Insulin-Like Growth Factor-I and Insulin as Growth and Differentiation Factors in Chicken Embryogenesis FLORA DE PABLO,1 HENRI L. ROBCIS,2 TRINIDAD CAUCUS, JORGE ALEMANY, LOUIS SCAVO, and JOSE SERRANO Section on Receptors and Hormone Action, Diabetes Branch, and Veterinary Resources Program, National Center for Research Resources, National Institutes of Health, Bethesda, Maryland 20892 (Received for publication August 5, 1990)

1991 Poultry Science 70:1790-1796 INSULIN AND INSUUN-L1KE GROWTH FACTOR-I MESSENGER RIBONUCLEIC ACID IN DEVELOPING EMBRYOS

Although in adult vertebrates the main source of insulin is the pancreas and the main source of insulin-like growth factor-I (IGF-I) is the liver, the sources in fetal life are much less restricted (Daughaday and Rotwein, 1989; De Pablo et al, 1990). Insulin mRNA transcripts have been demonstrated in the chick embryo liver during midembryogenesis (Days 12 to 18) (Serrano et al, 1989) (Figure 1). The size of the main transcript is similar to that found in the pancreas, although its abundance is much lower. The content of insulin mRNA increases at the end of embryogenesis in the pancreas, but it decreases by Day 21 (hatching) in the liver. Interestingly, IGF-I mRNA is virtually undetectable in liver, even by extremely sensitive techniques such as the polymerase chain reaction (PCR), until the end of embryogenesis (Days 18 and hatching) (Serrano et al, 1990). (Figure 2). In a search for organs that may be a source of IGF-I

To whom correspondence should be addressed. Veterinary Resources Program.

during embryogenesis the present authors looked for IGF-I mRNA in other tissues. In embryos on Days 12 and 13, IGF-I transcripts were found in the. legs, eye, heart, stomach, and brain as well as in the whole embryo (Figure 3). These organs may all potentially contribute to the circulating IGF-I at that age. The pancreas also has detectable levels of IGFI mRNA during midembryogenesis (Serrano et al, 1990); this organ, thus, may synthesize IGF-I and secrete it into the bloodstream. Further, even before organogenesis begins and blood circulation starts in the embryo, during gastrulation and neurulation, the present authors found IGF-I and insulin transcripts. Thus, in addition to the typical endocrine-type of production of insulin and IGF-I in late embryogenesis and the postnatal period, a paracrine-type action of these peptides is likely to play a role in early embryogenesis, at least in the chicken. INSULIN-LIKE GROWTH FACTOR-I CONCENTRATIONS IN EMBRYOS WITH NORMAL AND DELAYED GROWTH

Although insulin serum levels in embryos are low and increase only slightly from the time the pancreas develops until hatching, there was no information on serum levels of IGF-I during chicken ontogeny. Recently, the 1790

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ABSTRACT The avian embryo has been a useful model system for studies on the role of insulin and its close relative insulin-like growth factor-I (IGF-I) in development. The unfertilized chicken egg contains both peptides from maternal origin, and the embryo expresses insulin and IGF-I before the major organs are formed. Insulin receptors and IGF-I receptors are found in the blastoderm and in all tissues examined during organogenesis. When exogenous insulin or IGF-I are added to the embryo, growth and differentiation events are stimulated. By contrast, insulin antibodies and insulin receptor antibodies retard embryo development. In embryos cultured ex ovo, in which growth is impaired, the levels of serum IGF-I are decreased. (Key words: insulin-like growth factor-I, insulin, receptors, development, chicken embryo)

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SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT TABLE 1. Insulin-like growth factor-I serum levels in chicken embryos Day of incubation Incubation group

10

12

14

15

16

17.9 2.5

8.3 3.7

(ng/mL) In ovo Ex ovo

in

4.0 4.1

13.5 4.3

•in

'For methodological details see Robcis et al. (1991).

bryogenesis IGF-I peak was totally blunted (Robcis et al., 1991). Previously, De Pablo et al. (1982) reported the presence of insulin in the unfertilized egg; more recently, IGF-I has been found. The concentration of IGF-I is in the range of that of insulin or slightly higher, at least in the yolk (Scavo et al., 1989).

Liver I Embryo age 12

Leg Pancreas 1

18 21

12

3weeks posth. -28S

-18S «f

Total R N A L

50/ig-

30Mg

FIGURE 1. An RNA blot of insulin in the developing liver. Total RNA (50 (Xg) from embryonic livers of the days indicated was fractionated in a formaldehyde gel and transferred lo a nylon membrane. The blot was hybridized with a 48-bp oligomer specific for chicken insulin. Total RNA from lower limbs (leg) of embryos at Day 12 and from the pancreas (30 ug) of a chicken at 3-wk posthatch (posth.) were used as negative and positive controls, respectively. The autoradiogram was exposed for 15 days at -70 C. The arrows indicate the major and minor pancreatic transcripts detected in this overexposed autoradiogram. In the liver, only the major transcript was detectable. For methodological details see Serrano et al. (1989).

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present authors measured IGF-I in serum from Day 4 of embryogenesis onward. The IGF-I was low, but detectable, by Day 6, increased progressively during the 2nd wk of embryogenesis, peaked at Day 15, and then decreased until hatching (Table 1). In embryos cultured ex ovo that are growth retarded, the midem-

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DE PABLO ET AL.

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RECEPTORS FOR INSULIN-LIKE GROWTH FACTOR-I DOMINATE IN MULTIPLE EMBRYONIC TISSUES OVER INSULIN RECEPTORS

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In all tissues studied, using membrane preparations and typical labeled ligand binding-competition assays, both insulin receptors

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^^^ days of development FIGURE 3. Polymerase chain reaction (PCR) analysis of insulin-like growth factor-I (IGF-I) gene expression in tissues from Days 12 to 13 embryos. Total RNA (10 |ig) from the tissues indicated (all from Day 12 embryos except the eye which was from a Day 13 embryo) was reversed transcribed and processed by PCR as described in the legend of Figure 2. The R indicates an aliquot of RNA treated with ribonuclease, used as a negative control. The whole E. indicates an RNA sample from the whole embryo. The arrow indicates the specific hybridizing band obtained with the DNA, amplified using oligomers corresponding to exon HI of chicken IGF-I gene. For methodological details see Serrano et al. (1990).

FIGURE 4. Binding of labeled peptides to chicken embryo membranes during ontogeny. [ I]-labeled insulin-like growth factor-I (IGF-I) (•), IGF-U" (O), and insulin (A) were incubated with membrane preparations obtained from developing heart (top), liver (middle), and limb buds (bottom) of the designated days of development. The specific binding, after subtracting the nonspecific binding from the total ligand bound, is shown. B/T 100 represents the percentage of specific binding relative to total counts per minute. The [ IS I]IGF-I binding is highest in heart and limb buds, and [ I]insulin binding predominates in liver. For methodological details see Bassas et al. (1988).

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FIGURE 2. Polymerase chain reaction (PCR) analysis of IGF-I gene expression in the developing liver. The total RNA (10 ^g) from livers of embryos at Day 18 (L18), neonate (Lip), and 50-day-old chicken (L50p) was reversed transcribed and the complementary cDNA was amplified by PCR using chicken insulin-like growth factor-I (IGF-I) specific oligomers as primers. A tube without RNA (Lane N) and a tube with genomic DNA (Lane G) were used as negative controls. A) Amplified products were run in an agarose gel and stained by ethidium bromide. A photograph taken under ultraviolet light is shown. B) The products were transferred to a nylon membrane that was hybridized to a random primed P-Iabeled chicken IGF-I genomic fragment. The autoradiogram shown was obtained after 1 day of exposure. For methodological details see Serrano et al. (1990).

SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT

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TABLE 2. Effects of anti-insulin and anti-insulin receptor antibodies on early chick embryos

Parameters

Anti-insulin antibody

Anti-insulin receptor antibody

Death rate Growth retardation Weight Total protein Total DNA Total RNA Creatine kinase Creatine kinase MB Triglycerides Phospholipids Cholesterol

Increased Increased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decrease.:;' Decreased

Increased Increased Decreased Decreased Decreased Decreased Decreased Unchanged Decreased Decreased Decreased

Very small change.

for both ligands were visualized (Figure 5). In head and brain, for example, they formed distinct patterns of distribution localized to well-defined anatomical structures (Bassas et al., 1989). However, earlier in embryogenesis (Days 6 to 12) the anatomical distribution of insulin receptors and IGF-I receptors overlapped. By adapting the autoradiographic approach to whole mounted young embryos Girbau et al. (1989) were able to detect binding of both ligands in the neurulating embryo, specifically in ectodermal structures. The receptors in young embryos contain an active tyrosine kinase sensitive to insulin and

FIGURE 5. Autoradiographic analysis of [125I]insulin binding and [I25I]insulin-like growth factor-I (IGF-I) binding to coronal sections of head and brain. Adjacent sections (15 urn) of a day 12 embryo head (top left and center) and an adult brain (lower left and center) were incubated with [125I)jnsulin (left top and left bottom) or [125rjIGF-I (center top and center bottom) under standard binding conditions. Autoradiograms were generated and photographs were taken under identical conditions. The drawings top and bottom right are schemes showing approximate planes at which serial sections were taken for the study. Note that f 25 I]IGF-I binding is more intense in the embryo head, but [125I]insulin binding is much higher than [125I]IGF-I binding in adult brain. For methodological details see Bassas et al. (1989).

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and IGF-I receptors were identified (Figure 4) (Bassas et al., 1987, 1988). These tissues included brain, heart, muscle, liver, limb buds, and the lens of the eye. Labeled IGF-II appears to bind exclusively to IGF-I receptors in chick embryo membranes. In the developing organs, distinctive patterns of binding were found. Labeled IGF-I binding was more prominent than insulin binding in all tissues with the exception of liver in which insulin binding was higher. The IGF-I receptors were also more prominent in the whole embryo during gastrulation and neurulation (Girbau et ai, 1989). By using direct tissue autoradiography, receptors

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IGF-I. Autophosphorylation of the (J-subunit of the receptors is demonstrable in embryos by Day 2 (unpublished observation). The ability to phosphorylate an exogenous substrate is developmentally regulated, increasing markedly in young embryos between Day 2 and Day 4 (Girbau et al., 1989).

IGF-I (M) 0

10~9

1 0 ' 8 10~7

Day 4 Embryos

pCP1

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10 9

10 8

10 7

Hormone Concentration (M)

10

50

100

1000

PEPTIDE DOSE (ng/embryol

FIGURE 6. Effect of insulin, insulin-like growth factor-I (IGF-I), proinsulin, and desoctapeptide insulin (D.O.P) on developing whole embryos. Different concentrations of the peptides were applied to Day 2 chick embryos; at Day 4 the embryos were weighed and homogenized and the content of protein, total creatine kinase (CK), and its isozyme creatine-kinase MB (CKMB) were measured. All the values are expressed as the percentage above control (100%) obtained with embryos injected with vehicle. For methodological details see Girbau et al. (1987).

FIGURE 7. Stimulation of 5l-crystallin promoter activity by insulin-like growth factor-I (IGF-I) and insulin. Top: Lens epithelial cells from Days 6 and 7 chicken embryo were cultured for 24 to 30 h, then transfected with a plasmid DNA (pCPl) containing a deletion of the 51crystallin promoter (-120 to +23) and the chloramphenicol acetyl transferase gene (CAT) in an expression vector. After washing, the cells were cultured for 24 h with IGF-I at the concentrations indicated and harvested; CAT activity was determined by separation of acetylated and unacetylated chloramphenicol in the cell lysate using thin layer chromatography; an autoradiogram of a plate is shown. Bottom: Transfected cells were incubated either with insulin or IGF-I as described in A, and the relative CAT activity, with respect to untreated cultures, was calculated. For methodological details see Alemany et al. (1990).

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SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT INSULIN AND INSULIN-LIKE GROWTH FACTOR-I EFFECTS ON GROWTH AND DIFFERENTIATION

ACKNOWLEDGMENTS

The authors wish to thank Jesse Roth for support to this project, Maxine Lesniak for

critical reading of the manuscript, and Esther Bergman for secretarial assistance. The studies reviewed here were partially supported by the U.S. - Spain Joint Committee for Scientific and Technological Cooperation. REFERENCES Alemany, J., T. Borras, and F. De Pablo, 1990. Transcriptional stimulation of the 8-crystallin gene by insulin-like growth factor I and insulin requires DNA m-elements in chicken. Proc. Natl. Acad. Sci. USA 87:3353-3357. Alemany, J., P. Zelenka, J. Serrano, and F. De Pablo, 1989. Insulin-like growth factor I and insulin regulate 8-crystallin gene expression in developing lens. J. Biol. Chem. 264:17,559-17,563. Bassas, L., F. De Pablo, M. A. Lesniak, and J. Roth, 1987. The Insulin receptors of chick embryo show tissuespecific structural differences which parallel those of the IGF-I receptors. Endocrinology 121:1468-1476. Bassas, L., M. Girbau, M. A. Lesniak, J. Roth, and F. De Pablo, 1989. Development of receptors for insulin and insulin-like growth factor I in head and brain of chick embryos: autoradiographic localization. Endocrinology 125:2320-2327. Bassas, L., M. A. Lesniak, J. Serrano, J. Roth, and F. De Pablo, 1988. Developmental regulation of insulin and type I IGF receptors and absence of type II IGF receptors in chick embryo tissues. Diabetes 37: 637-644. Beebe, D. C, M. H. Silver, K. S. Belcher, J. J. Van Wyk, M. E. Svoboda, and P. S. Zelenka, 1987. Lentropin a protein that controls lens fiber formation, is related functionally and immunologically to the insulin-like growth factors. Proc. Natl. Acad. Sci. USA. 84: 2327-2330. Daughaday, W. H., and P. Rotwein, 1989. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations. Endocr. Rev. 10:68-91. De Pablo, F., M. Girbau, J. A. Gomez, E. Hernandez, and J. Roth, 1985. Insulin antibodies retard and insulin accelerates growth and differentiation in early embryos. Diabetes 34:1063-1067. De Pablo, F., J. Roth, E. Hernandez, and R. M. Pruss, 1982. Insulin is present in chicken eggs and early chick embryos. Endocrinology 111:1909-1916. De Pablo, F., L. A. Scott, and J. Roth, 1990. Insulin and insulin-like growth factor I in early development: Peptides, receptors and biological events. Endocr. Rev. 11:558-577. Girbau, M., L. Bassas, J. Alemany, and F. De Pablo, 1989. In situ autoradiography and ligarid-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor-I receptors in prepancreatic chicken embryos. Proc. Natl. Acad. Sci. USA 86:5868-5872. Girbau, M., J. A. Gomez, M. A. Lesniak and F. De Pablo, 1987. Insulin and IGF-I both stimulate metabolism and growth in the postneurula chick embryo. Endocrinology 121:1477-1482. Girbau, M., M. A. Lesniak, J. A. Gomez, and F. De Pablo, 1988. Insulin action in early embryonic life: antiinsulin receptor antibodies retard chicken embryo

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Insulin, IGF-I, and, to a lesser extent, proinsulin, can stimulate growth of chick embryos between Day 2 and Day 4 of development (Girbau et al., 1987) (Figure 6). The physiological role of endogenous insulin in overall development of the embryo is further supported by the experiments in which embryos of similar age were treated with antiinsulin antibodies or anti-insulin receptor antibodies (De Pablo et al, 1985; Girbau et al, 1988). A significant percentage of embryos died and the survivors were biochemically and morphologically retarded (Table 2). Thus far, similar experiments with anti-IGF-I antibodies have not been conducted. To explore further the effect of insulin and IGF-I in differentiation the present authors have studied the lens of the eye. This isolated, avascular organ is bathed by the vitreous humor and formed by a unique type of cells. The front layer consists of dividing epithelial cells, and the bulk of the lens is formed by elongated, differentiated fiber cells. A factor isolated from chicken embryo vitreous humor, named lentropin, as well as purified IGF-I and insulin, all promote fiber cell elongation (Beebe et al, 1987). The present authors have confirmed that vitreous humor from embryos at Day 6 and older contains IGF-I (unpublished results). One of the markers that reflects differentiation of epithelial cells into fiber cells is the accumulation of 6-crystallin and its mRNA. Alemany et al. (1989) have shown that 6crystallin mRNA levels increase in primary cultures of lens cells treated with insulin and IGF-I. More recently, Alemany et al. (1990) have done DNA transfections of the 8crystallin gene promoter into epithelial lens cells. In this system, IGF-I is more active than insulin in stimulating transcription of the 5crystallin gene, as reported by a chloramphenicol acetyl transferase assay (Figure 7). Further studies include characterization of exacting DNA elements and nuclear transacting factors that may mediate the effects of insulin and IGF-I in the regulation of 8-crystallin expression in the developing lens.

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growth but not muscle differentiation in vivo. Biochem. Biophys. Res. Commun. 153:142-148. Robcis, H. L., T. Caldes, and F. De Pablo, 1991. Insulinlike growth factor I serum levels show a midembryogenesis peak in chicken that is absent in growth retarded embryos cultured ex ovo. Endocrinology (in press). Scavo, L., J. Alemany, J. Roth, and F. De Pablo, 1989. Insulin-like growth factor I activity is stored in the yolk of the avian egg. Biochem. Biophys. Res.

Commun. 162:1167-1173. Serrano, J., C. L. Bevins, W. S. Young, and F. De Pablo, 1989. Insulin gene expression in chicken ontogeny: Pancreatic, cxtranpancreatic and prepancreatic. Develop. Biol. 132:410-418. Serrano, J., A. R. Shuldiner, C. T. Roberts, Jr., D. LeRoith, and F. De Pablo, 1990. The insulin-like growth factor I (IGF-I) gene is expressed in chick embryo during organogenesis. Endocrinology 127:1547 -1549.

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Insulin-like growth factor-I and insulin as growth and differentiation factors in chicken embryogenesis.

The avian embryo has been a useful model system for studies on the role of insulin and its close relative insulin-like growth factor-I (IGF-I) in deve...
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