF-II and insulin. Each of these ligands has preferred receptors, and there are at least six IGF-binding proteins. For normal embryonic development to proceed, both IGF-I and IGF-II and their preferred receptors are apparently necessary (3). IGF-II is present earlier in gestation than IGF-I, and although its receptor is also present early in development (42, 43), as in other systems IGF-II actions are thought to occur primarily through the IGF-I receptor during early stages of development (5). Although less abundant than the IGF receptors, the insulin receptor is also present during early embryonic development (44). Insulin gene expression is not apparent in early embryos (44); however, the embryonic insulin receptor responds to insulin and may be acted upon by maternal insulin (42). In addition, IGF-II can act on the embryonic insulin receptor, perhaps making this receptor a mediator of IGF-II growth regulatory functions (45). The transcripts for both IGFs and their receptors have been localized in the embryo (44, 46). However, extraembryonic tissues, including placental trophoblasts and the maternal circulation, have been suggested as additional sources of IGFs and IGF regulators interacting with the embryo during gestation (47).

Little is known about the coordination of the complex events involved in the regulation of embryonic growth during the early postimplantation period of development. However, in addition to VIP and its secreted protein, ADNF, recent work has implicated numerous factors, including early pregnancy factor (48, 49), TGFß, TNF{alpha}, and TNFß (50), epidermal growth factor, fibroblast growth factor (51), parathyroid-related hormone (52), protease nexin-1 (53), and sonic hedgehog (54). During embryogenesis, the floor plate of the neural tube is a known regulatory site coordinating development through the release of diffusable and contact-mediated growth factors (54, 55). The floor plate also has abundant VIP-binding sites during early postimplantation embryogenesis, which is consistent with a role for this peptide in the release of growth factors (9, 10, 13, 14). The current study reveals that IGF-I may be among the mediators of VIP/ADNF actions at this time and additionally that VIP may function as a regulator of IGF-I gene expression during early embryogenesis.


   Acknowledgments
 
We are grateful to Dr. Gordon Glazner for technical guidance and critical discussion, and to Mr. Daniel Abebe for expert technical assistance in these studies.


   Footnotes
 
This work was supported in part by the NICHD intramural program, the Howard Hughes-NIH Research Scholars Program (S.J.S.), the Israeli Science Foundation, a U.S.-Israel binational grant, a Neufeld Memorial Award, and the Lily and Avraham Gildor Chair for the Investigation of Growth Factors.

Abbreviations: ADNF, Activity-dependent neurotrophic factor; E, embryonic day.

Received February 2, 2001.

Accepted for publication April 30, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rotwein P, Pollock KM, Watson M, Milbrandt JD 1987 Insulin-like growth factor gene expression during rat embryonic development. Endocrinology 121:2141–2144[Abstract/Free Full Text]
  2. Kaufman MH 1992 The atlas of mouse development. New York: Academic Press
  3. Baker J, Liu J-P, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  4. Powell-Braxton L, Hollingshead P, Warburton C, et al. 1993 IGF-I is required for normal embryonic growth in mice. Genes Dev 7:2609–2617[Abstract/Free Full Text]
  5. Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice encoding null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type I IGF receptor (IgfIr). Cell 75:59–72[Medline]
  6. Mathews LS, Hammer RE, Behringer RR, et al. 1988 Growth enhancement of transgenic mice expressing human insulin-like growth factor-I. Endocrinology 123:2827–2833[Abstract/Free Full Text]
  7. Gressens P, Hill JM, Gozes I, Fridkin M, Brenneman DE 1993 Growth factor function of vasoactive intestinal peptide in whole cultured mouse embryos. Nature 362:155–158[CrossRef][Medline]
  8. Gressens P, Hill JM, Paindaveine B, Gozes I, Fridkin M, Brenneman DE 1994 Severe microcephaly induced by blockade of vasoactive intestinal peptide function in the primitive neuroepithelium of the mouse. J Clin Invest 94:2020–2027
  9. Hill JM, Agoston DV, Gressens P, McCune SK 1994 Distribution of VIP mRNA and two distinct VIP binding sites in the developing rat brain: Relation to ontogenic events. J Comp Neurol 342:186–205[CrossRef][Medline]
  10. Hill JM, McCune SK, Alvero RJ, et al. 1996 Maternal vasoactive intestinal peptide and the regulation of embryonic growth in the rodent. J Clin Invest 97:202–208[Medline]
  11. Dibbern D, Glazner GW, Gozes I, Brenneman DE, Hill JM 1997 HIV envelope protein gp120 inhibits embryonic growth. J Clin Invest 99:2837–2841[Medline]
  12. Gressens P, Paindaveine B, Hill JM, Evrard P, Brenneman DE 1998 Vasoactive intestinal peptide shortens both G1 and S phases of cell cycle in whole cultured mouse embryos. Eur J Neurosci 10:1734–1742[CrossRef][Medline]
  13. Hill JM, Lee SJ, Dibbern DA, Fridkin M, Gozes I, Brenneman DE 1999 Pharmacologically distinct vasoactive intestinal peptide binding sites: CNS localization and role in embryonic growth. Neuroscience 93:783–7910[CrossRef][Medline]
  14. Spong CY, Lee SJ, McCune SK, et al. 1999 Maternal regulation of embryonic growth: The role of vasoactive intestinal peptide. Endocrinology 140:917–924[Abstract/Free Full Text]
  15. Glazner GW, Gressens P, Lee SJ, et al. 1999 Activity-dependent neurotrophic factor: a potent regulator of embryonic growth and development. Anat Embryol 200:65–71[CrossRef][Medline]
  16. Gozes I, Meltzer E, Rubinrout S, Brenneman DE, Fridkin M 1989 Vasoactive intestinal peptide potentiates sexual behavior: inhibition by novel antagonist. Endocrinology 125:2945–2949[Abstract/Free Full Text]
  17. Gozes, I, McCune SK, Jacobsen L, Warren D, Moody TW, Fridkin M, Brenneman DE 1991 An antagonist to vasoactive intestinal peptide: effects on cellular functions in the central nervous system. J Pharmacol Exp Ther 257:959–966[Abstract/Free Full Text]
  18. Brenneman DE, Neale EA, Foster GA, d’Autrement S, Westbrook GL 1987 Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide. J Cell Biol 104:1603–1610[Abstract/Free Full Text]
  19. Brenneman DE, Hill JM, Glazner GW, Gozes I, Phillips TW 1995 Interleukin-1{alpha} and vasoactive intestinal peptide: enigmatic regulation of neuronal survival. Int J Dev Neurosci 13:187–200[CrossRef][Medline]
  20. Brenneman DE, Gozes I 1996 A femtomolar-acting neuroprotective peptide. J Clin Invest 97:2299–2307[Medline]
  21. Festoff BE, Nelson PG, Brenneman DE 1996 Prevention of activity-dependent neuronal death: vasoactive intestinal polypeptide stimulates astrocytes to secrete the thrombin-inhibiting, neurotrophic serpin, protease nexin 1. J Neurobiol 30:255–266[CrossRef][Medline]
  22. Brenneman DE, Westbrook GL, Fitzgerald SF, et al. 1988 Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide. Nature 335:639–642[CrossRef][Medline]
  23. Brenneman DE, Hauser J, Neale E, et al. 1998 Activity-dependent neurotrophic factor: Structure-activity relationships of femtomolar-acting peptides. J Pharmacol Exp Ther 285:619–627[Abstract/Free Full Text]
  24. Blondel O, Collin C, McCarran WJ, et al. 2000 A glia-derived signal regulating neuronal differentiation. J Neurosci 20:8012–8020[Abstract/Free Full Text]
  25. White DM, Walker S, Brenneman DE, Gozes I 2000 CREB contributes to the increased neurite outgrowth of sensory neurons induced by vasoactive intestinal polypeptide and activity-dependent neurotrophic factor. Brain Res 868:31–38[CrossRef][Medline]
  26. New DAT 1971 Methods for the culture of postimplantation embryos of rodents. In: Daniel Jr JC, ed. Methods in mammalian embryology. San Francisco: Freeman; 305–319
  27. Van Maele-Fabry G, Debras JP, Francois I, Boucau M, Picard JJ 1988 Sera as culture media for 8.5 days whole mouse embryos. Arch Biol 99:431–453
  28. Brown NA, Fabro S 1981 Quantitation of rat embryonic development in vitro: a morphological scoring system. Teratology 24:65–78[CrossRef][Medline]
  29. Bradford M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  30. Burton K 1956 Study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–3239[Medline]
  31. Munro HN 1968 The determination of nucleic acids. Methods Biochem Anal 14:113–176
  32. Chomczynski P, Sacchi N 1989 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef]
  33. Kawasakai ES, Wang AM 1989 Detection of gene expression. In: Erlich HA, ed. PCR technology: principles and applications of DNA amplifications. New York: Stockton Press; 89–97
  34. Piatak Jr M, Luk KC, Williams B, Lifson JD 1993 Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. BioTechniques 14:70–81[Medline]
  35. Deleted in proof
  36. Deleted in proof
  37. Slabaugh MB, Lieberman ME, Rutledge JJ, Gjorski J 1982 Ontogeny of growth hormone and prolactin gene expression in mice. Endocrinology 110:1489–1497[Abstract/Free Full Text]
  38. Flandez B, Alvarez E, Blazquez E 1986 Delayed appearance of liver growth hormone binding sites and of growth hormone-induced somatomedin production during rat development. Biochem Biophys Res Commun 136:38–44[CrossRef][Medline]
  39. DiCicco-Bloom E, Black IB 1988 Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts. Proc Natl Acad Sci USA 85:4066–4070[Abstract/Free Full Text]
  40. Baserga R 1994 Oncogenes and the strategy of growth factors. Cell 79:927–930[CrossRef][Medline]
  41. Sell C, Dumenil G, Deveaud D, et al. 1994 Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts. Mol Cell Biol 14:3604–3612[Abstract/Free Full Text]
  42. Rappolee DA, Strum K, Schultz GA, Pederson RA, Werb Z 1990 The expression of growth factor ligands and receptors in preimplantation mouse embryos. In: Heyer S, Wiley L, eds. Early embryo development and paracrine relationships. New York: Wiley-Liss; 11–25
  43. Heyner S, Smith RM, Schultz GA 1989 Temporally regulated expression of insulin and insulin-like growth factors and their receptors in early mammalian development. BioEssays 11:171–176[CrossRef][Medline]
  44. Telford NA, Hogan A, Franz CR, Schultz GA 1990 Expression of genes for insulin and insulin-like growth factors and receptors in early postimplantation mouse embryos and embryonal carcinoma cells. Mol Reprod Dev 27:81–92[CrossRef][Medline]
  45. Czeck MP 1989 Signal transmission by the insulin-like growth factors. Cell 59:235–238[CrossRef][Medline]
  46. Bondy CA, Werner H, Roberts JCT, LeRoith D 1990 Cellular pattern of insulin-like growth factor-I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression. Mol Endocrinol 4:1386–1398[Abstract/Free Full Text]
  47. Murphy LJ, Baron DJ 1993 The IGFs and their binding proteins in murine development. Mol Reprod Dev 35:376–381[CrossRef][Medline]
  48. Athanasas-Platsis A, Quinn KA, Wong T-Y, Rolfe BE, Cavanagh AC, Morton H 1989 Passive immunization of pregnant mice against early pregnancy factor causes loss of embryonic viability. J Reprod Fertil 87:495–502[Abstract/Free Full Text]
  49. Lee SJ, Servoss SJ, Cavanagh AC, et al. 1996 Early pregnancy factor regulates brain growth of the early postimplantation (E9) mouse embryo. Soc Neurosci Abstr 22:743
  50. Kohchi C, Noguchi K, Tanabe Y, Mizuno D, Soma G 1994 Constitutive expression of TNF-{alpha} and -ß genes in mouse embryo: roles of cytokines as regulator and effector on development. Int J Biochem 26:111–119[CrossRef][Medline]
  51. Hebert JM, Basilico C, Goldfarb M, Haub O, Martin GR 1990 Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev Biol 138:454–463[CrossRef][Medline]
  52. Karperien M, Lanser P, de Laat LW, Boonstra J, Defize LH 1996 Parathyroid hormone related peptide mRNA expression during murine postimplantation development: evidence for involvement in multiple differentiation processes. Int J Dev Biol 40:599–608[Medline]
  53. Mansuy IM, van der Putten H, Schmid P, Meins M, Bjotteri FM, Monard D 1993 Variable and multiple expression of protease nexin-1 during mouse organogenesis and nervous system development. Development 119:1119–1134[Abstract]
  54. Echelard Y, Epstein DJ, St Jacques B, et al. 1993 Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75:1417–1430[CrossRef][Medline]
  55. Jessell TM, Dodd J 1992 Floor plate-derived signals and the control of neural cell pattern in vertebrates. Harv Lect 86:87–128



This article has been cited by other articles:


Home page
 EndocrinologyHome page
K. Tamura, T. Hara, M. Kutsukake, K. Iwatsuki, M. Yanagida, M. Yoshie, and H. Kogo
Expression and the Biological Activities of Insulin-Like Growth Factor-Binding Protein Related Protein 1 in Rat Uterus during the Periimplantation Period
Endocrinology, November 1, 2004; 145(11): 5243 - 5251.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Servoss, S. J.
Right arrow Articles by Hill, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Servoss, S. J.
Right arrow Articles by Hill, J. M.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals