help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2008-0534
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sang, X.
Right arrow Articles by Wood, A. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sang, X.
Right arrow Articles by Wood, A. W.
Endocrinology Vol. 149, No. 10 5035-5042
Copyright © 2008 by The Endocrine Society

Paracrine Insulin-Like Growth Factor Signaling Influences Primordial Germ Cell Migration: In Vivo Evidence from the Zebrafish Model

Xianpeng Sang, Matthew S. Curran and Antony W. Wood

Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; and Harvard Stem Cell Institute, Cambridge, Massachusetts 02138

Address all correspondence and requests for reprints to: Antony W. Wood, Ph.D., 55 Fruit Street, THR 933, Boston, Massachusetts 02114. E-mail: awwood{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF signaling has been shown to stimulate migration of multiple cell types in vitro, but few studies have confirmed an equivalent function for IGF signaling in vivo. We recently showed that suppression of IGF receptors in the zebrafish embryo disrupts primordial germ cell (PGC) migration, but the mechanism underlying these effects has not been elucidated. We hypothesized that PGCs are intrinsically dependent upon IGF signaling during the migratory phase of development. To test this hypothesis, we first examined the spatial expression patterns of IGF ligand genes (igf1, igf2a, and igf2b) in the zebrafish embryo. In situ analyses revealed distinct expression patterns for each IGF ligand gene, with igf2b mRNA expressed in a spatial pattern that correlates strongly with PGC migration. To determine whether PGC migration is responsive to IGF signaling in vivo, we synthesized gene hybrid expression constructs that permit conditional overexpression of IGF ligands by PGCs into the PGC microenvironment. Conditional overexpression of IGF ligands consistently disrupted PGC migration, confirming that PGC migration is sensitive to local aberrations in IGF signaling. Finally, we show that conditional suppression of IGF signaling, via PGC-specific overexpression of a mutant IGF-I receptor, disrupts PGC migration, confirming that zebrafish PGCs intrinsically require IGF signaling for directional migration in vivo. Collectively, these studies confirm an in vivo role for IGF signaling in cell migration and identify a candidate ligand gene (igf2b) regulating PGC migration in the zebrafish.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CELL MIGRATION is a fundamental feature of animal development. It plays a critical role during embryonic organogenesis, and maintains importance during postnatal development as a mediator of tissue homeostasis, wound healing, and immune system function. In addition, many pathological conditions involve disruptions to normal cell migration dynamics. For example, an essential feature of the metastatic cascade is the acquired ability of tumor cells to detach and migrate into new tissues, leading to de novo tumor formation (1, 2, 3). Thus, identifying factors that regulate cell migration remains an important goal in both basic and applied biology.

Primordial germ cell (PGC) migration provides an interesting and informative in vivo model of directed cell migration. PGCs are the embryonic progenitors of the gametes (eggs and sperm), and in most species are specified in an extragonadal location, after which they proliferate and migrate to the embryonic genital ridges. Failure of PGCs to migrate correctly can lead to insufficient germ cell engraftment in the embryonic gonad, compromising future reproductive development, and possibly leading to the formation of extragonadal germ cell tumors (4, 5).

As with other cell migration paradigms, PGC migration is influenced by numerous cell signaling factors and pathways, though relatively few have been described in detail. Among the most well known are the CXCL-family chemokines (e.g. CXCL12) that, through activation of specific G protein-coupled receptors (e.g. CXCR4), have been identified as critical mediators of PGC migration in mice, zebrafish, and chick (6, 7, 8, 9). Other factors include TGF-β family cytokines (10), Kit ligand (11), cell adhesion molecules (12), and lipid phosphate phosphatases (13). However, much remains to be learned regarding the mechanisms by which these and other signaling factors coordinately regulate PGC migration in vivo.

The zebrafish embryo has proven to be an exceptional in vivo model for the study of PGC migration. Zebrafish PGCs are segregated from the somatic lineage very early in embryonic development, and shortly thereafter begin a characteristic pattern of migration toward the genital ridges (14, 15). This process is completed within the first 24 h of development, and due to the optical transparency of zebrafish embryos, can be studied at high resolution using simple microscopy. Furthermore, the zebrafish is highly amenable to genetic manipulation, with significant relevance to higher vertebrates, making it an exceptional model in which to explore genes and pathways regulating key developmental processes.

Using the zebrafish model, we recently showed that signaling through a specific IGF-I receptor (IGF-IR) is required for PGC migration (16). Loss-of-function studies targeting the duplicate zebrafish IGF-IRs (igf1ra and igf1rb) revealed that a loss of igf1rb specifically led to aberrant PGC migration, such that fewer PGCs engrafted in the genital ridges. These data revealed novel functions for IGF signaling in the development of the germ cell lineage, however, the identity of cells producing and/or responding to IGF signaling in this context remained unclear. Numerous published reports have demonstrated a positive chemotactic response to IGF signaling by multiple cell types (17, 18, 19, 20), leading us to hypothesize a novel function for embryonic IGF signaling: paracrine regulation of PGC migration.

In this study we tested this hypothesis by: 1) examining the spatial expression patterns of the zebrafish homologs of mammalian IGF-I and IGF-II (igf1, igf2a, and igf2b) during PGC migration; and 2) observing the migratory behavior of PGCs in response to conditional gain or loss of IGF signaling at the PGC surface. We show that the expression of igf2b in vivo is temporally and spatially consistent with a paracrine role in PGC development, PGC migration is affected by experimental alterations in local IGF ligand expression, and PGC migration is compromised after PGC-specific expression of a mutant (dominant-negative) IGF-IR. These studies identify a novel, cell-intrinsic role for paracrine IGF signaling in the regulation of zebrafish PGC migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Zebrafish (Tübingen A/B strain) were maintained at 28.5 C according to standard methods, and fed twice daily with pelleted food (Adult Zebrafish Diet; Zeigler Brothers, Inc., Gardners, PA) and live brine shrimp. Embryos were derived from natural crosses, and staged according to Kimmel et al. (21). All experiments were conducted in accordance with guidelines established by the Subcommittee on Research Animal Care at Massachusetts General Hospital.

Chemical and reagents
Cloning reagents [pCRII-TOPO, TOP10 One-shot chemically competent cells, Platinum Taq polymerase, and Superscript II reverse transcriptase (RT)] were purchased from Invitrogen Corp. (Carlsbad, CA). Reagents for in vitro synthetic mRNA transcription were from Ambion (Palo Alto, CA). Reagents for in situ hybridization [digoxigenin (DIG)-labeled ribonucleotide triphosphates, alkaline phosphatase-conjugated anti-DIG antibody, and nitroblue tetrazolium/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt)] were purchased from Roche (Indianapolis, IN). All other reagents were from Thermo Fisher Scientific Inc. (Waltham, MA), or Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Plasmid DNA encoding the 3'-untranslated region (UTR) of the zebrafish nanos1 gene was generously provided by Erez Raz (Institute of Cell Biology, Zentrum für Molekularbiologie der Entzündung, Münster, Germany).

RT-PCR
RT-PCR was used to determine relative mRNA expression levels for each ligand (igf1, igf2a, and igf2b) in whole embryos at three different stages of development PCR. Briefly, 500 ng total RNA was purified from a pool of 10 embryos at 0, 12, and 24 h after fertilization (hpf), and reverse transcribed to first-strand cDNA using Superscript II RT. Uniform aliquots (5 µl) of cDNA were PCR amplified under defined conditions (94 C, 30 sec; 58 C, 30 sec, 72 C, 45 sec) using gene-specific primers targeted to the 3'-UTR of each target gene (Table 1Go). cDNA encoding ornithine decarboxylase 1 (odc1) was used as an internal reference standard (22). All primer sets were designed with a commercial software package (Vector NTI Advance 9.1; Invitrogen), using identical parameters to generate amplicons of similar size. The number of PCR cycles for each target gene was empirically determined to ensure that amplification was terminated within the linear phase. PCR products were separated by agarose electrophoresis, stained with ethidium bromide, and visualized under UV.


View this table:
[in this window]
[in a new window]

  		TABLE 1. List of oligonucleotide primers used in the study

 
Riboprobe synthesis
DIG-labeled riboprobes for each ligand were synthesized using cDNA template derived from the respective 3'-UTR primers (Table 1Go). Nucleotide sequences among the ligands are highly divergent in this region (<5% identity), precluding cross-reactivity among riboprobes. Total RNA extracted from adult brain using Tri Reagent (Sigma-Aldrich) was used as template for RT; 500 ng total RNA was reverse transcribed to first-strand cDNA using Superscript II RT as described above. Each target cDNA was then PCR amplified, subcloned into pCRII-TOPO, sequenced to confirm accuracy and insert direction, linearized by restriction digest, and used as template for DIG-labeled riboprobe synthesis as previously described (23).

In situ hybridization
Embryos were raised in egg water (+0.003% propylthiouracil to inhibit pigmentation) at 28.5 C until the desired stage of development, manually dechorionated, fixed overnight in 4% PBS-buffered paraformaldehyde, and stored in 100% methanol at –20 C for a minimum of 24 h. After rehydration into PBS [containing 0.1% Tween 20 (PBS-T)], embryos were permeabilized with proteinase K, and in situ hybridizations were performed as described (23). Images were collected with a digital camera (Nikon CoolPix 5500) attached to a Nikon LMZ-1000 dissecting scope (Nikon Corp., Tokyo, Japan).

PGC-specific IGF ligand expression
RT-PCR was used to generate cDNA sequences corresponding to the open reading frames of each IGF ligand (using gene-specific primers (Table 1Go). Unique restriction endonuclease sites were introduced where required to permit directional insertion into plasmid vectors used for subsequent in vitro transcription of synthetic mRNA.

PCR products for each open reading frame were first subcloned into pCRII-TOPO and sequenced to confirm identity and accuracy. The cloned sequences were then ligated into pCS2+ (24, 25), and the vector was further modified by replacing the SV40 polyadenylation site with the nanos1 3'-UTR. Specific sequence regions within the nanos1 3'-UTR confer stability of upstream mRNA sequences specifically in germ cells, while promoting their Dicer-mediated degradation in somatic cells (26, 27, 28, 29). Thus, each IGF:nanos1 hybrid vector encoded for a specific IGF ligand with PGC-specific expression.

PGC-specific dominant-negative IGF-IR
Sequence encoding a truncated IGF-IRb was generated by PCR amplification of cDNA using a primer pair that spans the open reading frame of IGF-IRb, including the transmembrane domain, but that excludes the tyrosine kinase domain. The amplified sequence (dn954) encodes a 954-residue fragment of IGF-IRb, and is virtually identical in design to dominant-negative constructs previously described for use in zebrafish (16, 30) and mammalian cells (31, 32). dn954 was ligated into pcs2+GFP, yielding a dominant-negative receptor-GFP fusion protein construct (dn954:SV40) containing the SV40 polyadenylation site (permitting ubiquitous expression in embryos). A gene-hybrid variant of this construct (dn954:nanos1) was made by replacing the SV40 polyadenylation site with the nanos1 3'-UTR, as described previously, to confer PGC-specific expression and, therefore, PGC-specific suppression of IGF signaling.

In vitro transcription
Hybrid expression constructs were linearized by NotI digestion, electrophoretically separated, and purified by gel extraction (QiaQuick; QIAGEN Inc., Valencia, CA). Linearized plasmid (1–2 µg) was used as template for the synthesis of capped synthetic mRNA, using the SP6 MEGAscript transcription kit (Ambion). Synthetic mRNA was precipitated by centrifugation in LiCl and its integrity confirmed by formaldehyde-agarose electrophoresis. Stock synthetic mRNA solutions were quantified by absorbance and diluted to desired concentrations in 1x Danieau buffer for microinjection.

Templates for riboprobe synthesis were linearized by restriction enzyme digestion, electrophoretically separated, purified by gel extraction, and resuspended in sterile ddH2O. DIG-labeled riboprobes for target genes were synthesized as described (23).

In vivo overexpression
Synthetic capped mRNA was injected into one-cell stage zebrafish embryos derived from natural crosses. For ligand overexpression experiments, treatment embryos were coinjected with gfp:nanos1 mRNA (50 pg/embryo) and either igf1:nanos1, igf2a:nanos1 or igf2b:nanos1 (100–200 pg/embryo). Control embryos were injected with gfp:nanos1 mRNA alone (50 pg/embryo), and wild-type (WT) embryos received no injection. In overexpression experiments with dominant-negative IGF receptors, treatment embryos were coinjected with gfp:nanos1 mRNA (50 pg/embryo) and either dn954:SV40 or dn954:nanos1. Coinjection with gfp:nanos1 was deemed necessary for the dn954 experiments because native GFP expression from the dn954:nanos1 expression constructs was difficult to detect in the PGCs during later development.

Embryos were reared in egg water at 28.5 C until the desired stage of development. Injected embryos (treatments and controls) were examined by fluorescence microscopy for GFP expression, which becomes visible within 5–6 hpf after successful injection. Those without detectable GFP expression were considered to be failed injections and discarded.

Successfully injected embryos were collected at the prim-5 stage (~24 hpf) and fixed in phosphate-buffered paraformaldehyde (4%) overnight at 4 C. The embryos were then transferred to 100% methanol, and stored at –20 C until analysis for PGC development.

PGC development analyses
PGC development was analyzed in embryos at the prim-5 stage, when, under normal circumstances (i.e. in WT embryos), greater than 95% of PGCs have migrated into the genital ridge region (33, 34). Embryos were serially rehydrated to 1 x PBS-T and incubated overnight with rabbit anti-Vasa antiserum. After extensive washes in PBS-T, embryos were incubated for 1 h in secondary antibody (AlexaFluor 488-conjugated goat antirabbit IgG) as previously described (15). The embryos were then washed extensively in PBS-T, immersed in glycerol:PBS-T (70:30), visualized with fluorescence microscopy, and PGC development was analyzed by enumerating the number of PGCs on either side of the genital ridges, and the number of ectopic PGCs. During normal development, zebrafish PGCs converge into two distinct clusters (left and right) in the genital ridges; thus, "ectopic" PGCs were defined as those that were distinctly separate from the genital ridge clusters. At analysis, the observer was blinded with respect to the assignment (WT, treatment, or control) of each embryo group.

Data analyses
Total PGC numbers represent the sum total of genital ridge and ectopic PGCs. Mean total PGCs and mean ectopic PGCs were statistically compared among treatment (igf:nanos1 plus gfp:nanos1; dn954:nanos1), control (gfp:nanos1), and WT embryos using one-way ANOVA, followed by Tukey’s test where appropriate. Significance was accepted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distinct and variable expression of IGFs in zebrafish embryos
RT-PCR analyses revealed distinct variations in expression among the three IGF ligand genes during the first 24 h development (Fig. 1Go). igf1 mRNA was barely detectable after 35 PCR cycles at all stages examined; igf2a mRNA was slightly more abundant than igf1 in one-cell embryos (0 hpf), and increased marginally during the first 24 h development. Expression levels of igf2b were similar to those of igf2a up to 12 hpf, but markedly increased between 12 and 24 hpf. These patterns were reproducible among separate pools of embryos, and all three primer sets abundantly amplified their respective target sequences from cDNA of adult brain and ovary (data not shown), confirming that variations in embryonic expression were not due to different primer efficiencies.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 1. A, RT-PCR analyses showing distinct and variable mRNA expression of igf1, igf2a, and igf2b relative to a housekeeping gene (odc1) in zebrafish embryos during the first 24 hpf. Each lane shows cDNA from a distinct pool of 10 embryos. B, Quantitative summary of relative expression levels of igf1, igf2a, and igf2b mRNA in zebrafish embryos from 0–24 hpf. Expression levels of each ligand are normalized to odc1 levels in equivalent-stage embryos and, thus, presented as arbitrary units (A.U.).

 
We next used in situ hybridization to determine the spatial expression patterns of each igf ligand gene in zebrafish embryos. As expected from the low levels of expression detected by RT-PCR, mRNA for igf1 and igf2a exhibited notably restricted patterns of expression. In 18-somite (18S) embryos, igf1 mRNA was detected exclusively in opposing regions of the lateral midbrain corresponding to the trigeminal ganglia (Fig. 2AGo), whereas expression of igf2a mRNA was detected primarily in the notochord (Fig. 2CGo). In contrast, igf2b showed a distinctly broader pattern of expression (Fig. 2EGo); strong expression was detected in ventral prosencephalon and nephron primordia, with lower levels of expression detected in the floor plate and hypochord along the body axis, and among cells in the vicinity of the urogenital ridges.


Figure 2
View larger version (81K):
[in this window]
[in a new window]

  		FIG. 2. In situ hybridization analyses showing distinct (tissue-specific) expression patterns of igf1 (A and B), igf2a (C and D), and igf2b (E and F) in 18S (A, C, and E) and prim-5 stage (B, D, and F) zebrafish embryos. igf1 mRNA is detected only in trigeminal ganglia (TG). igf2a mRNA is detected in caudal regions of notochord (NC); igf2b mRNA is detected in ventral prosencephalon (VP), nephron primordia (NP), floor plate (FP), hypochord (HC), and the urogenital ridges (UGR). Inset (E and F) shows a dorsal view of igf2b mRNA expression in paired nephron primordia (arrows) in both 18S and prim-5 stage embryos.

 
At the prim-5 stage (24 hpf), igf1 mRNA remained detectable only in the region of the trigeminal ganglia (Fig. 2BGo), whereas igf2a mRNA was increasingly restricted to the caudal tip of the notochord (Fig. 2DGo). However, the pattern of igf2b mRNA in prim-5 embryos differed from that observed in 18S embryos. Although mRNA expression in the floor plate and hypochord was diminished relative to 18S embryos, expression remained abundant in ventral prosencephalon and nephron primordia, and greatly increased in the urogenital ridges (Fig. 2FGo). This pattern in expression is significant because both nephron primordia and the urogenital ridges represent, respectively, intermediate and final migration targets of zebrafish PGCs, within the 24-h window of development examined.

All respective sense controls showed no detectable hybridization signal (data not shown).

PGC-specific expression of IGF ligands affects PGC migration
To determine whether PGC migration is responsive to aberrations in paracrine IGF signaling, we designed hybrid mRNA expression constructs designed to confer PGC-specific expression of the zebrafish homologs of IGF-I and IGF-II (IGF-I, IGF-IIa, IGF-IIb; Fig. 3AGo). We independently tested three IGF:nanos1 hybrids, reasoning that if effects were mediated through the IGF-IR, then similar results would be observed regardless of which ligand was overexpressed.


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 3. Structure of synthetic hybrid mRNA constructs (A) designed to conditionally overexpress GFP and IGF ligands in a PGC-specific manner (B). C, Fusion of a target gene to the nanos1-3'-UTR (nos1) confers PGC-specific expression; inset shows high-magnification image of GFP-expressing PGCs in live embryo. As predicted, PGC migration was disrupted in embryos coexpressing IGF ligands and GFP (e.g. IGF:nanos1) relative to control (GFP:nanos1). D, Mean total PGC numbers were unchanged among control (GFP:nanos1) and treatment embryos. E, A significant increase in the mean number of ectopic PGCs was observed in treatment embryos (IGF-I, IGF-IIa, and IGF-IIb) relative to control (GFP) and WT embryos. The asterisk indicates a statistically significant difference from control-injected embryos (ANOVA, Tukey’s, P < 0.05).

 
Treatment embryos were coinjected with mRNA encoding a specific IGF:nanos1 hybrid and GFP:nanos1, whereas control embryos were injected with mRNA for GFP:nanos1 alone. IGF ligands are secreted proteins; therefore, PGC-specific expression of IGF ligands is predicted to create a zone of high IGF ligand expression in the PGC microenvironment (Fig. 3BGo). If PGC migration is sensitive to IGF ligand signaling, a disruption to PGC migration should be observed after these alterations in local IGF ligand expression.

Microinjection of gfp:nanos1 mRNA (~50 pg/embryo) in control embryos resulted in detectable expression of GFP, in a PGC-specific pattern (Fig. 3CGo). Overexpression of GFP:nanos1 did not affect PGC survival because the mean total number of PGCs in GFP:nanos1 embryos (31.1 ± 0.8; n = 63) was similar to that in WT embryos (29.2 ± 0.8; n = 55; P = 0.22; Fig. 3DGo). PGC migration was also unaffected by overexpression of GFP:nanos1; the mean number of ectopic PGCs in GFP:nanos1 embryos (2.5 ± 0.3; n = 63) did not differ from the mean number of ectopic PGCs in WT embryos (2.7 ± 0.4; n = 55; P = 0.29; Fig. 3EGo).

In embryos coexpressing GFP:nanos1 and IGF-I:nanos1, the mean total number of PGCs (32.2 ± 1.8; n = 24) was similar to that of GFP:nanos1 embryos (P > 0.05), indicating that IGF-I:nanos1 expression does not influence PGC specification or survival (Fig. 3DGo). However, a significant increase in the mean number of ectopic PGCs (Fig. 3EGo) was observed in IGF-I:nanos1 embryos (5.3 ± 1.0; n = 24; P < 0.05), relative to GFP:nanos1 embryos.

Similarly, the mean total number of PGCs in embryos coinjected with GFP:nanos1 and IGF-IIa:nanos1 mRNA (32.9 ± 1.7; n = 20) did not differ from that of GFP:nanos1 (control) embryos (P > 0.05; Fig. 3DGo). However, there was a significant increase (P < 0.01) in the mean number of ectopic PGCs (6.1 ± 0.8; n = 20; Fig. 3EGo) relative to GFP:nanos1 embryos. Similar results were observed in embryos coinjected with mRNA for IGF-IIb:nanos1 and GFP:nanos1; the mean total number of PGCs (28.3 ± 2.0; n = 12; Fig. 3DGo) did not differ from GFP:nanos1-injected embryos, but a significant increase was observed in the mean number of ectopic PGCs (7.4 ± 1.3; n = 12; P < 0.01; Fig. 3EGo). All treatment embryos were morphologically indistinguishable from GFP:nanos1-injected embryos, suggesting that the observed PGC migration defects were not the result of patterning defects induced by IGF ligand overexpression.

PGC-specific suppression of IGF signaling disrupts PGC migration
To determine whether PGCs intrinsically require IGF signaling for migration, we synthesized dominant-negative type 1 IGF receptors (Fig. 4AGo) designed for either ubiquitous (dn954:SV40; Fig. 4BGo) or PGC-specific (dn954:nanos1; Fig. 4CGo) expression in embryos. Injection of dn954:SV40 resulted in detectable GFP expression in blastula-stage embryos (Fig. 4Go, D and E), confirming expression of the receptor-GFP fusion protein. Although GFP expression became increasingly difficult to detect beyond gastrulation, the intensity of GFP expression correlated strongly with somatic growth defects, which were similar to those after combined knockdown of igf1ra and igf1rb (30, 33). Whereas GFP-injected embryos (Fig. 4GGo) were morphologically indistinguishable from WT embryos (Fig. 4FGo), dn954:SV40-injected embryos exhibited proportionally stunted growth (Fig. 4HGo). By Western immunoblot analysis (data not shown), we also detected a reduction in Akt phosphorylation in dn954:SV40-injected embryos at 12 hpf, indicating successful suppression of intracellular signaling pathways downstream of the IGF receptor.


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 4. A, Full-length zebrafish IGF-IRb, with arrows indicating primer binding sites to amplify a truncated (dominant-negative) IGF-IR. B, Truncated IGF-IRb fused to GFP (dn954), with SV40 polyadenylation site for ubiquitous translation in embryos. C, Truncated IGF-IRb (dn954) fused to GFP and 3'-UTR of the nanos1 gene (dn954:nanos1); nanos1 confers PGC-specific translation of the dn954 receptor. D, Bright-field image of a noninjected blastula-stage embryo (left embryo) and a blastula-stage embryo injected with dn954:SV40 mRNA (right embryo). E, Fluorescent image of the same embryos, showing abundant GFP expression in dn954:SV40-injected embryo. F, WT (noninjected) embryo at 24 hpf. G, Control-injected embryo at 24 hpf. H, dn954:SV40-injected embryo at 24 hpf, with gross growth/development defects. I, dn954:nanos1-injected embryo at 24 hpf, showing an absence of growth defects (contrast with H). J, Vasa-positive PGCs detected in the genital ridges (arrowhead) of a gfp:nanos1-injected (control) embryo at 24 hpf. K, Vasa-positive PGCs detected in both genital ridges (arrowhead) and ectopic locations (arrows) of dn954:nanos1-injected embryo (24 hpf). L, Quantitative summary of mean total and mean ectopic PGCs in WT (noninjected), control-injected, and treatment (dn954:nanos1) embryos at 24 hpf. The asterisk indicates a statistically significant difference from control-injected embryos (ANOVA, Tukey’s, P < 0.05). TKD, Tyrosine-kinase domain; TM, transmembrane domain.

 
Embryos injected with dn954:nanos1 did not exhibit any gross somatic development defects (Fig. 4IGo), indicating that the nanos1 was successful in preventing expression of the dominant-negative receptor in somatic cells of the embryo. Despite the lack of somatic defects, overexpression of dn954:nanos1 resulted in pronounced disruptions to PGC migration (Fig. 4KGo), relative to gfp:nanos1-injected embryos (Fig. 4JGo). A statistically significant increase (P < 0.01) in the mean number of ectopic PGCs (Fig. 4LGo) was detected in dn954:nanos1 embryos (8.8. ± 1.6, n = 16), relative to gfp:nanos1-injected (2.5 ± 0.3, n = 63) and WT embryos (2.7 ± 0.4, n = 55). As with the ligand overexpression experiments, there were no detectable changes in mean total PGC numbers among treatment groups, suggesting that IGF signaling is not intrinsically required by PGCs for specification or proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides three lines of in vivo evidence demonstrating that paracrine IGF signaling influences PGC migration in the zebrafish embryo. These findings expand upon our earlier work showing that nonconditional (whole body) suppression of a specific type-1 IGF receptor disrupted zebrafish PGC migration (16). A significant limitation to our earlier study was that the gene knockdown approaches used did not target specific tissues or cell types. We were consequently unable to determine whether global suppression of IGF signaling affected PGC migration directly, or whether the observed effects resulted from the compromised development of IGF-dependent somatic tissues whose integrity is essential to promote normal PGC migration. In this study, we used conditional (cell specific) methods to manipulate paracrine IGF signaling levels in the PGC microenvironment, specifically within the window of development corresponding to PGC migration. This approach revealed that (1) PGC migration is distinctly sensitive to alterations in paracrine IGF signaling, and (2) PGC-specific suppression of IGF signaling leads to aberrant PGC migration. Together with the expression patterns of IGF ligand genes revealed by in situ analyses, our findings support the hypothesis that IGF signaling functions in vivo to modulate the migratory behavior of PGCs. Furthermore, we identified a candidate ligand (igf2b), whose pattern of embryonic expression is spatially and temporally consistent with a role in PGC development.

The first published report identifying IGF ligands in zebrafish identified two sequences encoding the putative zebrafish homologs of mammalian IGF-I and IGF-II, and reported ubiquitous expression of each during embryogenesis (34). However, a zebrafish igf2 paralog (termed igf2b) was subsequently identified and its expression confirmed during heart regeneration (35). In this study we confirmed embryonic expression of igf2b in zebrafish embryos, revealing it to be (1) maternally inherited in embryos, and (2) the most abundantly and dynamically expressed of the known zebrafish IGF-I/II homologs during early development.

A spatial analysis of expression by in situ hybridization revealed distinct (tissue-specific) expression patterns for each igf gene, contrary to the findings of Maures et al. (34). Because the genes encoding type-1 IGF receptors are expressed ubiquitously in vertebrate embryos, including zebrafish (16, 34, 36), our findings support the notion that the pleiotropic effects of IGF signaling during early animal development are driven largely by divergent expression of individual IGF ligands and related regulatory genes such as IGF binding proteins (20, 23, 37, 38). Therefore, exploring the functions of IGF ligands on a case-by-case basis should provide valuable insights into the diversity and evolution of the IGF signaling axis.

Of great interest to our primary hypothesis was the spatial and temporal expression pattern of igf2b. In 18S embryos we detected igf2b mRNA in ventral forebrain, nephron primordia, floor plate mesoderm, hypochord, and in a small number of cells in the urogenital ridges. Expression of igf2b mRNA in the floor plate and hypochord tapered off by the end of the segmentation period (prim-5 stage), whereas expression in the urogenital ridges markedly increased at this time. Notably, this period of development corresponds directly to the final convergence of PGCs in the embryonic urogenital ridges (14, 15, 33, 34). The observed expression pattern of igf2b is also strikingly similar to that of cxcl12a (39), the most well-defined chemokine regulating zebrafish PGC migration, with the exception that igf2b is not expressed detectably in somites (where PGCs rarely migrate).

To determine whether PGC migration is responsive to paracrine IGF signaling in vivo, we used gene hybrid expression constructs designed to overexpress IGF ligands specifically in the PGC microenvironment. This approach took advantage of the properties of a germ cell-specific gene (nanos1) that achieves cell-specific translation via specific sequence elements in the 3'-UTR (26, 28, 29). Therefore, fusion of IGF ligand genes to the nanos1 3'-UTR (nanos1) provides PGC-specific expression and secretion of IGF ligands, predicted to create an abundance of IGF ligand in the PGC microenvironment. The efficacy of this approach in our studies is demonstrated by the specificity of expression of a coinjected reporter gene (GFP). Although this surrogate approach is less desirable than direct detection of the exogenously delivered gene product, the lack of specific antibodies for each zebrafish IGF ligand precludes the latter approach.

The results of these overexpression studies support the hypothesis that PGC migration in zebrafish embryos is sensitive to disruptions in IGF signaling in the PGC microenvironment. PGC-specific overexpression of any one of the three IGF ligands had no effect on total PGC numbers, but potently disrupted their migration, leading to large numbers of mismigrated PGCs. The fact that all three ligands elicited a similar effect is important because this argues that the effect is likely mediated through classic type-1 IGF receptors. The underlying mechanism at this point in time remains clear. One possibility is that overexpression of IGF ligands results in physiological down-regulation of type-1 IGF receptors, either in PGCs or juxtaposing cells, thereby compromising PGC migration behavior. Such a mechanism implies a central role for type-1 IGF receptors in mediating PGC migration.

To explore specifically this possibility, we sought to determine whether IGF receptor function was intrinsically required by PGCs for normal migration. This was achieved by suppressing IGF receptor function specifically in the PGCs, while maintaining normal IGF receptor function in somatic cells. This was accomplished by overexpressing a dominant-negative IGF-IR (dn954) under the translational control of the nanos1 3'-UTR. The specificity of this approach was confirmed, in that PGC-specific suppression of IGF signaling had no detectable effects on somatic tissues. However, we observed deleterious effects on directional PGC migration, thereby supporting the notion that PGCs intrinsically require IGF signaling to migrate correctly. Interestingly, the PGCs do not require IGF signaling for proliferation or survival because total PGC numbers did not differ among treatment, control, and WT embryos. These finding suggest a cellular function (migration) for IGF signaling that is wholly separate from its more widely known roles in promoting cell proliferation and survival.

The ultimate fate of mismigrated PGCs in these embryos is unknown, but it is likely that they will be eliminated by apoptosis later in development, possibly as a method to avoid the development of extragonadal germ cell tumors (5). Future studies will seek to explore this, using transgenic lines of fish (e.g. vasa:EGFP) that exhibit stable reporter gene expression in the germ lineage (40) and, thus, allow long-term tracing of the germ cell lineage.

Although we demonstrate a cell-intrinsic response of PGCs to IGF signaling, it is highly likely that IGF signaling plays other indirect roles in PGC development. For example, it is likely that IGF signaling is also required for the development of somatic tissues in the urogenital tract, particularly considering the strong expression of igf2b in the nephron primordia and the presumptive pronephric ducts. In fact, we feel it is likely that defective development of urogenital tract tissues contributed to the migratory defects of PGCs after whole body suppression of IGF signaling (16). The importance of somatic tissue development for PGC migration was clearly demonstrated by analyses of zebrafish mutants with specific defects in mesoderm development (15, 41). For example, PGCs fail to aggregate at their intermediate clustering site (pronephric mesoderm) in one-eyed-pinhead;notail (oep;ntl) and spadetail;notail (spt;ntl) double mutants, a defect that was attributed to improper differentiation of pronephric tissues. Similarly, PGCs in hands off (han) mutants, which exhibit defects in gonadal mesoderm development, fail to migrate from the intermediate target site to the embryonic gonad (15). In each case, aberrant PGC migration could ultimately be attributed to the failed development of somatic tissues from which required chemotactic signals emanate (15, 39). Future studies are planned to identify the role played by IGF signaling in the development of the urogenital tract. Based on expression it appears likely that igf2b is important for the development of the pronephros; targeted loss-of-function studies will likely reveal the precise requirements for each IGF ligand in the development of specific embryonic tissues, while providing novel perspectives on the functional evolution of this important growth factor signaling axis.

It will also be of interest to explore further the downstream signaling pathways activated by IGF signaling in PGCs. Although IGF signaling is known to be mediated via both MAPK and phosphatidylinositol 3-kinase/Akt signaling pathways, it would be of interest to determine whether one or both of these pathways mediate PGC migration. It will also be important to explore the question of cross talk between IGF and other signaling axes (e.g. CXCL-family chemokines), as suggested by recent in vitro studies of metastatic breast cancer cells (19). Demonstrating cross talk in vivo remains a formidable challenge but may be achievable using emerging models such as zebrafish.

In summary, we have provided new evidence that suggests an in vivo role for paracrine IGF signaling in zebrafish germline development, in part via cell-intrinsic effects on PGC migration. These findings provide new information on the pleiotropic functions of the IGF signaling axis and may help explain why deficiencies in IGF signaling are often associated with compromised reproductive development.


   Acknowledgments
 
We thank Drs. Jose Teixeira, Hideo Sakamoto, Peter J. Schlueter, and James K. Pru for their thoughtful comments, and Ms. Petra Sergeant for animal care.


   Footnotes
 
The study was supported financially by Vincent Memorial Research Funds. X.S. received a fellowship from the Program for Research in Science and Engineering at Harvard University, and was supported by the Harvard Undergraduate Research Program. X.S. and M.S.C. were further supported by the Harvard Stem Cell Institute Summer Undergraduate Research Program.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2008

Abbreviations: CXCL, C-X-C motif ligand; DIG, digoxigenin; GFP, green fluorescent protein; hpf, h after fertilization; IGF-IR, IGF-I receptor; odc1, ornithine decarboxylase 1; PBS-T, PBS containing 0.1% Tween 20; PGC, primordial germ cell; RT, reverse transcriptase; 18S, 18-somite; UTR, untranslated region; WT, wild type.

Received April 15, 2008.

Accepted for publication June 11, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Li F, Tiede B, Massague J, Kang Y 2007 Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res 17:3–14[CrossRef][Medline]
  2. Arya M, Bott SR, Shergill IS, Ahmed HU, Williamson M, Patel HR 2006 The metastatic cascade in prostate cancer. Surg Oncol 15:117–128[CrossRef][Medline]
  3. Albelda SM 1993 Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Lab Invest 68:4–17[Medline]
  4. Hoei-Hansen CE, Sehested A, Juhler M, Lau YF, Skakkebaek NE, Laursen H, Rajpert-de Meyts E 2006 New evidence for the origin of intracranial germ cell tumours from primordial germ cells: expression of pluripotency and cell differentiation markers. J Pathol 209:25–33[CrossRef][Medline]
  5. Schneider DT, Schuster AE, Fritsch MK, Hu J, Olson T, Lauer S, Gobel U, Perlman EJ 2001 Multipoint imprinting analysis indicates a common precursor cell for gonadal and nongonadal pediatric germ cell tumors. Cancer Res 61:7268–7276[Abstract/Free Full Text]
  6. Stebler J, Spieler D, Slanchev K, Molyneaux KA, Richter U, Cojocaru V, Tarabykin V, Wylie C, Kessel M, Raz E 2004 Primordial germ cell migration in the chick and mouse embryo: the role of the chemokine SDF-1/CXCL12. Dev Biol 272:351–361[CrossRef][Medline]
  7. Knaut H, Werz C, Geisler R, Nüsslein-Volhard C 2003 A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor. Nature 421:279–282[CrossRef][Medline]
  8. Molyneaux KA, Zinszner H, Kunwar PS, Schaible K, Stebler J, Sunshine MJ, O’Brien W, Raz E, Littman D, Wylie C, Lehmann R 2003 The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival. Development 130:4279–4286[Abstract/Free Full Text]
  9. Schier AF 2003 Chemokine signaling: rules of attraction. Curr Biol 13:R192–R194
  10. Chuva de Sousa Lopes SM, van den Driesche S, Carvalho RL, Larsson J, Eggen B, Surani MA, Mummery CL 2005 Altered primordial germ cell migration in the absence of transforming growth factor β signaling via ALK5. Dev Biol 284:194–203[CrossRef][Medline]
  11. Farini D, La Sala G, Tedesco M, De Felici M 2007 Chemoattractant action and molecular signaling pathways of Kit ligand on mouse primordial germ cells. Dev Biol 306:572–583[CrossRef][Medline]
  12. De Felici M, Scaldaferri ML, Farini D 2005 Adhesion molecules for mouse primordial germ cells. Front Biosci 10:542–551[Medline]
  13. Sano H, Renault AD, Lehmann R 2005 Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2. J Cell Biol 171:675–683[Abstract/Free Full Text]
  14. Weidinger G, Wolke U, Köprunner M, Klinger M, Raz E 1999 Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells. Development 126:5295–5307[Abstract]
  15. Weidinger G, Wolke U, Koprunner M, Thisse C, Thisse B, Raz E 2002 Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target. Development 129:25–36[Abstract/Free Full Text]
  16. Schlueter PJ, Sang X, Duan C, Wood AW 2007 Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival. Dev Biol 305:377–387[CrossRef][Medline]
  17. Ponte AL, Marais E, Gallay N, Langonne A, Delorme B, Herault O, Charbord P, Domenech J 2007 The in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities. Stem Cells 25:1737–1745[CrossRef][Medline]
  18. Fiedler J, Brill C, Blum WF, Brenner RE 2006 IGF-I and IGF-II stimulate directed cell migration of bone-marrow-derived human mesenchymal progenitor cells. Biochem Biophys Res Commun 345:1177–1183[CrossRef][Medline]
  19. Akekawatchai C, Holland JD, Kochetkova M, Wallace JC, McColl SR 2005 Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells. J Biol Chem 280:39701–39708[Abstract/Free Full Text]
  20. Firth SM, Baxter RC 2002 Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 23:824–854[Abstract/Free Full Text]
  21. Kimmel CB, Ballard WH, Kimmel SR, Ullmann B, Schilling TF 1995 Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310[Medline]
  22. Draper BW, Morcos PA, Kimmel CB 2001 Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis 30:154–156[CrossRef][Medline]
  23. Wood AW, Schlueter PJ, Duan C 2005 Targeted knockdown of insulin-like growth factor binding protein-2 disrupts cardiovascular development in zebrafish embryos. Mol Endocrinol 19:1024–1034[Abstract/Free Full Text]
  24. Farah MH, Olson JM, Sucic HB, Hume RI, Tapscott SJ, Turner DL 2000 Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127:693–702[Abstract]
  25. Turner DL, Weintraub H 1994 Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8:1434–1447[Abstract/Free Full Text]
  26. Ramasamy S, Wang H, Quach HN, Sampath K 2006 Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells. Dev Biol 292:393–406[CrossRef][Medline]
  27. Mishima Y, Giraldez AJ, Takeda Y, Fujiwara T, Sakamoto H, Schier AF, Inoue K 2006 Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr Biol 16:2135–2142[CrossRef][Medline]
  28. Blaser H, Eisenbeiss S, Neumann M, Reichman-Fried M, Thisse B, Thisse C, Raz E 2005 Transition from non-motile behaviour to directed migration during early PGC development in zebrafish. J Cell Sci 118:4027–4038[Abstract/Free Full Text]
  29. Köprunner M, Thisse C, Thisse B, Raz E 2001 A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev 15:2877–2885[Abstract/Free Full Text]
  30. Schlueter PJ, Royer T, Farah MH, Laser B, Chan SJ, Steiner DF, Duan C 2006 Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors. FASEB J 20:1230–1232[Abstract/Free Full Text]
  31. Prager D, Li HL, Asa S, Melmed S 1994 Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant. Proc Natl Acad Sci USA 91:2181–2185[Abstract/Free Full Text]
  32. Prager D, Yamasaki H, Weber MM, Gebremedhin S, Melmed S 1992 Human insulin-like growth factor I receptor function in pituitary cells is suppressed by a dominant negative mutant. J Clin Invest 90:2117–2122[Medline]
  33. Schlueter PJ, Peng G, Westerfield M, Duan C 2007 Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression. Cell Death Differ 14:1095–1105[CrossRef][Medline]
  34. Maures T, Chan SJ, Xu B, Sun H, Ding J, Duan C 2002 Structural, biochemical, and expression analysis of two distinct insulin-like growth factor I receptors and their ligands in zebrafish. Endocrinology 143:1858–1871[Abstract/Free Full Text]
  35. Schebesta M, Lien CL, Engel FB, Keating MT 2006 Transcriptional profiling of caudal fin regeneration in zebrafish. Sci World J 6(Suppl 1):38–54
  36. Ayaso E, Nolan CM, Byrnes L 2002 Zebrafish insulin-like growth factor-I receptor: molecular cloning and developmental expression. Mol Cell Endocrinol 191:137–148[CrossRef][Medline]
  37. Wood AW, Duan C, Bern HA 2005 Insulin-like growth factor signaling in fish. Int Rev Cytol 243:215–285[CrossRef][Medline]
  38. Kajimura S, Aida K, Duan C 2005 Insulin-like growth factor-binding protein-1 (IGFBP-1) mediates hypoxia-induced embryonic growth and developmental retardation. Proc Natl Acad Sci USA 102:1240–1245[Abstract/Free Full Text]
  39. Doitsidou M, Reichman-Fried M, Stebler J, Koprunner M, Dorries J, Meyer D, Esguerra CV, Leung T, Raz E 2002 Guidance of primordial germ cell migration by the chemokine SDF-1. Cell 111:647–659[CrossRef][Medline]
  40. Krovel AV, Olsen LC 2002 Expression of a vas::EGFP transgene in primordial germ cells of the zebrafish. Mech Dev 116:141–150[CrossRef][Medline]
  41. Wolke U, Weidinger G, Koprunner M, Raz E 2002 Multiple levels of posttranscriptional control lead to germ line- specific gene expression in the zebrafish. Curr Biol 12:289–294[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
Y. A. R. White, J. T. Kyle, and A. W. Wood
Targeted Gene Knockdown in Zebrafish Reveals Distinct Intraembryonic Functions for Insulin-Like Growth Factor II Signaling
Endocrinology, September 1, 2009; 150(9): 4366 - 4375.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sang, X.
Right arrow Articles by Wood, A. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sang, X.
Right arrow Articles by Wood, A. W.

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