The insulin-like growth factors (IGFs) stimulate ontogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-H. At all ages from early post-implantation through midgestation, the expression of IGFBP-2 was highly complementary to IGF-H. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-H expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-H expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-ell, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes. IGFBP-2 and IGF-H expression continued to be complementary throughout many non-neural tissues following gastrulation: IGFBP-2 was expressed at high levels in the surface ectoderm of the branchial arches while IGF-H was expressed at high levels in the mesenchyme of the branchial arches and at lower levels in the surface ectoderm; IGFBP-2 mRNA was prominent in the dorsal region of the developing foregut and throughout the hindgut; while IGF-H mRNA was prominent in cells of the ventral foregut but not detectable in the hindgut, and finally, IGFBP-2 mRNA was expressed in a restricted set of mesodermal tissues that did not express IGF-H including the notochord (especially when embedded in or adjacent to the dorsal gut), mesonephric tubules, and the anterior splanchnic mesodermal plate (ASMP) adjacent to the foregut. By el2 and through midgestation stages, IGFBP-2 expression was undetectable in the notochord and in caudal regions of the neural tube except for the floor plate, where expression was initiated at el3. The expression of IGFBP-2 either in populations of rapidly dividing cells (such as the epiblast and ventricular zone of rostral neuroepithelium) or in regions that direct the growth and differentiation of neighboring cells and tissues (including the surface ectoderm of the branchial arches, notochord, and the ASMP) suggests that IGFBP-2 may have important roles during development of numerous fetal tissues either by modulating IGF action or by acting independently of the IGFs as a constituent of the extracellular matrix.

The insulin-like growth factors, IGF-I and IGF-II, are mitogenic polypeptides synthesized by a wide variety of cell types in both adult and embryonic tissues (Humbel, 1984; Froesch et al., 1985; Daughaday and Rotwein, 1989; Rechler and Nissley, 1990). IGF-II is the predominant IGF in fetal tissues (Brown et al., 1986; Soares et al., 1986; Gray et al., 1987; Stylianopoulou et al., 1988a) and drops to low or undetectable levels in most adult tissues except the choroid plexus and leptomeninges of the brain (Stylianopoulou et al., 1988b; Hynes et al., 1988). IGF-II is now known to modulate fetal growth in vivo, since mice heterozygous (when the gene is inherited from the paternal genome) or homozygous for a mutated IGF-II gene exhibit a growth deficiency of both the embryo and the placenta at least as early as embryonic day (e) 16 (DeChiara et al., 1990; DeChiara et al., 1991). In contrast, IGF-I is expressed at lower levels than IGF-II during fetal development (Rotwein et al., 1987) and in a different expression pattern at mid-gestational ages (Bondy et al., 1990; Streck et al., unpublished data), but at high levels after puberty when it mediates the action of growth hormone (Rechler and Nissley, 1990).

In all serum and tissue fluids examined, both IGF-I and IGF-II exist as high relative molecular mass complexes that include specific IGF binding proteins (IGFBPs; Baxter and Martin, 1989; Rechler and Nissley, 1990; Clemmons, 1990). Six IGF binding proteins that likely mediate IGF actions have thus far been cloned and sequenced (Julkunen et al., 1988; Brewer et al., 1988; Brinkman et al., 1988; Wood et al., 1988; Brown et al., 1989; Margot et al., 1989; Murphy et al., 1990; Shimasaki et al., 1990; Shimasaki et al., 1991). At least one of these binding proteins, IGFBP-2, is expressed in high abundance prenatally and, like IGF-II, is reduced dramatically in most adult tissues (Brown et al., 1989; Orlowski et al., 1990). Although IGFBPs were initially isolated as proteins circulating in the serum, most contain an RGD or electrostatically similar sequence commonly found on extracellular matrix components. Therefore, it is possible that matrix or cell surface binding sites for IGFBPs localize these molecules following their secretion, and both in vitro and in vivo studies support this possibility (Elgin et al., 1987; Hill et al., 1989).

During early post-implantation mouse development, the IGF-II gene is expressed exclusively in extraembryonic tissues until the onset of gastrulation and the formation of the primitive streak, when expression begins first in newly formed extraembryonic mesoderm and then in embryonic mesoderm (Lee et al., 1990). Initial observations limited to the mid-gestational period of rat development demonstrated that the IGF-II and IGFBP-2 expression patterns are distinct and generally complementary at that stage (Wood et al., 1990). Thus, while IGF-II mRNA is prominent in most mesoderm derivatives from el3.5-el5 (Stylianopoulou et al., 1988a; Wood et al., 1990), IGFBP-2 is expressed instead in many ectoderm and endoderm derivatives including ectodermal placodes, the epithelial fining of the developing digestive tract, and specific sites in the central nervous system including the floor plate and rostral regions of neuroepithelium (Wood et al., 1990).

Since there is a direct role for IGF-II in embryonic and fetal growth, and since the IGF binding proteins likely mediate the actions of IGFs on target tissues, we have extended our analysis of IGFBP-2 gene expression during rat embryogenesis to early post-implantation ages and have continued to compare directly its expression with the known expression pattern of IGF-II. We report here that the IGFBP-2 gene is expressed in early post-implantation embryos, that the expression pattern is strikingly complementary to IGF-II in derivatives of all germ layers, and that IGFBP-2 expression characterizes many tissues that are either rapidly proliferating or that direct the outgrowth and proliferation of neighboring cells. Finally, we demonstrate unique sites of IGF-II expression in limited regions of surface ectoderm and neuroectoderm.

Preparation of embryos

Decidua from timed pregnant rats of gestational ages 7 to 9 days were dissected, separated and fixed in 4% formaldehyde (freshly made from paraformaldehyde) in PBS. Decidua were equilibrated with 20% sucrose, embedded in OCT (Miles), and cryostat sectioned as described (Lugo et al., 1989). Two or four decidua in different orientations were embedded in each block. The grain distributions over sections from a minimum of six embryos from at least two litters were examined after hybridization with IGFBP-2 or IGF-II. At ages >e10, 3–4 embryos from at least two litters were dissected and processed individually, and examined in either transverse or sagittal planes.

In situ hybridization

Conditions for the in situ hybridizations, washes, autoradiography, exposure to photoemulsion, development and counterstaining with hematoxylin/eosin were as described (Lugo et al., 1989) except that the hybridizations used 35S-labeled probes and that the hybridization buffer contained 0.1% sodium dodecyl sulfate and 10 mM dithiothreitol, while all wash solutions contained 1% sodium thiosulfate, 0.05% sodium pyrophosphate and 0.1% 2-mercaptoethanol, as described (Wood et al., 1990; Lee et al., 1990). The hybridization patterns were evaluated initially by X-ray film autoradiography prior to exposure to nuclear track emulsion and histological examination. Following autoradiography, photographic slides of all sections were made and examined to expedite reconstruction of the embryos. Hybridization with control (sense) RNA yielded very low backgrounds in all cases.

35S-labeled RNA transcripts for IGFBP-2 and IGF-II were synthesized from the pGem3 plasmids pGrBP2-2-ll and pGrIGF-II-7a, respectively. pGrBP2–2–ll contains a HindIII-SacI fragment (nucleotides 502–1087) of the rIGFBP-2 Cdna clone isolated by Brown et al. (1989). pGrIGF-II-7a contains a 551 bp Psii-BamHi fragment corresponding to the 5′ end of the prIGF-II-1 clone of Whitfield et al. (1984). Both plasmid DNAs were linearized with HindIII and incubated with T7 polymerase in the presence of CTP, GTP, ATP and 35S-UTP. The resulting RNA transcripts (about 590 nucleotides for IGFBP-2 and 560 nucleotides for IGF-II) were purified on Sephadex G-50 (Boehringer-Mannheim) and used without hydrolysis as described (Wood et al., 1990). In order to investigate the onset of neurogenesis with a molecular marker, selected sections were hybridized with an antisense probe to the neuronal neurofilament gene alpha-internexin (Fliegner et al., 1990). RNA probes to internexin included antisense transcripts synthesized from a pGem3 vector containing a 1553 bp alpha-internexin cDNA insert (Fliegner et al., 1990).

The expression patterns of the IGFBP-2 and IGF-II genes were compared in serial sections that spanned entire embryos beginning at early post-implantation ages. Initial experiments performed with saturating probe concentrations demonstrated that the relative abundance of IGF-II was much greater (about 10-fold, on the average) than IGFBP-2 at all early postimplantation and mid-gestation ages examined. Thus, in some experiments, the amount of IGF-II probe incubated with individual sections was lowered, so that adjacent sections on the same slide hybridized with IGFBP-2 or IGF-II gave comparable grain densities after emulsion autoradiography. The qualitative pattern of IGF-II expression was not altered by this procedure.

IGFBP-2 and IGF-II expression are complementary at the egg cylinder stage

IGFBP-2 expression was detected at the earliest age examined, e7 (early egg cylinder), and was restricted to the embryonic epiblast (Fig. 1A). Given the limits of resolution of in situ hybridization using radiolabeled probes, it appeared that all cells of the embryonic epiblast contained IGFBP-2 mRNA. The IGFBP-2 expression pattern was essentially complementary to that of rat IGF-II, which instead was detected in the columnar visceral endoderm and all extraembryonic ectoderm as has also been described in the mouse (Lee et al., 1990; Fig. 1B). At e8 (late egg cylinder), the expression patterns of IGFBP-2 and IGF-II were unchanged from those at e7 (Fig. 1C,D); again, all epiblast cells appeared labeled when hybridized with IGFBP-2 (Fig. 1C), while IGF-II mRNA was undetectable in the epiblast but was detected at high levels in most extraembryonic cells (Fig. 1D).

Fig. 1.

Pattern of IGFBP-2 and IGF-II gene expression at e7 (A-B) and e8 (C-D). Sagittal sections were hybridized with 35S-labeled antisense IGFBP-2 (A,C) or IGF-II (B,D) RNA probes. IGFBP-2 mRNA is detected only in the epiblast (A,C) while IGF-II mRNA is detected in extraembryonic ectoderm, visceral endoderm and the ectoplacental cone (B,D). Abbreviations: ec, ectopiacental cone; ee, extraembryonic ectoderm; ep, epiblast; ve, visceral endoderm. Bar=100 micrometers.

Fig. 1.

Pattern of IGFBP-2 and IGF-II gene expression at e7 (A-B) and e8 (C-D). Sagittal sections were hybridized with 35S-labeled antisense IGFBP-2 (A,C) or IGF-II (B,D) RNA probes. IGFBP-2 mRNA is detected only in the epiblast (A,C) while IGF-II mRNA is detected in extraembryonic ectoderm, visceral endoderm and the ectoplacental cone (B,D). Abbreviations: ec, ectopiacental cone; ee, extraembryonic ectoderm; ep, epiblast; ve, visceral endoderm. Bar=100 micrometers.

IGFBP-2 and IGF-II expression change reciprocally as mesoderm forms

Following primitive streak formation, there was a dramatic alteration in the expression of IGFBP-2 in epiblast derivatives. Specifically, IGFBP-2 expression could not be detected in either the embryonic or extraembryonic mesoderm (Fig. 2A,C) at stages either concurrent with, or slightly later than, the time when these cell types delaminate from the epiblast. In contrast, IGF-II expression by this stage had appeared in both extraembryonic (e.g. allantois; Fig. 2D) and embryonic (e.g developing heart; Fig. 2B,D) mesoderm. IGFBP-2 expression persisted in cells of the embryonic ectoderm that had not contributed to the primitive streak. For example, IGFBP-2 transcripts were present throughout the rostral-caudal extent of the presumptive neuroepithelium (Fig. 2A,C), although slightly higher expression levels in this tissue were detected reproducibly in the rostral regions of the embryo (Fig. 2A). In addition, low but detectable IGFBP-2 expression was detected in the surface ectoderm, particularly that surrounding the heart (arrowheads, Fig. 2A,C), lining the oral cavity (stomo-deal ectoderm; data not shown), and in the epithelium of the newly forming anterior gut (Fig. 2C). Close examination of multiple embryos allowed us to determine that IGF-II was co-expressed with IGFBP-2 in cells of the surface ectoderm around the heart (arrowheads; Fig. 2B,D) and lining the ventral surface of the oral cavity (data not shown).

Fig. 2.

Pattern of IGFBP-2 and IGF-II gene expression at e9. Sagittal (A,B) or oblique transverse (C,D) sections were hybridized with IGFBP-2 (A,C) or IGF-II (B,D) RNA probes. IGFBP-2 gene expression is detected in the neuroectoderm extending from the anterior head fold region (A,C) throughout the neural tube (A) and in surface ectoderm over the developing heart (arrowheads, A,C) and gut epithelium (C). IGF-II expression is seen in developing heart (B,D), surface ectoderm surrounding the heart (arrowheads B,D), head mesenchyme (B), embryonic mesoderm (B) and lateral (but not dorsal) foregut (D) in addition to the visceral yolk sac (B,D), amnion (B,D), extraembryonic mesoderm (B) and allantois (D). Abbreviations: al, allantois; am, amnion; em, extraembryonic mesoderm; fg, foregut; h, heart; hf, head fold; hm, head mesenchyme; m, embryonic mesoderm; ne, neuroectoderm; so, somite; vys, ventral yolk sac. Bar=100 micrometers.

Fig. 2.

Pattern of IGFBP-2 and IGF-II gene expression at e9. Sagittal (A,B) or oblique transverse (C,D) sections were hybridized with IGFBP-2 (A,C) or IGF-II (B,D) RNA probes. IGFBP-2 gene expression is detected in the neuroectoderm extending from the anterior head fold region (A,C) throughout the neural tube (A) and in surface ectoderm over the developing heart (arrowheads, A,C) and gut epithelium (C). IGF-II expression is seen in developing heart (B,D), surface ectoderm surrounding the heart (arrowheads B,D), head mesenchyme (B), embryonic mesoderm (B) and lateral (but not dorsal) foregut (D) in addition to the visceral yolk sac (B,D), amnion (B,D), extraembryonic mesoderm (B) and allantois (D). Abbreviations: al, allantois; am, amnion; em, extraembryonic mesoderm; fg, foregut; h, heart; hf, head fold; hm, head mesenchyme; m, embryonic mesoderm; ne, neuroectoderm; so, somite; vys, ventral yolk sac. Bar=100 micrometers.

CNS IGFBP-2 expression becomes restricted and is complementary to alpha-internexin

Between e9 and e10, IGFBP-2 expression in regions of the nervous system formed during primary neurulation became restricted and was no longer detected in the spinal cord (Fig. 3B,D,E) and myelencephalon (not shown) but continued in rostral CNS regions through ell (Fig. 4A-E). Close examination showed that the grain densities among these regions were not uniform but instead were slightly higher in the telencephalon, ventral diencephalon and mesencephalon (Fig. 4A,C,D), while lower in the lateral extensions of the optic vesicles (Fig. 4D). By el3, IGFBP-2 expression in these rostral regions was restricted to the rapidly dividing neuroblasts of the ventricular zone (Fig. 5A). As these neuroblasts became post-mitotic and entered the mantle zone, IGFBP-2 expression decreased or disappeared. Concurrently, as illustrated in the mesencephalon and myelencephalon at el3.5, these cells began to accumulate alpha-internexin mRNA (Fig. 5B). This neurofilament-related protein is the earliest marker of neuronal differentiation thus far described (Fliegner et al., unpublished data). The onset of IGFBP-2 expression in most other CNS regions that express IGFBP-2 during mid-gestation (for example, infundibulum and choroid plexus epithelium; Wood et al., 1990) was coincident with the initial appearance of these structures. In contrast, IGFBP-2 expression in the floor plate (Wood et al., 1990) was not detectable until el2-el3, at least two days later than the appearance of most other floor plate markers at comparable stages of chick development (Yamada et al., 1991).

Fig. 3.

Pattern of IGFBP-2 (B,D,E,F,H,I) and IGF-II (C,G) gene expression at elO (B-E) and at e10.5 (F-I). Panel A drawn from an elO embryo section shows the level of sections in Panels B-I. IGFBP-2, but not IGF-II, is detected at high levels in the anterior splanchnic mesodermal plate (black arrows; compare B and C), in the posterior, but not anterior, notochord (compare D,F,H with B), and in the mesonephric ducts and tubules (compare F,H,1 with G). IGFBP-2 is also present in the posterior region of the spinal cord at e10.5 (H,I). Expression of both IGFBP-2 and IGF-II can be detected in the liver primordium (B and C), in the genital ridge (F and G) and in gut epithelium (B-I). Abbreviations: ge, gut epithelium; gr, genital ridge, L, liver; md, mesonephric duct; nt, notochord; sc, spinal cord. Bar=100 micrometers.

Fig. 3.

Pattern of IGFBP-2 (B,D,E,F,H,I) and IGF-II (C,G) gene expression at elO (B-E) and at e10.5 (F-I). Panel A drawn from an elO embryo section shows the level of sections in Panels B-I. IGFBP-2, but not IGF-II, is detected at high levels in the anterior splanchnic mesodermal plate (black arrows; compare B and C), in the posterior, but not anterior, notochord (compare D,F,H with B), and in the mesonephric ducts and tubules (compare F,H,1 with G). IGFBP-2 is also present in the posterior region of the spinal cord at e10.5 (H,I). Expression of both IGFBP-2 and IGF-II can be detected in the liver primordium (B and C), in the genital ridge (F and G) and in gut epithelium (B-I). Abbreviations: ge, gut epithelium; gr, genital ridge, L, liver; md, mesonephric duct; nt, notochord; sc, spinal cord. Bar=100 micrometers.

Fig. 4.

Pattern of IGFBP-2 (A-E) and IGF-II (F) gene expression at ell. Panels A and B arc sagitta) and parasagittal sections respectively while panels C-F are transverse sections through ell embryos. IGFBP-2 expression is detected in surface ectoderm surrounding the maxillary process (not shown), mandibular arch and hyoid arch (A,B,F), in the otic (C), pituitary (D) and nasal (E) placodes and in neuroepithelium of the rostral brain (A-E) as well as in the posterior region of the spinal cord (A.B), but is undetectable in neuroepithelium of the myelencephalon (B-E). IGF-II expression is detected in surface ectoderm and mesoderm of the maxillary process, mandibular arch and hyoid arch but is undetectable in neuroepithelium at any level (F). Abbreviations: dn, diencephalon; gc, gut epithelium; ha, hyoid arch; L, liver; ma, mandibular arch; ms, mesencephalon; mt, metencephalon; my, myelencephalon; np, nasal placode; oc, oral cavity; op, otic placode; pt, presumptive anterior pituitary or Rathke’s pouch; sc, spinal cord; SP, anterior sphlanchnic mesodermal plate; tn, telencephalon. Bar= l millimeter (A-B), or 300 micrometers (C-F).

Fig. 4.

Pattern of IGFBP-2 (A-E) and IGF-II (F) gene expression at ell. Panels A and B arc sagitta) and parasagittal sections respectively while panels C-F are transverse sections through ell embryos. IGFBP-2 expression is detected in surface ectoderm surrounding the maxillary process (not shown), mandibular arch and hyoid arch (A,B,F), in the otic (C), pituitary (D) and nasal (E) placodes and in neuroepithelium of the rostral brain (A-E) as well as in the posterior region of the spinal cord (A.B), but is undetectable in neuroepithelium of the myelencephalon (B-E). IGF-II expression is detected in surface ectoderm and mesoderm of the maxillary process, mandibular arch and hyoid arch but is undetectable in neuroepithelium at any level (F). Abbreviations: dn, diencephalon; gc, gut epithelium; ha, hyoid arch; L, liver; ma, mandibular arch; ms, mesencephalon; mt, metencephalon; my, myelencephalon; np, nasal placode; oc, oral cavity; op, otic placode; pt, presumptive anterior pituitary or Rathke’s pouch; sc, spinal cord; SP, anterior sphlanchnic mesodermal plate; tn, telencephalon. Bar= l millimeter (A-B), or 300 micrometers (C-F).

Fig. 5.

Pattern of IGFBP-2 (A) and alpha-inteniexin (B) gene expression at el3-el4. The expression of IGFBP-2 in the neuroectoderm is restricted to the ventricular zone of the rostral brain as seen in a sagittal section of an el3.5 embryo (A). An adjacent section (B) hybridized with alpha-internexin shows expression in cells that have left the ventricular zone in the rostral brain and additional expression in the myelencephalon, cranial ganglia and caudal spinal cord. IGFBP-2 hybridization to the pituitary, otic and nasal placodes, choroid plexus and liver are also apparent (see also Wood et al., 1990). (C) Transverse section through an el4 embryo showing the expression of IGF-11 in an outpocketing of the dorsal diencephalon which will form the epiphysis or pineal gland. IGFBP-2 is not detected in this restricted region of neuroepithelium (data not shown). (D) Transverse sections through an el3 embryo showing expression of IGFBP-2 in striated muscle near the developing sympathetic ganglia as well as in the esophageal and tracheal epithelium. IGFBP-2 is no longer detectable in the notochord at this age. Abbreviations: cp, choroid plexus; dn, diencephalon; ep, epiphysis; es, esophagus; he, heart; L, liver; ms, mesencephalon; my, myelencephalon; np, nasal placode; nt, notochord; op, otic placode; s, stomach; sc, spinal cord; sg, sympathetic ganglia; sm, striated muscle; tn, telencephalon; tr, trachea. Bar=l millimeter (A-B), 300 micrometers (C), or 100 micrometers (D).

Fig. 5.

Pattern of IGFBP-2 (A) and alpha-inteniexin (B) gene expression at el3-el4. The expression of IGFBP-2 in the neuroectoderm is restricted to the ventricular zone of the rostral brain as seen in a sagittal section of an el3.5 embryo (A). An adjacent section (B) hybridized with alpha-internexin shows expression in cells that have left the ventricular zone in the rostral brain and additional expression in the myelencephalon, cranial ganglia and caudal spinal cord. IGFBP-2 hybridization to the pituitary, otic and nasal placodes, choroid plexus and liver are also apparent (see also Wood et al., 1990). (C) Transverse section through an el4 embryo showing the expression of IGF-11 in an outpocketing of the dorsal diencephalon which will form the epiphysis or pineal gland. IGFBP-2 is not detected in this restricted region of neuroepithelium (data not shown). (D) Transverse sections through an el3 embryo showing expression of IGFBP-2 in striated muscle near the developing sympathetic ganglia as well as in the esophageal and tracheal epithelium. IGFBP-2 is no longer detectable in the notochord at this age. Abbreviations: cp, choroid plexus; dn, diencephalon; ep, epiphysis; es, esophagus; he, heart; L, liver; ms, mesencephalon; my, myelencephalon; np, nasal placode; nt, notochord; op, otic placode; s, stomach; sc, spinal cord; sg, sympathetic ganglia; sm, striated muscle; tn, telencephalon; tr, trachea. Bar=l millimeter (A-B), 300 micrometers (C), or 100 micrometers (D).

As slightly older embryos were examined, a unique site of CNS IGF-II expression was noted at el4 in an outpocketing of the dorsal diencephalon that will ultimately form the epiphysis or pineal gland (Fig. 5C). This region was denoted also by the absence of IGFBP-2 expression that is present in other regions of the diencephalon at this age (data not shown). IGF-II expression in the pineal had been noted previously at el8 (Ayer-le Lievre et al., 1991) but not at earlier ages (Stylianopoulou et al., 1988a; Ayer-le Lievre et al., 1991).

IGFBP-2 is expressed during secondary neurulation

Neural tissue arises not only from the neural plate but also from posterior mesenchyme during secondary neurulation. During the early stages of this process, tail bud mesenchyme condenses into a cluster (“rosette formation”), and a canal subsequently forms within this condensation that becomes continuous with the spinal canal (Schoenwolf, 1984). Reciprocal changes in IGF-II and IGFBP-2 expression occur as the tail mesenchyme condenses. IGF-II mRNA disappears (not shown) while IGFBP-2 mRNA appears in the newly forming rosette (Fig. 6A,C,D,F). IGFBP-2 expression continues in these cells as the spinal canal enlarges, and is retained transiently in the caudal spinal cord that originated during this process (Fig. 3I; 4A,B; 6D). IGFBP-2 expression is retained in the most caudal neuroepithelium until el4, after completion of secondary neurulation.

Fig. 6.

IGFBP-2 is re-expressed in the tail bud during secondary neurulation. B shows the level of the sections from one e10.5 embryo in A and C and E shows the level of the sections from a second embryo in D and F. IGFBP-2 is detected in the most caudal regions of the spinal cord (D) and in the medullary rosette in the tail bud during secondary neurulation (A,C,D,F). C is a section just caudal to that shown in A where the lumen of the medullary rosette is reduced. Similarly, D shows a section through the rosette that has expanded to form a lumen while the more caudal section in F has not yet formed a clear lumen in the medullary rosette. Abbreviations: ge, gut epithelium; md, mesonephric duct; mr, medullary rosette; nt, notochord; sc, spinal cord. Bar=100 micrometers.

Fig. 6.

IGFBP-2 is re-expressed in the tail bud during secondary neurulation. B shows the level of the sections from one e10.5 embryo in A and C and E shows the level of the sections from a second embryo in D and F. IGFBP-2 is detected in the most caudal regions of the spinal cord (D) and in the medullary rosette in the tail bud during secondary neurulation (A,C,D,F). C is a section just caudal to that shown in A where the lumen of the medullary rosette is reduced. Similarly, D shows a section through the rosette that has expanded to form a lumen while the more caudal section in F has not yet formed a clear lumen in the medullary rosette. Abbreviations: ge, gut epithelium; md, mesonephric duct; mr, medullary rosette; nt, notochord; sc, spinal cord. Bar=100 micrometers.

Post-gastrulation IGFBP-2 and IGF-II expression patterns are often complementary in non-neural tissues

Ectoderm

The IGFBP-2 expression noted in surface ectoderm following gastrulation continued through mid-gestation. The levels of expression were not uniform and grain densities in the surface ectoderm surrounding all branchial arches (Fig. 4A,B,E), the lining of the oral cavity (Fig. 4A,B,E), and all developing placodes (for example, otic, Fig. 4C, and nasal, Fig. 4E) were at least as high as or higher than in the CNS. Both the initial invagination of the pituitary primordium, Rathke’s pouch (Fig. 4A,D), and the surface ectoderm anterior to this region (Fig. 4A) were highly positive at ell, although IGFBP-2 expression decreased dramatically just caudal to the pouch (Fig. 4A). Although, as previously described, most surface ectoderm does not express IGF-II (Stylianopoulou et al., 1988a), specific regions were found here to express both the IGF-II and IGFBP-2 genes at earlier ages (e10-el2) than those reported previously. These included the ectoderm surrounding the branchial arches and lining the oral cavity (Fig. 4F) as well as Rathke’s pouch (data not shown).

Endoderm

Following gastrulation, IGFBP-2 expression also continued in the epithelium of the gut (Fig. 3B,D,E,F,H,I; Fig. 4B; Fig. 6A,C,D,F). The relative level of IGFBP-2 expression in the dorsal and ventral regions of gut epithelium was correlated with the proximity of the notochord to the dorsal gut wall. IGFBP-2 mRNA levels were higher in dorsal gut epithelium at elO when the dorsal gut wall is still closely apposed to the notochord throughout most of its length (Fig. 3B,D). In contrast, IGFBP-2 was relatively uniformly expressed through the gut after the notochord and the dorsal wall of the foregut have separated, as most readily observed in caudal regions of e10.5 embryos (Fig. 3H; Fig 6A,C,D,F). IGF-II expression in the gut, when detected, was again complementary to IGFBP-2. Thus, when IGFBP-2 expression was elevated in the dorsal foregut, IGF-II was prominent in the ventral gut and was not detected in dorsal gut (Fig. 3C). Further, when IGFBP-2 was expressed throughout the gut epithelium (Fig. 3E, F, H), IGF-II was undetectable (Fig. 3G). IGF-II and IGFBP-2 expression did overlap in other endodermal derivatives such as in the fiver primordium (compare Figs 3B and C) and in the lining of the pharynx and pharyngeal pouches (data not shown).

Mesoderm

Although IGFBP-2 mRNA was not detected in most mesoderm derivatives, several tissue types represented exceptions which expressed IGFBP-2 but not IGF-II, especially from e10-el2. These tissues included the mesonephric ducts and tubules (Fig. 3F,H,I), the notochord (particularly in more posterior regions; Fig. 3D,F,H), and the anterior splanchnic mesodermal plate (ASMP), a region of mesoderm which has thickened to form epithelial-like plates lateral to the anterior gut (arrowheads, Fig. 3B; Fig. 4A). These mesodermal derivatives all expressed IGFBP-2 but not IGF-II (Fig. 3C,G and data not shown). By el2, IGFBP-2 was not detectable in the notochord at any level and had disappeared in the region of the ASMP concurrent with the disappearance of this structure. In a small subset of mesodermal tissues, IGFBP-2 and IGF-II were coexpressed, these included the newly forming genital ridge (Fig. 3F,G) and newly forming striated muscle located near the sympathetic ganglia (Fig. 5D; Stylianopoulou et al., 1988a).

We have demonstrated that distinct and highly complementary patterns of expression for the IGFBP-2 and IGF-II genes are established by early post-implantation stages of rat embryogenesis and are maintained in most tissues into mid-gestational ages. The IGFBP-2 expression pattern itself is thus far distinct among genes expressed during early prenatal mammalian development and suggests potential roles for the IGFBP-2 protein in mediating growth and/or morphogenesis by either modulating IGF action or acting independently of IGFs as discussed below.

Complementary patterns of IGFBP-2 and IGF-II gene expression during germ layer formation

We have shown that the IGFBP-2 gene is expressed at least as early as the egg cylinder stage when its expression is restricted to the epiblast and thus is complementary to IGF-II. Since IGFBP-2 is expressed in inner cell mass-derived ES cells in vitro (Pintar et al., 1991), it is conceivable that IGFBP-2 expression in vivo begins even earlier. During and immediately following gastrulation, IGFBP-2 continues to be expressed in ectoderm but is not detectable in mesoderm. In contrast, shortly after gastrulation, most endoderm and several mesoderm (notochord, mesonephros, ASMP) derivatives express IGFBP-2 in patterns usually complementary to IGF-II. We do not know whether these tissues arise from a still undetected subset of cells that retain IGFBP-2 expression while moving through the primitive streak or whether IGFBP-2 expression ceases in all cells that enter the streak and is re-initiated in a subset of cells that form these specific tissues. Some of the present data are most consistent with continued expression; for example, IGFBP-2 mRNA is observed in both the notochord and gut immediately following gastrulation and could reflect continued expression in derivatives of the adjacent epiblast regions that give rise to these tissues (Tam, 1989). It is unlikely, however, that all genes expressed in the endodermal lineage soon after gastrulation are expressed in the epiblast since IGF-II is not detected in the anterior gut until after gastrulation (Fig. 3C; see also Lee et al., 1990).

The mouse Oct-3 gene, which encodes a POU domain transcription factor, is the only other gene thus far examined that is also expressed in the embryonic ectoderm and briefly in the neural ectoderm following gastrulation, but is not detected in newly forming mesoderm (Rosner et al., 1990; Scholer et al., 1990). Although it is tempting to speculate that Oct-3 activates IGFBP-2, no octomer binding motif is present in the 5′ 1300 bases of the IGFBP-2 gene (Brown et al., 1990); moreover, IGFBP-2 expression continues much later than Oct-3, which becomes undetectable (except in germ cells) soon after gastrulation. Nonetheless, the identical expression patterns of Oct-3 and IGFBP-2 in early post-implantation embryos represent a remarkable contrast to IGF-II, which first becomes detectable in the embryo only as IGFBP-2 and Oct-3 expression disappear, and illustrate the specificity with which multiple genes are regulated during gastrulation and as different mesoderm lineages arise.

Althoügh the mechanisms that mediate the complementary IGFBP-2 and IGF-II expression patterns remain unknown, it is notable that the pattern can be reversed during secondary neurulation. In this instance, the IGFBP-2-expressing cells in newly formed neural tissue have arisen not from the epiblast directly, but from mesenchyme (Schoenwolf, 1984) whose ancestors had first expressed IGFBP-2 in the epiblast and then transiently expressed IGF-II. It is further notable that IGFBP-2 expression thus characterizes most, if not all, cells that are at a very early stage of neural commitment, even when these progenitors arise at different times from quite distinct precursors.

Potential relationships between IGFBP-2 and autocrine or paracrine growth

Numerous sites of IGFBP-2 fetal expression represent cell populations that are proliferating more rapidly than their neighbors or descendents and suggest that IGFBP-2 may modulate growth of at least some fetal cells. For example, the cell cycle time of epiblast cells may be as rapid as 4–5 hrs (Snow, 1977), while mesodermal cells derived from the epiblast (and not expressing IGFBP-2) divide appreciably slower. In addition, as neuroblasts leave the ventricular zone and begin to differentiate into neurons, both cell division and IGFBP-2 expression cease. We suggest that secreted IGFBP-2 may be bound near its sites of synthesis via its RGD sequence (Brown et al., 1989) and may interact with IGFs secreted from distinct cell types or absorbed from CSF in a manner that maximally stimulates DNA synthesis in IGFBP-2 expressing cells. In vitro studies on IGFBP-1, which also contains an RGD sequence, support such a model. For example, one form of human IGFBP-1, the major IGF binding protein in human placenta and amniotic fluid, acts synergistically with IGF-I to increase the mitotic rate of fibroblasts and smooth muscle cells (Elgin et al., 1987). Other experiments using IGF analogues with reduced affinity for IGFBP-1 have shown that both IGFBPs and IGF receptors are necessary to produce maximal mitotic effects in cultured cell fines (Clemmons et al., 1990), possibly in conjunction with other serum or CSF factors (Clemmons and Gardner, 1990).

IGFBP-2 may alternatively, or in addition, direct growth processes in neighboring cells. Two specific examples (outgrowth of the facial processes and limbs) are consistent with the possibility that epithelially-derived IGFBP-2 interacts with adjacent mesenchyme. We have shown here that high levels of IGFBP-2 expression characterize the tips of branchial arch epithelia adjacent to the region of subepithelial mesenchyme where proliferation is highest (Minkoff, 1980; Bailey et al., 1988). In the limb, the highest rate of proliferation in limb mesenchyme occurs just under the distal apical ectodermal ridge (AER; Reiter and Solursh, 1982) that is also characterized by high IGFBP-2 expression (Pintar et al., 1991; Streck et al., unpublished data). Since these two epithelia can substitute for each other in experimental paradigms (Saber et al., 1989), it is possible that both the AER and the epithelia overlying the developing maxillary process are producing an identical factor (potentially IGFBP-2) that is essential for mesenchyme viability and growth. In contrast to IGFBP-2, IGF-I and IGF-II mRNA are present within the mesenchyme of both the facial processes and limb during early development of these structures (Bondy et al., 1990; Streck et al., unpublished data). Recent immunolocalization of IGFs in developing chick limb demonstrated that the IGFs are concentrated in peripheral limb mesenchyme just under the surface ectoderm and suggested a somewhat analogous localization in the facial processes (Ralphs et al., 1990). In both cases, then, the location of IGFs (and presumably their major site of action) are adjacent to epithelia with high levels of IGFBP-2 expression and may represent sites of IGFBP-2 localization. This possibility is consistent with results from the midgestation human fetus, where sites of IGF accumulation are distinct from sites of IGF synthesis (Han et al., 1987) and often correlate with sites of IGFBP-1 immunoreactivity (Hill et al., 1989). Finally, the ASMP represents another example where IGFBP-2 expression occurs in a tissue thought to mediate growth of an adjacent region. In this case, analysis of the mouse mutant Dominant hemomelia has shown that the growth of the stomach and spleen from adjacent gut epithelium is severely retarded when the ASMP does not develop (Green, 1967), which conceivably could result from low regional levels of IGFBP-2.

The functional significance, if any, of the complementary IGFBP-2 and IGF-II expression patterns remains unknown. Interaction between IGFBP-2 and IGF-II does not appear to be an essential method for directing autocrine or paracrine growth since the complete genetic ablation of IGF-II (DeChiara et al., 1990, 1991) does not lead to the severe deficits that would be expected near IGFBP-2 expressing tissues. It is possible that IGF-I can compensate for the loss of IGF-II, especially in the outgrowth of the facial processes and limbs. IGF-I is produced locally in these regions and could occupy IGFBP-2 sites in the ECM that would normally be occupied by IGF-II. A fetal source of IGF-I as a potential ligand for IGFBP-2 during the earliest post-implantation ages remains unlikely, however, since IGF-I has not been detected by PCR before e8 (Rappolee and Werb, 1991) or by in situ hybridization before day 10 (Streck et al., unpublished data). It remains possible that IGFBPs may have still unknown functions independent of IGF binding, since the only assays for IGFBP activity have been their ability to modulate IGF mitogenic effects in cultured cells (see Rechler and Nissley, 1990).

IGFBP-2 involvement in cell-matrix interactions

Although IGFBP-2 expression is often associated with proliferating cell populations, some IGFBP-2 expressing cells, including the floor plate (Jessell et al., 1989) and infundibulum (Wood and Pintar, unpublished), instead divide quite slowly. The possibility that IGFBP-2 may associate with the cell surface or extracellular matrix through interaction with its carboxyl RGD sequence, and be involved in tissue interactions known to occur at these sites has been previously discussed (Wood et al., 1990). The present results suggest additional regions where high IGFBP-2 expression characterizes interactions between neighboring tissues that are not necessarily growth related. For example, IGFBP-2 expression in newly forming notochord and gut suggests its participation in notochord formation, its subsequent detachment from the roof of the gut and/or separation of the basal laminae surrounding these structures (Jurand, 1974; Larners et al., 1987). IGFBP-2 expression in the notochord also coincides with times when this tissue has the capacity to induce the formation and differentiation of the floor plate (van Straaten et al., 1988; Jessell et al., 1989) and is subsequently initiated in floor plate cells themselves (see Results). Since both the notochord and the floor plate are sources of polarizing signals that determine cell pattern along the dorsal-ventral axis of the developing nervous system (Yamada et al., 1991), it is conceivable that IGFBPs participate in this process.

Expression of IGF binding proteins may be a particularly critical element of notochord/floor plate development, since an additional binding protein, IGFBP-5, is expressed in both the floor plate and notochord at ell (Green, Wood, Streck and Pintar, unpublished data). Definitive immunolocalization of the IGFBPs during these processes will provide an initial test for these possibilities.

In summary, the precise role(s) of IGFBP-2 in modulating growth or morphogenetic processes remain unclear and await immunolocalization of the IGFBP-2 protein and in vivo alteration of its expression pattern. Even at present, however, the unique association of IGFBP-2 expression with cells or tissues in particularly dynamic periods of growth or morphogenesis indicate multiple potential roles for the IGFBP-2 protein that could result either by altering IGF availability or bioactivity or by ECM-mediated processes possibly unrelated to IGF binding.

The authors are pleased to acknowledge the collaboration of Karsten Fliegner and Ron Liem during the alpha-internexin experiments and the expert technical assistance of Paul Hsu and Roberta Bivins during the course of this project. We would also like to thank Dr. M. Rechler for providing the rat IGFBP-2 and IGF-II cDNA clones. This work was supported by Grant NIH-NS-21970 (JP). T.W. was supported in part by NIH Training Grant T7734 (to Georgiana Jagiello); R.S. was supported by NIH Training Grant NS-07062-14 (to Michael D. Gershon).

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