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The role of exogenous growth-promoting factors and their receptors in embryogenesis
Reproductive Toxicology, Vol. 12, No. 2, pp. 201-207, 1998 0 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0890-6238/98 $19.00 + .OO
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THE ROILE OF EXOGENOUS GROWTH-PROMOTING FACTORS AND THEIR RECEPTORS IN EMBRYOGENESIS MARGARET K. PRA~EN Department of Human Anatomy and Cell Biology, The Medical School, Queens Medical Centre, Nottingham, United Kingdom Abstract - During organogenesis, the cells of the embryo may require growth factors that promote a cascade of intracellular events. An absolute requirement for exogenous insulin by presomite 9.5-d rat embryos grown in culture has been demonstrated. The uptake and processing of insulin and insulin-like growth factor-I showed
different uptake and localization patterns. When epidermal growth factor (EGF) or “long EGF” is added to media depleted of low molecular weight material, a dose-dependent improvement in growth is observed. Furthermore, thle specific EGF receptor signal transduction inhibitor Tyrphostin 47 can inhibit embryonic growth when it is administered in culture. When Tyrphostin 47 was microinjected into embryos on Day 11 and their growth and differentiation evaluated on Day 12 of gestation, a dose-dependent decrease in developmental score was observed. Thus, exogenous growth factors may be essential to normal rat development and these may be synthesized locally in the decidua or placental tissues. Perturbations to ligand-receptor interactions may be a mechanism for dysmorphogenesis. 0 1998 Elsevier Science Inc. Key Words: embryo culture; yolk sac; microcannulation;
growth factors.
quiescent state to enter the cell cycle (competence factors) e.g., platelet-derived growth factor and fibroblast growth factor (FGF). Others are concerned with the cell progressing through the stages of the cell cycle (progression factors), e.g., epidermal growth factor (EGF), insulin-like growth factor I (IGF-I; Reference 3). It has been suggested that perturbations in certain aspects of ligand-receptor interaction for growth factors may provide the mechanism for some congenital abuormalities. Very early studies showed that EGF could cause premature eyelid opening and incisor eruption (4) and more recently, it has been shown that the development of the teeth in mice involves both EGF and FGF-4. In this latter study, an interplay between the growth factors and the process of apoptosis (programmed cell death) has also been suggested (5). EGF is also involved in fusion of the palatal shelves (6), and binding sites for this molecule have been demonstrated in palatal meseuthyme and epithelium (7). Therefore, it has been suggested that perturbations of the interaction of the EGF receptor and this ligand, or TGFa, may be important in the generation of cleft lip and palate (8). Serum IGF-I levels have been correlated with birth size and therefore linked to intrauterine growth retardation, as well as macrosomia in diabetic pregnancy (9,lO). Transforming growth factor p has been shown by gene knockout studies to play au important role in vasculogenesis and hematopoiesis, particularly in the rodent yolk sac (11). A highly specific role for several of the
INTRODUCTION
The cells and tissues of the post-implantation embryo undergo several processes that include proliferation, differentiation, migration, and active removal of cells by apoptosis. These events may be controlled by extra- and intracellular signaling,, and growth factors might act as one such signal. It ~LSbelieved that many maternally derived growth factors are involved in the regulation of embryonic growth (e.g., References 1,2). The mechanism by which these molecules act as regulators could be by alterations in the level of expression of receptors within the conceptus, because the levels of growth factors in the maternal circulation, in most cases, do not correlate with different stages of gestation. However, it is possible that tissues local to the embryo, such as the decidua, placenta, or extraembryonic membranes, may up-regulate the synthesis of growth factors and these may act in a paractiue fashion. Most growth factors mediate their effects through specific receptors that often possess tyrosine kiuase activity and promote a cascade of intracellular signals that usually lead to cell division. Some such molecules permit cells to move from a Presented as part of l.he Vth International Symposium on Vertebrate Whole Embryo Culture: Clinical and Genetic Implications, held in Jerusalem, Israel, l-4 April, 1997. Address correspondence to Dr Margaret K. Pratten, Department of Human Anatomy and Cell Biology, The Medical School, Queens Medical Centre, Nottingham NG7 ZUH, UK. E-mail: Margaret. [email protected]. 201
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FGF family, in conjunction with various other signals, has been elucidated in pattern formation of the chick limb bud (as reviewed by Reference 12), although other researchers have shown that IGF-I and insulin may also play an important role (13). In addition, the apoptosis necessary for the degeneration of interdigital material can be inhibited by local application of FGF-2 or FGF-4 in the embryonic chick (14). There is evidence that some growth factors are produced within the embryo itself (2,15-l@. For example, detailed studies using techniques such as Northern blotting and in situ hybridization have indicated that in the embryonic, fetal and neonatal stages of development, mRNA for several growth factors is produced by developing tissues (e.g., IGF II: References 19-22; transforming growth factor CKReferences 23,24; and transforming growth factor (Y and p: Reference 25). Recent studies using gene knockout as a tool have indicated that in many cases the lack of expression of a single growth factor gene has relatively little effect on growth and survival, in a few cases only causing some small abnormalities. This has led to the theory that many functions of the growth factor families are subject to redundancy and that just because a gene is highly conserved and abundantly expressed does not mean it is essential (26). This gene redundancy may also extend to the ability of the maternal system to compensate for deficits in the embryonic make-up or indicate that certain factors are normally supplied exogenously in utero (1). MATERIALS
AND METHODS
The role of insulin, IGF-I, and EGF in embryos has been studied using whole embryo culture, supported by anembryonic yolk sac culture and intravitelline injection of rat embryos. Culture of whole rat embryos Wistar rats were mated overnight and their pregnancy timed from midnight of the night producing a vaginal plug. Conceptuses were explanted around noon on Day 9 by the method of New (27). Early head fold embryos were cultured in rat serum, or fractions of it, in bottles in a roller incubator with appropriate gassing daily. After 48 h, the embryos were removed and assessed morphologically by the method of Van Maele Fabry et al. (28). The protein content of the embryos was assessed (29). Culture of rat anembryonic yolk sacs Conceptuses were explanted as described above on Day 9 and the embryonic pole removed before culture in bottles in a roller incubator with appropriate gassing daily (30). After usually 6 d of culture, the anembryonic
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yolk sacs were harvested and measured, content was assessed (29).
and the protein
Intravitelline injection Embryos were cultured for 48 h as described above and then evaluated for the presence of a beating heart and vigorous vitelline circulation. Porential inhibitors of growth factor receptors were injected into the vitelline vessels by the method of Cumberland et al. (31). The role of low molecular weight factors and their receptors Early studies showed that during embryo culture certain biopolymers are specifically depleted from the serum. Such “exhausted” serum could be partially replenished by supplementation with physiologic concentrations of insulin or EGF (32). Using serum subjected to ultrafiltration to remove low molecular weight material, substances in the molecular weight range of 5 to 30 thousand were shown to be essential to normal embryonic growth. RESULTS
AND DISCUSSION
The role of insulin and IGFs and their receptors Maternally derived insulin may be required for normal embryogenesis. There is evidence for the receptors for insulin in early post-implantation embryos (33, 34) but very little evidence exists for synthesis of insulin either by immunocytochemistry (35) or in situ hybridization (20). It had been indicated that the growthpromoting properties of serum depleted by repeated culture of rat conceptuses could be partially restored by the addition of insulin at physiologic concentrations (32). An absolute requirement for exogenous insulin by presomite 9.5-d rat embryos grown in culture was demonstrated using serum specifically depleted of insulin by affinity chromatography (36). Using affinity chromatography to deplete insulin specifically from the culture serum to levels less than 0.5 ng/mL, as measured by radioimmunoassay, it was shown that such serum cannot support normal embryonic growth. The addition of 10 ng/mL exogenous porcine insulin can restore normal growth-promoting properties to the serum. Also, when guinea pig serum was used as a culture medium for post-implantation rat embryo culture, the embryos were found to be dysmorphogenic, with over 85% of embryos abnormal, compared to no abnormalities recorded when embryos were cultured in rat serum. This was thought to be caused by the difference in structure and potency of guinea pig insulin compared to rat insulin. Indeed, when insulin was added at physiologic concentrations to guinea pig serum, the serum showed an improved ability to support normal growth of rat embryos (37). A similar
Growth-promoting factors in rat embryogenesis 0 M. K.
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the embryo by circumventing the pinocytic/lysosomal digestion system. The uptake and processing of insulin and IGF-I were studied in the visceral yolk sac of the rat conceptus. These two ligands are very similar structurally, and both possess receptors that are dimers with tyrosine kinase activity. Although either ligand can interact with either receptor, they possess much greater affinity for their own receptor. In the case of the growing rat conceptus, it is not clear whether both receptors or only one is involved in uptake and processing of these ligands. Uptake of insulin appeared to differ from that of IGF-I when 17.5-d visceral yolk sacs taken directly from the mother were employed and given radiolabeled ligands (Figure 1; Reference 42). Also, it has been found when insulin was tagged with fluorescein and IGF-I was tagged with rhodamine and they were presented simultaneously, that the two molecules seem to be located in different vesicles and those containing IGF-I only were found deeper in the cell (43). Anembryonic yolk sac culture, in which the yolk sac is cultured as a closed vesicle, which permits the study of vectorial transport of molecules (44), has confirmed this. When the two ligands were presented to such anembryonic yolk sacs, the vesicles containing IGF-I were the only ones that were found toward the base of the cell and therefore able to be presented to the embryonic face of the yolk sac (Figure 2). Thus material could be delivered into the vitelline circulation for transfer to the embryonic circulation. Therefore, this may suggest that the two molecules,
Fig. 1. Uptake and processing of ‘*‘I-labeled insulin or IGF-I by anembryonic rat visceral yolk sacs. Yolk sacs were incubated at 37°C in Medmm 199 in the presence of 5 pg/mL ‘251-labeled insulin (a) or 5 PglmL IGF-I. Results are expressed as a clearance and the rate of uptake expressed in /.LL of medium processed per mg protein.
effect was observed when IGF-I was used to supplement guinea pig serum, suggesting that the action of insulin in this case might be mediated by the IGF Type 1 receptor. It has also been shown by Sadler and his colleagues (38,39) that mouse conceptuses cultured in the presence of a somatomedin inhibitor, isolated from diabetic rats, demonstrated developmental abnormalities and growth retardation. Further studies indicated that this effect was mediated via an effect on pinocytic uptake and processing of protein by the visceral yolk sac (40), which caused nutritional deprivation in the embryos. Furthermore, it was shown that the effect of somatomedin inhibitors could be at least partly reversed by the addition of exogenous essential amino acids to the culture medium (41), which would provide the necessary substrates for
Fig. 2. Uptake and processing of fluorescein isothiocyanate (FITC)-labeled insulin and tetra ethyl rhodamine isothiocyanate (TRITC)-labeled IGF I by anembryonic rat visceral yolk sacs. Yolk sacs were incubated at 37°C in Medium 199 in the simultaneous presence of FITC-labeled insulin (5 pg/mL) and TRITC-labeled IGF-I (5 pg/mL) for 1 h. The tissue was then rinsed in saline, fixed, snap-frozen, cryostat sectioned, and wet mounted in an antiphotobleaching mountant. It was viewed under a confocal microscope. The micrograph shows images for FITC-labeled insulin (left) and TRITC-labeled IGF-I (right). Scale bar, 50 pm.
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WRS
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Fig. 3. The role of EGF and the EGF receptor during early organogenesis: effect on morphologic score of rat embryos cultured in vitro. Conceptuses were explanted on Gestational Day 9 and cultured in serum that had been subjected to ultrafiltration to remove material below 30,000 molecular weight in the presence or absence of various concentrations of EGF (a) or “long EGF” (b). In c, further embryos were cultured in whole rat serum in the presence of Tyrphostin 47 or Tyrphostin 1. Embryos were assessed by the scoring system of Van Maele Fabry et al. (28). In d, embryos were explanted on Day 9 and cultured in whole rat serum until Day 11, when they were microinjected into the vitelline circulation with 200 nL of medium containing Tyrphostin 47 or Tyrphostin 1.They were cultured for a further 24 h and scored by using the method of Cumberland et al. (31). All results shown are the median value and interquartile range of scores for at least 10 embryos. WRS, whole rat serum; 30kR, retenate containing molecules with MW > 30,000; DMSO, DMSO vehicle control containing 0.05% DMSO; A47, Tyrphostin 47; Al, Tyrphostin 1. *, significant difference in growth from 30K retenate control (b), rat serum control (c), or from DMSO vehicle control (d) at P < 0.05. Data derived from Tebbs et al. (47). although
similar,
are actually
whereas
insulin
is processed
ma1 system,
handled
differently
and that
via the endosomal/lysoso-
IGF-I may be transported to the embryo intact. When antisera to either IGF-I or the Type 1 IGF receptor were included in the culture medium, the growth of embryos was abnormal (Karabulut and Pratten; Cowley and Pratten, 1997, unpublished observations). A role for the IGFs has been suggested during palatal closure (45). Gene knockout studies of mice carrying null mutations for the IGFs and the Type 1 and Type 2 receptor have shown that the Type 1 receptor is of greatest importance for maintenance of growth of the embryo, whereas Type 2 receptors or an additional unknown receptor, may play a role in the maintenance of the placenta (34). Null mutations of the IGF-I gene show a 60% growth retardation, and some of these die shortly
after birth. When the Type 1 receptor is deleted, the pups are only 45% of normal size and die of respiratory failure at birth, exhibiting general delays in muscle and skeletal development (46). The role of EGF and its receptor A role for EGF as a supplement to media depleted of low molecular weight material by the use of immersible filters or centrifugal concentrators with a cut off of 30,000 was shown. When EGF is added to such sera, a dose-dependent improvement in growth was observed. Further studies making use of the more stable molecule “long EGF,” which has an amino terminal extension of 53 amino acids, indicates that growth of embryos can be promoted by molecules of the EGF family. Furthermore, the specific EGF receptor signal transduction inhibitor
Growth-promoting
factors in rat embryogenesis
Tyrphostin 47 can inhibit embryonic growth when administered in culture. It has been possible to look at the direct effects on the embryo of various inhibitors of growth factor receptor function using microcannulation of the embryonic vasculature to bypass the metabolic effects of the yolk sac. When Tyrphostin 47 was microinjected into embryos on Day 11 and their growth and differentiation evaluated on Day 12 of gestation, a decrease in develop:mental score was observed with Tyrphostin 47 (Figure 3; Reference 47). Additionally, EGF has been shown to be avidly captured by the visceral yolk sac of the 11 S-d conceptus in culture (48) and also by anembryonic yolk sacs grown in culture. In the case of EGF, the receptor has been identified in embryonic tissues (49-52), but evidence indicates that there is no embryonic synthesis of the molecule until Day 19 in the rat (53,54). However, EGF is known to be produced by the decidual cells immediately surrounding the growing conceptus in the mouse (55) The receptor for EGF is known to be expressed even at the preimplantation stage of mouse development (56,57). Targeted disruption of the EGF receptor has indicated an important role for this molecule, particularly in midgestation in the mouse (.58,59). It has also been shown that gene knockout of TIGFcx, the embryonically produced EGF analogue, does not completely prevent development (Ferguson, 1997, personal communication), probably because of the capture of exogenous EGF at the time when the embryo would normally be self-sufficient. Mice with a null mutation of the TGFa gene have abnormalities of the skin and appendages, in particular curly whiskers and wavy hair (60,61). The similarity between this phenotype and that of the mutant waved-l mouse have led to the conclusion that this is a mutation of the EGF (TGFa) receptor gene (62). Could perturbations in growth factor function be a mechanism for teratogenesis? The results indicate that exogenous growth factors may be essential to normal rat development and that these may be provided in the maternal serum or synthesized locally in the decidua or placental tissues. Perturbations to ligand-rec’eptor interactions may be a mechanism for dysmorphogenesis. A role for growth factors has been suggested in the generation of small for date babies caused by intrauterine growth retardation. Therefore, it would be of interest to compare the uptake and processing of growth factors, as well as receptor distributions, in normal embryogenesis and embryos under various types of stress, such as nutritional deficit or in the presence of teratogens such as ethanol or valproate. The potential role for growth factor perturbations is outlined in Figure 4. Because of the complex role of the yolk sac, particularly its capacity to transport specific molecules as
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DEFECT Fig. 4. Some potential growth factor-signaling pathology.
mechanisms whereby pathways may cause
disruptions to developmental
well as digest exogenous material, it is not clear whether a growth factor is likely to interact with the yolk sac receptors in the usual way or interact with a receptor that permits transport of the molecule to the embryo intact. Thus, it is possible that the growth factor binds to a growth factor receptor at the surface, causes autophosphorylation of the tyrosine residues, and sets off a cascade of intracellular signals. These signals could result in the yolk sac expanding due to the triggering of cell division. Alternatively, the effect could be on the expression of various genes in the yolk sac that might result in altered uptake or transport functions. Indirectly, this could then have an effect on embryonic growth. On the other hand, because of the known transport routes that avoid the lysosomal compartment available in the yolk sac for certain specific molecules, it is conceivable that the growth factor receptors on the yolk sac are principally not for the transduction of signals at the yolk sac surface but for transport of these factors to the embryo intact. Such a routing would require a sorting mechanism within the endosomal compartment of the yolk sac cell so that the receptor-ligand complex could be directed to the base of the cell and thus into the vitelline circulation and to the embryo. Acknowledgmenfs - MKP would like to acknowledge the work performed while working in her research group both in the Department of Human Anatomy and Cell Biology, University of Nottingham, and in the Department of Anatomy, University of Leicester, by Peter Cumberland, Elizabeth Cowley, Karen Andrew& Caroline Tebbs, and Tim Jefferson.
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ELSEVIER
PII SOWO-6238(97)00146-9
THE ROILE OF EXOGENOUS GROWTH-PROMOTING FACTORS AND THEIR RECEPTORS IN EMBRYOGENESIS MARGARET K. PRA~EN Department of Human Anatomy and Cell Biology, The Medical School, Queens Medical Centre, Nottingham, United Kingdom Abstract - During organogenesis, the cells of the embryo may require growth factors that promote a cascade of intracellular events. An absolute requirement for exogenous insulin by presomite 9.5-d rat embryos grown in culture has been demonstrated. The uptake and processing of insulin and insulin-like growth factor-I showed
different uptake and localization patterns. When epidermal growth factor (EGF) or “long EGF” is added to media depleted of low molecular weight material, a dose-dependent improvement in growth is observed. Furthermore, thle specific EGF receptor signal transduction inhibitor Tyrphostin 47 can inhibit embryonic growth when it is administered in culture. When Tyrphostin 47 was microinjected into embryos on Day 11 and their growth and differentiation evaluated on Day 12 of gestation, a dose-dependent decrease in developmental score was observed. Thus, exogenous growth factors may be essential to normal rat development and these may be synthesized locally in the decidua or placental tissues. Perturbations to ligand-receptor interactions may be a mechanism for dysmorphogenesis. 0 1998 Elsevier Science Inc. Key Words: embryo culture; yolk sac; microcannulation;
growth factors.
quiescent state to enter the cell cycle (competence factors) e.g., platelet-derived growth factor and fibroblast growth factor (FGF). Others are concerned with the cell progressing through the stages of the cell cycle (progression factors), e.g., epidermal growth factor (EGF), insulin-like growth factor I (IGF-I; Reference 3). It has been suggested that perturbations in certain aspects of ligand-receptor interaction for growth factors may provide the mechanism for some congenital abuormalities. Very early studies showed that EGF could cause premature eyelid opening and incisor eruption (4) and more recently, it has been shown that the development of the teeth in mice involves both EGF and FGF-4. In this latter study, an interplay between the growth factors and the process of apoptosis (programmed cell death) has also been suggested (5). EGF is also involved in fusion of the palatal shelves (6), and binding sites for this molecule have been demonstrated in palatal meseuthyme and epithelium (7). Therefore, it has been suggested that perturbations of the interaction of the EGF receptor and this ligand, or TGFa, may be important in the generation of cleft lip and palate (8). Serum IGF-I levels have been correlated with birth size and therefore linked to intrauterine growth retardation, as well as macrosomia in diabetic pregnancy (9,lO). Transforming growth factor p has been shown by gene knockout studies to play au important role in vasculogenesis and hematopoiesis, particularly in the rodent yolk sac (11). A highly specific role for several of the
INTRODUCTION
The cells and tissues of the post-implantation embryo undergo several processes that include proliferation, differentiation, migration, and active removal of cells by apoptosis. These events may be controlled by extra- and intracellular signaling,, and growth factors might act as one such signal. It ~LSbelieved that many maternally derived growth factors are involved in the regulation of embryonic growth (e.g., References 1,2). The mechanism by which these molecules act as regulators could be by alterations in the level of expression of receptors within the conceptus, because the levels of growth factors in the maternal circulation, in most cases, do not correlate with different stages of gestation. However, it is possible that tissues local to the embryo, such as the decidua, placenta, or extraembryonic membranes, may up-regulate the synthesis of growth factors and these may act in a paractiue fashion. Most growth factors mediate their effects through specific receptors that often possess tyrosine kiuase activity and promote a cascade of intracellular signals that usually lead to cell division. Some such molecules permit cells to move from a Presented as part of l.he Vth International Symposium on Vertebrate Whole Embryo Culture: Clinical and Genetic Implications, held in Jerusalem, Israel, l-4 April, 1997. Address correspondence to Dr Margaret K. Pratten, Department of Human Anatomy and Cell Biology, The Medical School, Queens Medical Centre, Nottingham NG7 ZUH, UK. E-mail: Margaret. [email protected]. 201
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FGF family, in conjunction with various other signals, has been elucidated in pattern formation of the chick limb bud (as reviewed by Reference 12), although other researchers have shown that IGF-I and insulin may also play an important role (13). In addition, the apoptosis necessary for the degeneration of interdigital material can be inhibited by local application of FGF-2 or FGF-4 in the embryonic chick (14). There is evidence that some growth factors are produced within the embryo itself (2,15-l@. For example, detailed studies using techniques such as Northern blotting and in situ hybridization have indicated that in the embryonic, fetal and neonatal stages of development, mRNA for several growth factors is produced by developing tissues (e.g., IGF II: References 19-22; transforming growth factor CKReferences 23,24; and transforming growth factor (Y and p: Reference 25). Recent studies using gene knockout as a tool have indicated that in many cases the lack of expression of a single growth factor gene has relatively little effect on growth and survival, in a few cases only causing some small abnormalities. This has led to the theory that many functions of the growth factor families are subject to redundancy and that just because a gene is highly conserved and abundantly expressed does not mean it is essential (26). This gene redundancy may also extend to the ability of the maternal system to compensate for deficits in the embryonic make-up or indicate that certain factors are normally supplied exogenously in utero (1). MATERIALS
AND METHODS
The role of insulin, IGF-I, and EGF in embryos has been studied using whole embryo culture, supported by anembryonic yolk sac culture and intravitelline injection of rat embryos. Culture of whole rat embryos Wistar rats were mated overnight and their pregnancy timed from midnight of the night producing a vaginal plug. Conceptuses were explanted around noon on Day 9 by the method of New (27). Early head fold embryos were cultured in rat serum, or fractions of it, in bottles in a roller incubator with appropriate gassing daily. After 48 h, the embryos were removed and assessed morphologically by the method of Van Maele Fabry et al. (28). The protein content of the embryos was assessed (29). Culture of rat anembryonic yolk sacs Conceptuses were explanted as described above on Day 9 and the embryonic pole removed before culture in bottles in a roller incubator with appropriate gassing daily (30). After usually 6 d of culture, the anembryonic
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yolk sacs were harvested and measured, content was assessed (29).
and the protein
Intravitelline injection Embryos were cultured for 48 h as described above and then evaluated for the presence of a beating heart and vigorous vitelline circulation. Porential inhibitors of growth factor receptors were injected into the vitelline vessels by the method of Cumberland et al. (31). The role of low molecular weight factors and their receptors Early studies showed that during embryo culture certain biopolymers are specifically depleted from the serum. Such “exhausted” serum could be partially replenished by supplementation with physiologic concentrations of insulin or EGF (32). Using serum subjected to ultrafiltration to remove low molecular weight material, substances in the molecular weight range of 5 to 30 thousand were shown to be essential to normal embryonic growth. RESULTS
AND DISCUSSION
The role of insulin and IGFs and their receptors Maternally derived insulin may be required for normal embryogenesis. There is evidence for the receptors for insulin in early post-implantation embryos (33, 34) but very little evidence exists for synthesis of insulin either by immunocytochemistry (35) or in situ hybridization (20). It had been indicated that the growthpromoting properties of serum depleted by repeated culture of rat conceptuses could be partially restored by the addition of insulin at physiologic concentrations (32). An absolute requirement for exogenous insulin by presomite 9.5-d rat embryos grown in culture was demonstrated using serum specifically depleted of insulin by affinity chromatography (36). Using affinity chromatography to deplete insulin specifically from the culture serum to levels less than 0.5 ng/mL, as measured by radioimmunoassay, it was shown that such serum cannot support normal embryonic growth. The addition of 10 ng/mL exogenous porcine insulin can restore normal growth-promoting properties to the serum. Also, when guinea pig serum was used as a culture medium for post-implantation rat embryo culture, the embryos were found to be dysmorphogenic, with over 85% of embryos abnormal, compared to no abnormalities recorded when embryos were cultured in rat serum. This was thought to be caused by the difference in structure and potency of guinea pig insulin compared to rat insulin. Indeed, when insulin was added at physiologic concentrations to guinea pig serum, the serum showed an improved ability to support normal growth of rat embryos (37). A similar
Growth-promoting factors in rat embryogenesis 0 M. K.
A
Radiollabelled Insulin Uptake 1600
_ 1400 .S g 1200 =1000 g
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the embryo by circumventing the pinocytic/lysosomal digestion system. The uptake and processing of insulin and IGF-I were studied in the visceral yolk sac of the rat conceptus. These two ligands are very similar structurally, and both possess receptors that are dimers with tyrosine kinase activity. Although either ligand can interact with either receptor, they possess much greater affinity for their own receptor. In the case of the growing rat conceptus, it is not clear whether both receptors or only one is involved in uptake and processing of these ligands. Uptake of insulin appeared to differ from that of IGF-I when 17.5-d visceral yolk sacs taken directly from the mother were employed and given radiolabeled ligands (Figure 1; Reference 42). Also, it has been found when insulin was tagged with fluorescein and IGF-I was tagged with rhodamine and they were presented simultaneously, that the two molecules seem to be located in different vesicles and those containing IGF-I only were found deeper in the cell (43). Anembryonic yolk sac culture, in which the yolk sac is cultured as a closed vesicle, which permits the study of vectorial transport of molecules (44), has confirmed this. When the two ligands were presented to such anembryonic yolk sacs, the vesicles containing IGF-I were the only ones that were found toward the base of the cell and therefore able to be presented to the embryonic face of the yolk sac (Figure 2). Thus material could be delivered into the vitelline circulation for transfer to the embryonic circulation. Therefore, this may suggest that the two molecules,
Fig. 1. Uptake and processing of ‘*‘I-labeled insulin or IGF-I by anembryonic rat visceral yolk sacs. Yolk sacs were incubated at 37°C in Medmm 199 in the presence of 5 pg/mL ‘251-labeled insulin (a) or 5 PglmL IGF-I. Results are expressed as a clearance and the rate of uptake expressed in /.LL of medium processed per mg protein.
effect was observed when IGF-I was used to supplement guinea pig serum, suggesting that the action of insulin in this case might be mediated by the IGF Type 1 receptor. It has also been shown by Sadler and his colleagues (38,39) that mouse conceptuses cultured in the presence of a somatomedin inhibitor, isolated from diabetic rats, demonstrated developmental abnormalities and growth retardation. Further studies indicated that this effect was mediated via an effect on pinocytic uptake and processing of protein by the visceral yolk sac (40), which caused nutritional deprivation in the embryos. Furthermore, it was shown that the effect of somatomedin inhibitors could be at least partly reversed by the addition of exogenous essential amino acids to the culture medium (41), which would provide the necessary substrates for
Fig. 2. Uptake and processing of fluorescein isothiocyanate (FITC)-labeled insulin and tetra ethyl rhodamine isothiocyanate (TRITC)-labeled IGF I by anembryonic rat visceral yolk sacs. Yolk sacs were incubated at 37°C in Medium 199 in the simultaneous presence of FITC-labeled insulin (5 pg/mL) and TRITC-labeled IGF-I (5 pg/mL) for 1 h. The tissue was then rinsed in saline, fixed, snap-frozen, cryostat sectioned, and wet mounted in an antiphotobleaching mountant. It was viewed under a confocal microscope. The micrograph shows images for FITC-labeled insulin (left) and TRITC-labeled IGF-I (right). Scale bar, 50 pm.
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OJ
WRS
3okR
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A41 5a
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tyrphostin concentration (@vi) d
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Fig. 3. The role of EGF and the EGF receptor during early organogenesis: effect on morphologic score of rat embryos cultured in vitro. Conceptuses were explanted on Gestational Day 9 and cultured in serum that had been subjected to ultrafiltration to remove material below 30,000 molecular weight in the presence or absence of various concentrations of EGF (a) or “long EGF” (b). In c, further embryos were cultured in whole rat serum in the presence of Tyrphostin 47 or Tyrphostin 1. Embryos were assessed by the scoring system of Van Maele Fabry et al. (28). In d, embryos were explanted on Day 9 and cultured in whole rat serum until Day 11, when they were microinjected into the vitelline circulation with 200 nL of medium containing Tyrphostin 47 or Tyrphostin 1.They were cultured for a further 24 h and scored by using the method of Cumberland et al. (31). All results shown are the median value and interquartile range of scores for at least 10 embryos. WRS, whole rat serum; 30kR, retenate containing molecules with MW > 30,000; DMSO, DMSO vehicle control containing 0.05% DMSO; A47, Tyrphostin 47; Al, Tyrphostin 1. *, significant difference in growth from 30K retenate control (b), rat serum control (c), or from DMSO vehicle control (d) at P < 0.05. Data derived from Tebbs et al. (47). although
similar,
are actually
whereas
insulin
is processed
ma1 system,
handled
differently
and that
via the endosomal/lysoso-
IGF-I may be transported to the embryo intact. When antisera to either IGF-I or the Type 1 IGF receptor were included in the culture medium, the growth of embryos was abnormal (Karabulut and Pratten; Cowley and Pratten, 1997, unpublished observations). A role for the IGFs has been suggested during palatal closure (45). Gene knockout studies of mice carrying null mutations for the IGFs and the Type 1 and Type 2 receptor have shown that the Type 1 receptor is of greatest importance for maintenance of growth of the embryo, whereas Type 2 receptors or an additional unknown receptor, may play a role in the maintenance of the placenta (34). Null mutations of the IGF-I gene show a 60% growth retardation, and some of these die shortly
after birth. When the Type 1 receptor is deleted, the pups are only 45% of normal size and die of respiratory failure at birth, exhibiting general delays in muscle and skeletal development (46). The role of EGF and its receptor A role for EGF as a supplement to media depleted of low molecular weight material by the use of immersible filters or centrifugal concentrators with a cut off of 30,000 was shown. When EGF is added to such sera, a dose-dependent improvement in growth was observed. Further studies making use of the more stable molecule “long EGF,” which has an amino terminal extension of 53 amino acids, indicates that growth of embryos can be promoted by molecules of the EGF family. Furthermore, the specific EGF receptor signal transduction inhibitor
Growth-promoting
factors in rat embryogenesis
Tyrphostin 47 can inhibit embryonic growth when administered in culture. It has been possible to look at the direct effects on the embryo of various inhibitors of growth factor receptor function using microcannulation of the embryonic vasculature to bypass the metabolic effects of the yolk sac. When Tyrphostin 47 was microinjected into embryos on Day 11 and their growth and differentiation evaluated on Day 12 of gestation, a decrease in develop:mental score was observed with Tyrphostin 47 (Figure 3; Reference 47). Additionally, EGF has been shown to be avidly captured by the visceral yolk sac of the 11 S-d conceptus in culture (48) and also by anembryonic yolk sacs grown in culture. In the case of EGF, the receptor has been identified in embryonic tissues (49-52), but evidence indicates that there is no embryonic synthesis of the molecule until Day 19 in the rat (53,54). However, EGF is known to be produced by the decidual cells immediately surrounding the growing conceptus in the mouse (55) The receptor for EGF is known to be expressed even at the preimplantation stage of mouse development (56,57). Targeted disruption of the EGF receptor has indicated an important role for this molecule, particularly in midgestation in the mouse (.58,59). It has also been shown that gene knockout of TIGFcx, the embryonically produced EGF analogue, does not completely prevent development (Ferguson, 1997, personal communication), probably because of the capture of exogenous EGF at the time when the embryo would normally be self-sufficient. Mice with a null mutation of the TGFa gene have abnormalities of the skin and appendages, in particular curly whiskers and wavy hair (60,61). The similarity between this phenotype and that of the mutant waved-l mouse have led to the conclusion that this is a mutation of the EGF (TGFa) receptor gene (62). Could perturbations in growth factor function be a mechanism for teratogenesis? The results indicate that exogenous growth factors may be essential to normal rat development and that these may be provided in the maternal serum or synthesized locally in the decidua or placental tissues. Perturbations to ligand-rec’eptor interactions may be a mechanism for dysmorphogenesis. A role for growth factors has been suggested in the generation of small for date babies caused by intrauterine growth retardation. Therefore, it would be of interest to compare the uptake and processing of growth factors, as well as receptor distributions, in normal embryogenesis and embryos under various types of stress, such as nutritional deficit or in the presence of teratogens such as ethanol or valproate. The potential role for growth factor perturbations is outlined in Figure 4. Because of the complex role of the yolk sac, particularly its capacity to transport specific molecules as
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DEFECT Fig. 4. Some potential growth factor-signaling pathology.
mechanisms whereby pathways may cause
disruptions to developmental
well as digest exogenous material, it is not clear whether a growth factor is likely to interact with the yolk sac receptors in the usual way or interact with a receptor that permits transport of the molecule to the embryo intact. Thus, it is possible that the growth factor binds to a growth factor receptor at the surface, causes autophosphorylation of the tyrosine residues, and sets off a cascade of intracellular signals. These signals could result in the yolk sac expanding due to the triggering of cell division. Alternatively, the effect could be on the expression of various genes in the yolk sac that might result in altered uptake or transport functions. Indirectly, this could then have an effect on embryonic growth. On the other hand, because of the known transport routes that avoid the lysosomal compartment available in the yolk sac for certain specific molecules, it is conceivable that the growth factor receptors on the yolk sac are principally not for the transduction of signals at the yolk sac surface but for transport of these factors to the embryo intact. Such a routing would require a sorting mechanism within the endosomal compartment of the yolk sac cell so that the receptor-ligand complex could be directed to the base of the cell and thus into the vitelline circulation and to the embryo. Acknowledgmenfs - MKP would like to acknowledge the work performed while working in her research group both in the Department of Human Anatomy and Cell Biology, University of Nottingham, and in the Department of Anatomy, University of Leicester, by Peter Cumberland, Elizabeth Cowley, Karen Andrew& Caroline Tebbs, and Tim Jefferson.
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