SUMMARY

SH2-domain-containing inositol phosphatase 2 (SHIP2) belongs to a small family of phosphoinositide 5-phosphatases that help terminate intracellular signaling initiated by activated receptor tyrosine kinases. Mammalian SHIP2 is viewed primarily as an attenuator of insulin signaling and has become a prominent candidate target for therapeutic agents that are designed to augment insulin signaling. Despite this view, no signaling pathway has yet been demonstrated as being affected directly by SHIP2 function in vivo, and in vitro studies indicate that the protein may function in multiple signaling pathways. Here, we analyze the role of a SHIP2 family member in the early zebrafish embryo where developmental and gene expression defects can be used to assay specific signaling pathways. The zebrafish ship2a transcript is maternally supplied, and inhibiting the expression of its protein product results in the expansion of dorsal tissue fates at the expense of ventral ones. We show that the developmental defects are the result of perturbation of fibroblast growth factor (FGF) signaling in the early embryo. Loss of Ship2a leads to an increased and expanded expression of outputs of FGF-mediated signaling, including FGF-dependent gene expression and activated mitogen-activated protein kinase (MAPK) signaling. Our findings demonstrate that Ship2a attenuates the FGF signaling pathway in vivo and functions in the establishment of normal tissue patterning in the early embryo. We suggest that modulation of FGF signaling may be a principal function of SHIP2 in mammals.

INTRODUCTION

Negative feedback loops and constitutive dampening of the function of signal transduction components play important roles in both intracellular and intercellular signaling pathways. Attenuation of signaling has multiple functions, including resetting responsiveness, limiting the duration of signaling, or modulating the strength of a signal. Negative feedback loops are intrinsic to the mechanisms that maintain homeostasis, and the disruption of feedback mechanisms is a prominent causative component of the pathophysiology of metabolic disorders such as diabetes and growth control disorders such as cancer (Leslie and Downes, 2004; Engelman et al., 2006; Lazar and Saltiel, 2006; Muoio and Newgard, 2008). Negative feedback of signaling events also plays important roles in the patterning of tissue precursors in the embryo, contributing to the shaping of morphogen gradients and the establishment of positional information (Manahan et al., 2004; Affolter and Basler, 2007). The finding that signaling events are attenuated at multiple levels, including the sequestration or destruction of extracellular ligands and intracellular activators; dephosphorylation of members of kinase cascades; and competition for target promoter sites, indicates that modulation of signaling strength is a significant aspect of the ability of a cell to sense and respond to its environment (De Robertis et al., 2000; Logan and Nusse, 2004; Manahan et al., 2004; Tsang and Dawid, 2004; Affolter and Basler, 2007).

Phosphoinositides are both immediate effector components of receptor tyrosine kinase (RTK)-initiated signaling and major targets for the negative feedback modulation of RTK signaling. Activation of RTKs results in the recruitment and activation of phosphoinositide 3-kinase (PI3K) resulting in the local production of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3], which serves as a focus for the assembly and activation of signal transduction machinery (Lazar and Saltiel, 2006; Ooms et al., 2009). Multiple inositol phosphatases are recruited to remove PtdIns(3,4,5)P3; their activities generate derivative PtdIns, which may serve to terminate signaling or promote distinct signaling cascades. For example, the inositide 3-phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a primary attenuator of signaling in many contexts and consequently functions as a tumor suppressor (Leslie and Downes, 2004).

The src homology 2 (SH2)-domain-containing inositol 5-phosphatases (SHIPs) comprise a small family of proteins whose primary role appears to be downregulation of RTK signaling cascades (Backers et al., 2003). SHIPs are poised to interact with signaling cascades. They are expressed constitutively, facilitating their rapid activity-dependent recruitment to RTKs at the plasma membrane (Backers et al., 2003; Wang et al., 2004). SHIPs are multi-domain proteins that are capable of attenuating signaling in at least two ways: by virtue of their SH2 domain, which interacts directly with RTKs or with the adaptor molecules Shc and Grb2 and, thereby, presumably disrupts signaling interactions; or owing to their phosphatase activity, which catalyzes the dephosphorylation of PtdIns(3,4,5)P3 to phosphatidylinositol (3,4)-bisphosphate [PtdIns(3,4)P2] and antagonizes the PI3K-stimulated pathway (Pesesse et al., 1998; Wisniewski et al., 1999; Pesesse et al., 2001). As PtdIns(3,4)P2 also serves to support signaling through Akt (protein kinase B) and PDK1 (phosphoinositide-dependent kinase 1), SHIP2-dependent dephosphorylation may have modulatory functions in addition to attenuation of a signal (Ooms et al., 2009).

SHIPs attenuate signaling by mechanisms that are likely to affect multiple RTK pathways. Indeed, in vitro studies indicate that SHIPs can negatively regulate signaling events mediated by insulin, nerve growth factor (NGF), epidermal growth factor (EGF), c-Met, macrophage colony-stimulating factor (M-CSF) or platelet-derived growth factor receptor (PDGFR) (Pesesse et al., 2001; Wang et al., 2004; Koch et al., 2005; Aoki et al., 2007; Artemenko et al., 2009). Nevertheless, the true in vivo targets of SHIPs are unknown. In mammals, SHIP1 (also known as INPP5D) is expressed in a tissue-restricted manner, being present at high levels in cells of the hematopoietic lineage. Loss of Ship1 function in mice produces a mild lymphoproliferative phenotype in which both macrophages and B lymphocytes are hypersensitive to stimuli (Helgason et al., 1998; Liu et al., 1999). In contrast, SHIP2 (also known as INPPL1) is expressed broadly, and its immediate functions in vivo have yet to be defined (Muraille et al., 1999; Decker and Saltiel, 2005).

Whereas in vitro studies have implicated SHIP2 in multiple biological processes, many analyses have highlighted its role in insulin signaling (Clement et al., 2001; Sasaoka et al., 2001; Wada et al., 2001; Lazar and Saltiel, 2006). Overexpression of SHIP2 in humans or mice has been linked with susceptibility to type 2 diabetes and insulin resistance (Marion et al., 2002; Fukui et al., 2005; Kagawa et al., 2008). Consistent with these observations, the loss of function characterization of the Ship2/Inppl1 gene in mice has demonstrated a fundamental role of SHIP2 in the regulation of energy metabolism (Clement et al., 2001; Sleeman et al., 2005). Whereas one mutant allele affecting the Ship2 locus was associated with increased sensitivity to insulin (Clement et al., 2001), mice that were homozygous for a simple null allele of Ship2 were viable with mild skeletal defects; normal glucose levels and insulin tolerance; and resistance to high-fat diet-induced obesity (Sleeman et al., 2005). Although the cell types that mediate the physiological functions of SHIP2 and the molecular mechanism of SHIP2 action remain unresolved, these findings have been interpreted as indicating that both neural and non-neural functions of SHIP2 underlie the metabolism phenotype (Birnbaum, 2005; Decker and Saltiel, 2005; Lazar and Saltiel, 2006). As SHIP2 is viewed as a potential target for the development of new therapeutic agents to ameliorate the effects of diabetes (Lazar and Saltiel, 2006; Sasaoka et al., 2006), it is important to identify specific cellular signaling events that SHIP2 modulates in vivo.

As many tissue induction and patterning events in the vertebrate embryo have been linked to specific signaling pathways, we reasoned that an analysis of the earliest developmental defects associated with loss of SHIP2 function in vivo might indicate a signal transduction pathway that directly requires SHIP2 activity. We analyzed the consequences of loss of function of the zebrafish ship2a gene on the development of the zebrafish embryo and traced the origins of these defects to perturbation of a single signaling pathway. We show here that maternal Ship2a protein is needed to establish proper dorsoventral (DV) patterning in the zebrafish embryo. As interactions between the WNT, bone morphogenic protein (BMP) and fibroblast growth factor (FGF) signaling pathways establish and maintain the position-dependent identities of tissue precursors in the gastrula stage vertebrate embryo (De Robertis et al., 2000; Hammerschmidt and Mullins, 2002; Furthauer et al., 2004), we examined gene expression outputs dependent on each of these pathways in embryos deficient in Ship2a. We demonstrate that maternal Ship2a protein is required to establish proper DV patterning by virtue of its function as a negative regulator of FGF signaling in the pre-gastrula zebrafish embryo.

RESULTS

Identification and expression of zebrafish Ship2a

The human SHIP2 protein sequence was used in a BLAST homology search to identify expressed and genomic sequences that encoded the presumed zebrafish ortholog. A full-length zebrafish cDNA clone with a predicted open-reading frame encoding 1266 amino acids was generated by reverse transcription (RT)-PCR of mRNA isolated from 24-hour post-fertilization (hpf) embryos. The zebrafish protein, designated here as Ship2a, has the signature domains and the motif organization that unambiguously identify it as a SHIP2 family member, including an N-terminal SH2 domain, a central inositol polyphosphate 5-phosphatase catalytic (IPPc) domain, an NPXY motif, a proline-rich domain (PRD) and a C-terminal sterile alpha-motif (SAM), a specific motif that distinguishes SHIP2 from SHIP1 proteins (Fig. 1A) (see also the complete sequence and domain identification in supplementary material Fig. S1) (Backers et al., 2003). Ship2a is, overall, 65% identical to human SHIP2 and shares even greater identity with the functional domains of the human protein (Fig. 1A). The zebrafish genome also contains a second ship2 gene, ship2b (chromosome 14, zv7 scaffold contig BX088596.8), and a more distantly related gene, ship1 (chromosome 6, zv7 scaffold contig CR847938.10). Although the Ship2b protein sequence exhibits the typical SHIP2 domain structure, it is only approximately 44% identical to either human SHIP2 or zebrafish Ship2a. Comparative sequence analyses indicate that zebrafish Ship2a is the zebrafish protein that is most closely related to human and mouse SHIP2 (Fig. 1B; supplementary material Fig. S1). Moreover, zebrafish ship2a maps to the distal portion of chromosome 15 (zv7 scaffold 1537.2-1538.5), immediately adjacent to phox2a, which is a syntenic relation that is present in the mouse and human genomes, further supporting its identity as the true ortholog of mammalian SHIP2.

Zebrafish ship2a transcripts are broadly distributed throughout the embryo from the one-cell stage to 24 hpf (Fig. 1C–E and data not shown). Because ship2a is highly expressed in the one-cell zygote and in all blastomeres through the cleavage stages, we conclude that ship2a is a maternally supplied transcript that is ubiquitously expressed in the early embryo.

Maternal Ship2a protein is required for DV patterning

To identify an in vivo role of ship2a, we depleted the maternal Ship2a product from the embryo with antisense morpholinos (MOs) and examined how loss of Ship2 affected embryonic development. We verified that the sequence of the maternal transcript was identical to the one cloned from 24-hpf embryos and generated two translation-blocking MOs (ship2a MO1 and ship2a MO2), each complementary to a different non-overlapping sequence near the AUG start codon (see Methods). MOs were injected into the yolk of one-cell stage embryos shortly after fertilization, under conditions that enabled each MO to completely block the translation of a co-injected reporter mRNA, consisting of 5′ ship2a sequences fused to green fluorescent protein (GFP)-coding sequences (supplementary material Fig. S2). Furthermore, the effects of the MOs were highly target specific in that injection of either translation-blocking MO produced an identical phenotype that was uniformly expressed by treated gastrula-stage embryos, and distinct from that produced by effective splice blocking or other control MOs (Fig. 2J,L; supplementary material Fig. S3; and additional data not shown).

Embryos depleted of maternal Ship2a exhibited a characteristic dorsalization phenotype at the 5–6-somite stage, affecting multiple tissues and germ layers (Nguyen et al., 1998). Lateral expansion of somites (paraxial mesoderm) was readily visible (n=22/23 embryos), and lateral expansion of the hindbrain (neuroectoderm) was revealed by krox-20 expression in rhombomeres 3 and 5 (n=19/21 embryos) (Fig. 2A–D). In addition, as is often observed in dorsalized embryos, neural crest progenitors, which are present at the border between the neural and non-neural ectoderm and are indicated by foxd3 expression, were absent or generated in reduced numbers (n=24/27 embryos) (Fig. 2E,F). Most ship2 morphants died at segmentation stages; those that did survive to 24 hpf, which presumably represented weak versions of the prevailing phenotype, exhibited morphology consistent with a dorsalized patterning defect (Fig. 2G,H) (Stachel et al., 1993).

Fig. 1.

The zebrafish Ship2a protein and expression of maternalship2atranscripts. (A) The structure of the zebrafish Ship2a protein and the percentage identity between the signature domains of the zebrafish and human SHIP2 protein sequences. The zebrafish Ship2a protein contains motifs that are highly conserved among IPPc catalytic domains. SH2, src homology 2; IPPc, inositol polyphosphate 5-phosphatase catalytic domain; PRD, proline-rich domain; SAM, sterile alpha-motif. (B) Unrooted phylogenetic tree based on Clustal W protein sequence alignments, indicating the relatedness of zebrafish and human SHIP1 and SHIP2 proteins. (C–E) Zebrafish ship2a is maternally supplied and broadly expressed throughout early development. ship2a expression is shown in whole embryos (diameter ∼0.8 mm) at the one-cell (C), eight-cell (D) and 50% epiboly (5.3 hpf) (E) stages.

Fig. 1.

The zebrafish Ship2a protein and expression of maternalship2atranscripts. (A) The structure of the zebrafish Ship2a protein and the percentage identity between the signature domains of the zebrafish and human SHIP2 protein sequences. The zebrafish Ship2a protein contains motifs that are highly conserved among IPPc catalytic domains. SH2, src homology 2; IPPc, inositol polyphosphate 5-phosphatase catalytic domain; PRD, proline-rich domain; SAM, sterile alpha-motif. (B) Unrooted phylogenetic tree based on Clustal W protein sequence alignments, indicating the relatedness of zebrafish and human SHIP1 and SHIP2 proteins. (C–E) Zebrafish ship2a is maternally supplied and broadly expressed throughout early development. ship2a expression is shown in whole embryos (diameter ∼0.8 mm) at the one-cell (C), eight-cell (D) and 50% epiboly (5.3 hpf) (E) stages.

As ventrolateral expansion of dorsal tissues can be caused by perturbation of patterning in the gastrula-stage embryo (Hammerschmidt and Mullins, 2002), we analyzed patterning in terms of gene expression domains in Ship2a-depleted gastrulae. Analysis of foxb1.2 and eve1 expression at the sphere stage (4 hpf) revealed that gene expression domains marking prospective DV identities were already shifted in the early gastrula of ship2a-morphant embryos. foxb1.2 encodes a forkhead domain transcription factor that is normally expressed in the dorsolateral region; eve1 is a homeobox gene expressed in the ventroposterior region of the pre-gastrula embryo (Joly et al., 1993; Odenthal and Nusslein-Volhard, 1998). In Ship2a-depleted embryos, the foxb1.2 domain expanded ventrally so that it was expressed circumferentially (n=62/68 embryos) (Fig. 2I–L), and eve1 expression was severely reduced (n=18/19 embryos) (Fig. 2M,N and supplementary material Fig. S3E,F). Although identities were shifted along the DV axis, no alteration in the organizer was detected as assayed by goosecoid (gsc) expression (n=21/21 embryos) (Fig. 2O,P).

Fig. 2.

Knockdown of maternal Ship2a dorsalizes the embryo and affects patterning prior to the onset of gastrulation. (A–F) Lateral expansion of dorsal tissue fates in 5–6-somite stage embryos depleted for Ship2a protein. Maternal ship2a-morphant embryos have a lateral expansion of somites (A,B) and a lateral expansion of krox-20 expression in rhombomeres 3 and 5 (r3 and r5) (C,D) compared with WT controls. (E,F) foxd3 expression at the lateral border of the neural plate is reduced in maternal ship2a-morphant embryos. (G,H) ship2a morphants that survive to 24 hpf appear dorsalized. (I–N) foxb1.2 and eve1 expression at the sphere stage (4 hpf) in WT and maternal ship2a-morphant embryos. (I,K) foxb1.2 is expressed dorsally in WT embryos, and (J,L) its expression is expanded to the ventral side of the embryo in maternal ship2a morphants. (M) eve1 expression is restricted to the ventral side in WT embryos, and (N) ventral eve1 expression is lost in maternal ship2a morphants. (O,P) goosecoid (gsc) expression marking the dorsal organizer at 70% epiboly is unchanged between WT and maternal ship2a-morphant embryos. A and B, dorsal views, anterior to the left; C–F, dorsal views, anterior to top; G and H, lateral views; I–N, animal pole views with dorsal to the right; O and P, lateral views, dorsal to right, markings indicate blastoderm margin.

Fig. 2.

Knockdown of maternal Ship2a dorsalizes the embryo and affects patterning prior to the onset of gastrulation. (A–F) Lateral expansion of dorsal tissue fates in 5–6-somite stage embryos depleted for Ship2a protein. Maternal ship2a-morphant embryos have a lateral expansion of somites (A,B) and a lateral expansion of krox-20 expression in rhombomeres 3 and 5 (r3 and r5) (C,D) compared with WT controls. (E,F) foxd3 expression at the lateral border of the neural plate is reduced in maternal ship2a-morphant embryos. (G,H) ship2a morphants that survive to 24 hpf appear dorsalized. (I–N) foxb1.2 and eve1 expression at the sphere stage (4 hpf) in WT and maternal ship2a-morphant embryos. (I,K) foxb1.2 is expressed dorsally in WT embryos, and (J,L) its expression is expanded to the ventral side of the embryo in maternal ship2a morphants. (M) eve1 expression is restricted to the ventral side in WT embryos, and (N) ventral eve1 expression is lost in maternal ship2a morphants. (O,P) goosecoid (gsc) expression marking the dorsal organizer at 70% epiboly is unchanged between WT and maternal ship2a-morphant embryos. A and B, dorsal views, anterior to the left; C–F, dorsal views, anterior to top; G and H, lateral views; I–N, animal pole views with dorsal to the right; O and P, lateral views, dorsal to right, markings indicate blastoderm margin.

DV patterning of the gastrula specifically required Ship2a product derived from maternally supplied mRNA. Injection of 12–15 ng of a ship2a splice-blocking MO completely inhibited production of mature zygotic ship2a mRNA as assayed by RT-PCR, but failed to produce a dorsalized phenotype (data not shown).

Loss of maternal Ship2a affects the BMP but not WNT signaling pathway

Whereas maternal Ship2a did not seem to contribute to specification of organizer tissue, the altered expression of the foxb1.2 and eve1 transcription factor genes indicated that Ship2a was required before the onset of gastrulation to establish proper DV patterning of gene expression domains in the embryo. The BMP and WNT signaling pathways both contribute directly to assigning developmental fates along the DV axis of the early vertebrate embryo (Christian and Moon, 1993; Imai et al., 2001; Hammerschmidt and Mullins, 2002), and hence expression of components of these pathways was examined in embryos that were depleted of maternal Ship2a protein. Loss of maternal Ship2a resulted in substantially decreased expression of bmp7 in pre-gastrula and early gastrula-stage embryos. Normally, bmp7 is widely and abundantly expressed soon after the onset of zygotic transcription (30–40% epiboly) (Fig. 3A), and becomes restricted ventrally by the onset of gastrulation (50% epiboly) (Fig. 3C). However, depletion of maternal Ship2a caused a severe reduction in bmp7 expression at these stages (n=35/38 embryos) (Fig. 3B,D). Similarly, bmp4 expression was reduced in morphants (supplementary material Fig. S3A,B). By contrast, analysis of WNT signaling targets indicated that the WNT pathway was unaltered by loss of Ship2a. vox and vent are ventrally expressed genes that are directly regulated by WNT8 and encode transcriptional repressors that function to repress ventral expression of organizer genes (Imai et al., 2001; Ramel and Lekven, 2004). Embryos depleted of maternal Ship2a exhibited normal expression of vox (n=17/19 embryos) or vent (n=20/22 embryos) at 40% epiboly (Fig. 3E–H). Similarly, expression of bozozok (boz), one of the earliest zygotically expressed genes and a target of the dorsal WNT signaling pathway (Fekany et al., 1999; Koos and Ho, 1999), was not altered at the oblong-sphere stage (3.7–4 hpf) in ship2a-morphant embryos (n=27/28 embryos) (Fig. 3I,J). Together, these results indicate that, prior to the onset of gastrulation, BMP expression but not WNT signaling is dependent on maternal Ship2a.

Fig. 3.

Knockdown of maternal Ship2a alters BMP but not WNT signaling pathways. (A–D) Expression of bmp7 is reduced in 30–40% epiboly and 50% epiboly maternal ship2a morphants as compared with WT controls. (E–J) Gene expression that is dependent on the WNT signaling pathway is not affected in maternal ship2a-morphant embryos. Expression of vox (E,F) or vent (G,H) in 40% epiboly embryos, or bozozok (boz) (I–J) in oblong stage (3.7 hpf) embryos, is unchanged by depletion of maternal Ship2a. A–H, animal pole views with dorsal to the right; I and J, lateral views with animal pole up and dorsal to the right.

Fig. 3.

Knockdown of maternal Ship2a alters BMP but not WNT signaling pathways. (A–D) Expression of bmp7 is reduced in 30–40% epiboly and 50% epiboly maternal ship2a morphants as compared with WT controls. (E–J) Gene expression that is dependent on the WNT signaling pathway is not affected in maternal ship2a-morphant embryos. Expression of vox (E,F) or vent (G,H) in 40% epiboly embryos, or bozozok (boz) (I–J) in oblong stage (3.7 hpf) embryos, is unchanged by depletion of maternal Ship2a. A–H, animal pole views with dorsal to the right; I and J, lateral views with animal pole up and dorsal to the right.

Fig. 4.

Knockdown of maternal Ship2a affects the expression of FGF-dependent genes in the pre-gastrula embryo. (A,C) mkp3 is expressed dorsally in WT embryos at the oblong stage (3.7 hpf). (B,D) Knockdown of maternal Ship2a results in ventralward expansion of the mkp3 expression domain. (E,F) Expansion of the mkp3 expression domain persists at 50% epiboly (5.3 hpf) in maternal ship2a morphants. (G,H) Expression of erm, a direct target of the FGF signaling pathway, is increased in maternal ship2a morphants. A and B, lateral views with dorsal to the right; C–H, animal pole views with dorsal to the right.

Fig. 4.

Knockdown of maternal Ship2a affects the expression of FGF-dependent genes in the pre-gastrula embryo. (A,C) mkp3 is expressed dorsally in WT embryos at the oblong stage (3.7 hpf). (B,D) Knockdown of maternal Ship2a results in ventralward expansion of the mkp3 expression domain. (E,F) Expansion of the mkp3 expression domain persists at 50% epiboly (5.3 hpf) in maternal ship2a morphants. (G,H) Expression of erm, a direct target of the FGF signaling pathway, is increased in maternal ship2a morphants. A and B, lateral views with dorsal to the right; C–H, animal pole views with dorsal to the right.

FGF-dependent gene expression is elevated upon loss of maternal Ship2a

Two phenotypes observed in Ship2a-depleted embryos, dorsalization and diminished bmp gene expression, can be caused by the expansion of FGF signaling in the pre-gastrula embryo (Furthauer et al., 2001; Furthauer et al., 2004; Tsang et al., 2004). Dorsal-restricted expression of FGF in the blastula limits the expression of BMPs to the ventral side of the embryo (Furthauer et al., 2004). As FGF can function in a dose-dependent manner, precise regulation of spatial, temporal and quantitative aspects of FGF signaling is required for the correct DV patterning of gene expression and prospective tissue fates. DV patterning is so sensitive to FGF signaling that its perturbation has been used successfully to detect modifiers of the early embryonic FGF signaling cascade (for a review, see Tsang and Dawid, 2004; Thisse and Thisse, 2005).

The mitogen-activated protein kinase (MAPK) signaling pathway is required to mediate the effects of FGF in the early zebrafish embryo (Furthauer et al., 2001; Furthauer et al., 2004; Tsang et al., 2004). Signaling through the FGF receptor leads to increased expression of genes that encode both pathway mediators, including the Ets transcription factors Pea3 and Erm, and pathway inhibitors, including Mkp3, Sef and Sproutys (Raible and Brand, 2001; Roehl and Nusslein-Volhard, 2001; Tsang and Dawid, 2004; Tsang et al., 2004; Thisse and Thisse, 2005). As a measure of FGF signaling in maternal Ship2a-depleted embryos, we first analyzed the expression of mkp3, whose product MAP kinase phosphatase 3 (Dusp6) acts as a feedback attenuator of FGF signaling by dephosphorylating the active form of MAPK (Tsang et al., 2004). Shortly after the onset of zygotic gene expression, at the oblong-sphere stage (3.7–4 hpf), mkp3 expression is localized under the control of FGF signaling to the future dorsal side of wild-type (WT) embryos (Fig. 4A,C). Depletion of maternal Ship2a resulted in a dramatic expansion of mkp3 expression so that transcripts were distributed throughout the entire blastoderm in an apparently graded fashion at the oblong-sphere stage (n=14/17 embryos) (Fig. 4B,D). Over the following hour of development, between the dome stage and 50% epiboly (4.3–5.3 hpf), the normal mkp3 expression domain broadens circumferentially, and transcripts become distributed around the entire outer margin of the blastoderm, the site of mesoderm induction and a region of high FGF signaling (Fig. 4E; supplementary material Fig. S3C). In Ship2a-depleted embryos, ventral expansion of mkp3 expression occurred precociously and extended away from its normal domain at the edge of the blastoderm margin and deep into the internal portion of the embryo (n=18/19 embryos) (Fig. 4F; supplementary material Fig. S3D). Similarly, depletion of maternal Ship2a resulted in increased expression of erm, a target upregulated by FGF signaling (n=30/34 embryos) (Fig. 4G,H). The erm and mkp3 expression domains were not identical, indicating that the two genes may be responsive to different levels of FGF signaling, or that factors in addition to FGF regulate their transcription. In either case, the results indicated that loss of maternal Ship2a caused augmented and expanded expression of genes whose transcription responds to FGF signaling. These findings are consistent with the interpretation that FGF signaling was hyperactivated in embryos with reduced Ship2a.

Maternal Ship2a is required to attenuate FGF signaling

To measure directly the contribution of Ship2a to FGF signaling, we assessed the state of activation of the FGFR-Ras-MAPK pathway with an antibody specific to the active, diphosphorylated form of MAPK (pMAPK) (Shinya et al., 2001). In control embryos, cells with elevated levels of pMAPK are concentrated at the future dorsal region of the embryo at the oblong-sphere stage (3.7–4 hpf) (n=42/42 embryos) (Fig. 5A,B). In embryos that are depleted of maternal Ship2a, cells with high levels of pMAPK were distributed throughout the blastoderm (n=26/29 embryos, ship2a MO1) (Fig. 5C,D). Similar to the pattern of the expanded expression domains of mkp3 and erm, it appeared that cells with elevated FGF signaling were not randomly distributed following the loss of Ship2a, but rather that signaling extended aberrantly beyond its normal focus emanating from one portion of the blastoderm margin.

Fig. 5.

Maternal Ship2a attenuates FGF receptor-mediated pMAPK activation in the pre-gastrula embryo. (A–F) Anti-pMAPK immunoreactivity in oblong-sphere stage (3.7–4 hpf) WT, DMSO-treated control, maternal ship2a morphant and SU5402-treated embryos. (A,B) In WT and DMSO-treated control embryos, pMAPK is restricted to the dorsal side of the embryo. (C,D) Knockdown of maternal Ship2a causes a dramatic ventralward expansion of the pMAPK expression domain. (E,F) Inhibition of FGFR activity with 60 μM SU5402 completely inhibits pMAPK activation in both WT and maternal ship2a-morphant embryos. Confocal projections. Animal pole views with dorsal to the right.

Fig. 5.

Maternal Ship2a attenuates FGF receptor-mediated pMAPK activation in the pre-gastrula embryo. (A–F) Anti-pMAPK immunoreactivity in oblong-sphere stage (3.7–4 hpf) WT, DMSO-treated control, maternal ship2a morphant and SU5402-treated embryos. (A,B) In WT and DMSO-treated control embryos, pMAPK is restricted to the dorsal side of the embryo. (C,D) Knockdown of maternal Ship2a causes a dramatic ventralward expansion of the pMAPK expression domain. (E,F) Inhibition of FGFR activity with 60 μM SU5402 completely inhibits pMAPK activation in both WT and maternal ship2a-morphant embryos. Confocal projections. Animal pole views with dorsal to the right.

The results were consistent with the hypothesis that maternal Ship2a functions specifically to attenuate FGF signaling in the early embryo. In this model, the consequences of loss of Ship2 could be observed only in the context of effective intercellular FGF signaling. If maternal Ship2 indeed acts downstream of receptor activation, then inhibition of FGF receptor function should inhibit the activation of pMAPK regardless of whether or not maternal Ship2 is present. To test the hypothesis, FGF signaling in the embryo was blocked using SU5402, a small molecule that is a specific inhibitor of FGF receptors (Mohammadi et al., 1997). Incubation of embryos in 60 μM SU5402, beginning at the blastula stage (128-cell stage or ∼2.25 hpf), completely inhibited the expression of elevated pMAPK as measured at the oblong-sphere stage (n=24/24 embryos) (Fig. 5E). When embryos that were depleted of maternal Ship2 were also treated with SU5402, cells with elevated pMAPK were not observed (n=18/18 embryos) (Fig. 5F). Thus, loss of maternal Ship2a does not directly stimulate MAPK phosphorylation. Rather, these data indicate that Ship2a modulates the amount of pMAPK that is generated and/or maintained following activation of the FGF pathway.

Dorsal-restricted FGF signaling contributes to DV patterning of the embryo in part by repressing BMP expression and signaling on the future dorsal side of the embryo (Furthauer et al., 2004). Thus, the enhanced activity of FGF-dependent signaling that is observed in ship2a-depleted embryos may be responsible for the downregulation of bmp gene expression observed in ship2a MO-treated embryos, leading to their dorsalization (Figs 2 and 3). If Ship2a interacts specifically with the FGF pathway in the early embryo, and only indirectly affects BMP-dependent ventral identity development, then ventral identities might be restored to ship2a MO-treated embryos following ectopic induction of BMP signaling. We tested whether the tissue patterning defects caused by loss of maternal Ship2a function could be rescued by overexpression of bmp2b. Ventral identity was measured by eve1 expression, which is normally limited to the future ventral side in WT embryos at 50% epiboly (Fig. 6A). Injection of bmp2b mRNA at the one-cell stage caused a dorsalward expansion of the eve1 expression domain, indicated by circumferential expression of eve1 (Fig. 6B). Although eve1 expression was lost when maternal Ship2a function was blocked by MO1 or MO2 (Fig. 6C,E), co-injection of bmp2b mRNA with the MOs strongly enhanced eve1 expression, often restoring its WT expression pattern (Fig. 6D,F). These results indicate that the role of Ship2a in attenuating FGF signaling in the early embryo is sufficient to explain how loss of maternal Ship2a causes subsequent dorsalization of development.

DISCUSSION

An in vivo function of the SHIP2 protein

Defining the in vivo roles of, and requirements for, SHIP2 activity is important, as SHIP2 has been viewed as a regulator of glucose homeostasis and a favored potential molecular target in the development of therapies for type 2 diabetes (Decker and Saltiel, 2005; Lazar and Saltiel, 2006; Sasaoka et al., 2006). SHIP2 has been shown to be capable of modulating many pathways in vitro, and recent screens to identify components of the FGFR1 and FGFR2 signaling cascades found that the SHIP2 protein population responded dramatically to ligand presentation, becoming highly tyrosine-phosphorylated immediately upon FGF stimulation of mammalian cultured cells (Hinsby et al., 2004; Luo et al., 2009). Here we demonstrate that SHIP2 attenuates the response to FGF stimulation in vivo with the significant effect that it limits the spread of FGF signaling in the zebrafish embryo. We note that SHIP2 may have a similar role in mouse development, as Ship2 null mutant mice exhibit runting and skeletofacial anomalies (Sleeman et al., 2005); these defects may have arisen from aberrant regulation of FGF signaling (Muenke et al., 1994; Kan et al., 2002; Cohen, 2004). Conversely, the finding that Ship2a functions in FGF signaling in the pre-gastrula embryo does not preclude the possibility that it and/or Ship2b may function in additional signaling pathways in zebrafish.

FGF signaling is utilized in many different biological contexts, including regulation of cell proliferation, cell differentiation, cell migration, developmental fate patterning and metabolic homeostasis (Ornitz, 2000; Thisse and Thisse, 2005; Beenken and Mohammadi, 2009). Abnormal levels of FGF signaling play fundamental roles in the origins of birth defects and cancers, and can modulate metabolic rates and obesity (Turner and Grose, 2010; Beenken and Mohammadi, 2009). The work here shows that Ship2a functions to diminish FGF signaling. During early embryonic development a major role of FGF signaling is in assigning tissue fates, and negative regulation of the signal is key to this process (Tsang and Dawid, 2004; Thisse and Thisse, 2005). Following Ship2a depletion, the highest levels of FGF signaling-dependent gene expression and activated pMAPK were observed at their normal locations, indicating that sources of FGF in the embryo were probably not altered. Rather, in the absence of maternal Ship2a, the range of FGF signaling was extended in the pre-gastrula zebrafish embryo. Furthermore, overexpression of bmp2b mRNA in maternal Ship2a-depleted embryos could overcome the tissue patterning defects resulting from expanded FGF signaling. The ability of forced bmp gene expression to bypass the requirement for Ship2a in DV patterning indicates that the main function of Ship2a in the early embryo is probably restricted to its role in attenuating FGF signaling. In all, several co-expressed factors are dedicated to the regulation of FGF signal transduction intensity in the early embryo, although loss of Ship2a results in a far stronger dorsalization phenotype than is seen following depletion of either Sef or Sprouty4 (Furthauer et al., 2004). As the absence of any one component appears to have a readily detectable effect in the zebrafish embryo, it may be that there is considerable cross-dependence among the modulators.

Fig. 6.

Overexpression ofbmp2brestores DV patterning in embryos depleted of maternal Ship2a. (A–F) eve1 expression in WT or ship2a-morphant 50% epiboly embryos and similar embryos that had been injected with bmp2b mRNA. (A,C,E) eve1 expression is restricted to the ventral side of WT embryos and downregulated in ship2a MO1- or MO2-injected embryos. (B) Overexpression of bmp2b in WT embryos results in the dorsalward expansion and circumferential expression of eve1, indicating that ectopic bmp2b expression promotes ventralization. (D,F) Co-injection of bmp2b mRNA with either ship2a MO1 or MO2 restores the WT expression pattern of eve1.

Fig. 6.

Overexpression ofbmp2brestores DV patterning in embryos depleted of maternal Ship2a. (A–F) eve1 expression in WT or ship2a-morphant 50% epiboly embryos and similar embryos that had been injected with bmp2b mRNA. (A,C,E) eve1 expression is restricted to the ventral side of WT embryos and downregulated in ship2a MO1- or MO2-injected embryos. (B) Overexpression of bmp2b in WT embryos results in the dorsalward expansion and circumferential expression of eve1, indicating that ectopic bmp2b expression promotes ventralization. (D,F) Co-injection of bmp2b mRNA with either ship2a MO1 or MO2 restores the WT expression pattern of eve1.

SHIP2, FGFs and metabolism

Mice that are homozygous for a null mutation in Ship2/Inppl1 displayed resistance to obesity and an elevated basal metabolic rate when placed on a high-fat diet, but did not appear to exhibit altered glucose homeostasis or insulin sensitivity (Sleeman et al., 2005). In addition, the mice displayed runting and distinctive craniofacial skeletal anomalies. As a complex interplay of genes and molecular mechanisms affect regulation of obesity, lipid metabolism and insulin resistance, these phenotypic characteristics do not unambiguously implicate a single molecular pathway (Doria et al., 2008; Lusis et al., 2008; Muoio and Newgard, 2008); however, they are consistent with the effects resulting from augmentation of FGF signaling.

Although the majority of FGF proteins function as paracrine factors during development, a subfamily of FGFs (19, 21 and 23) act as endocrine factors regulating cholesterol, lipid, glucose, bile acid and phosphate metabolism (Beenken and Mohammadi, 2009). A link between FGF function and both regulation of obesity and insulin sensitivity comes from studies in which FGF19 or 21 was systemically administered or overexpressed in transgenic mice (Tomlinson et al., 2002; Kharitonenkov et al., 2005; Coskun et al., 2008; Xu et al., 2009). Transgenic mice expressing either FGF19 or 21 were resistant to dietary induced obesity, and systemic administration of FGF19 or 21 in ob/ob or db/db mice ameliorated obesity and hyperglycemia. As Ship2 null mice have normal insulin and glucose levels and tolerances, it is possible that the primary role of mammalian SHIP2 in vivo may be to affect metabolism through modulation of endocrine FGF signaling. This role need not exclude the possibility that SHIP2 also functions directly in the insulin-signaling pathway. Analysis of zygotic zebrafish Ship2a function may provide insight into its possible role in both FGF and insulin signaling in vivo.

Finally, the work presented here does not indicate the mechanism by which Ship2a downregulates FGF signaling in the embryo. It is somewhat unexpected to find that a protein that is best recognized for its phosphoinositide phosphatase activity clearly regulates the levels of activated MAPK present in cells responding to FGF. Nevertheless, it is consistent with previous studies that have shown that SHIP2 can modulate levels of activated MAPK in Chinese hamster ovary (CHO) cells stimulated with insulin (Blero et al., 2001; Kagawa et al., 2005). It is possible that Ship2a might function through its SH2 domain to compete with, and displace, molecules such as Shc and Grb2 from the activated FGF receptor and thereby reduce the number of active signaling complexes (Sasaoka et al., 2003). Consistent with this idea, SHIP1 or SHIP2 mutants lacking phosphatase activity are capable of inhibiting MAPK activity in 3T3-L1 cells stimulated with insulin or platelet-derived growth factor (PDGF) (Sharma et al., 2005; Artemenko et al., 2009). It is also possible that Ship2a attenuates the FGF pathway by functioning as a negative regulator of the PI3K signaling pathway (Wisniewski et al., 1999). Inhibition of PI3K activity in the early zebrafish embryo did not affect early dorosventral patterning even though it produced gastrulation defects (Montero et al., 2003). Thus, it is unlikely that loss of Ship2a-mediated modulation of PI3K signaling would be sufficient to account for the defects seen in Ship2a-depleted embryos. The ability of various forms of SHIP2 (i.e. IPPc mutants or SH2 mutants) to rescue any of the morphant phenotype will address the relative contributions of maternal Ship2a to different signal transduction pathways.

METHODS

Zebrafish maintenance

Zebrafish were maintained under standard conditions (Westerfield, 2000). Wild-type animals were of the AB strain. All experiments were performed in accordance with, and under the supervision of, the University of Utah’s Institutional Animal Care and Use Committee (IACUC).

Cloning and analysis of zebrafish ship2 sequences

Human SHIP2 (NP_001558) was used to BLAST search the zebrafish genome (www.ensembl.org/Danio_rerio/), and the putative translational start site and 3′ UTR of zebrafish ship2a were identified. The primers ship2F, 5′-CCGCGATGGCCGCTGTGGGTC-3′ and ship2R, 5′-GGAATTCCTCAGGTCAGATTCACCAGTGC-3′ were used to amplify the full coding sequence from WT AB strain cDNA, which was then subcloned into pGEM-T (Promega). The zebrafish ship2a and related ship2b sequences were deposited in GenBank (Accession numbers DQ272661 and DQ272662, respectively). Sequence alignment and phylogenetic analysis were performed using The Biology Workbench at the San Diego Supercomputer Center (http://workbench.sdsc.edu). Human SHIP2 (NP_001558), mouse SHIP2 (NP_034697), human SHIP1 (NP_001017915) and zebrafish Ship1 (XP_001923007) sequences were used in the comparison with the two zebrafish Ship2 proteins.

Morpholino oligonucleotide injections

Two non-overlapping antisense translation-blocking MOs were synthesized (Gene Tools): ship2a MO1 (−20 to +5 with respect to the translation initiation site), 5′-GCCATCGCGGAGACTCACACCGGAG-3′ (ship2a translational start site underlined) and ship2a MO2 (−75 to −50) 5′-CGACGCTCACCGCTGATCCTGCTTT-3′. Approximately 1 nl of morpholino was injected into the yolk of one-cell stage WT embryos.

Analysis of MO effectiveness

The 5′ UTR of ship2a (−109 to +6 with respect to the translation start site) was amplified from cDNA generated from 16–32-cell AB embryos and cloned into a CS2+ vector containing the gfp cDNA. 5′ UTR GFP mRNA was synthesized in vitro (Message Maker Kit, Epicentre Biotechnology) and either 400 pg chimeric mRNA or 400 pg chimeric mRNA plus ship2a MO1 or ship2a MO2 were injected into fertilized eggs. Embryos were collected at 4–5 hpf and processed for immunoblot analysis, or fixed in 4% paraformaldehyde and processed for immunohistochemistry (IHC).

Immunoblot analysis

Groups of 30 WT, 5′ UTR GFP mRNA-injected, 5′ UTR GFP mRNA plus ship2a MO1-injected, or 5′ UTR GFP mRNA plus ship2 MO2-injected embryos were harvested at 4–5 hpf and partially de-yolked with forceps. Embryos were then placed in 1 ml of cold PBSEE (PBS, 10 mM EDTA, 10 mM EGTA) and cells were dissociated with vigorous pipetting. Cells were centrifuged (1000 rpm, 5 minutes at 4°C), the supernatant was discarded, and the pellet was washed four times with cold PBSEE. Cells were finally centrifuged (3000 rpm, 1 minute at 4°C) and the pellet was resuspended and homogenized in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20, 0.05% NP-40) with protease inhibitors (complete protease inhibitor cocktail tablets, Roche Applied Science). The homogenized sample was centrifuged (10,000 rpm, 5 minutes), and the supernatant was stored at −80°C. 25 μg of total lysate (approximately 12 embryos) was resolved by electrophoresis through 4–20% polyacrylamide gradient gels (NuSep Inc.). Blots of the gels were probed with anti-GFP (1:4000, Molecular Probes) and anti-Paf1 (1:6500, Bethyl Laboratories, Inc.), and the signal was developed following incubation with enhanced chemoluminescence (ECL Plus, GE Healthcare).

Immunohistochemistry

Embryos for IHC were fixed with fresh 4% paraformaldehyle in 1× PBS buffer overnight at 4°C (Jurynec et al., 2008). Anti-GFP (1:1000; Molecular Probes) or anti-MAPK, activated (diphospho-ERK1 and 2) (pMAPK) antibody (1:10,000; Sigma M 8159) (Shinya et al., 2001) were diluted in DMSO-PBT-SS (PBS supplemented with 1% DSMO, 0.2% BSA, 0.1% Triton X-100 and 10% sheep serum). GFP immunoreactivity was detected by anti-rabbit Alexa 488 (1:200; Molecular Probes). Embryos were mounted in 75% glycerol and photographed under epifluorescence and bright field illumination. pMAPK immunoreactivity was detected by Alexa 488 tyramide amplification (Molecular Probes) following incubation with a goat anti-mouse IgG-HRP antibody (1:200, Molecular Probes). Embryos were mounted in 75% glycerol and images were obtained using a Leica DMRXE laser scanning confocal microscope.

SU5402 treatment

SU5042 was dissolved in DMSO and diluted in embryo-rearing medium (Westerfield, 2000). Embryos were treated continuously, beginning at the 128-cell stage. For the lot of SU5402 used here, 60 μM was the lowest concentration that was capable of inhibiting pMAPK expression completely, as judged by anti-pMAPK IHC at the oblong-sphere stages. Embryos were injected shortly after fertilization with MO and allowed to develop until the 128-cell stage, at which time MO-injected and control embryos were treated with either 60 μM SU5402 or DMSO. Embryos were processed for anti-pMAPK IHC at the oblong-sphere stages (3.7–4 hpf).

Acknowledgements

We thank Ken Poss for his kind gift of SU5042, Nathan Bahary for helping to isolate a YAC clone containing ship2a, and all of our Utah colleagues for constructive criticism. M.J.J. was supported by a Ruth L. Kirschstein NRSA predoctoral fellowship (F31 NS044665) and NIH postdoctoral fellowships (5T32NS07493 and 5T32HL079874). These studies were supported by NIH grants HD37572 and HD048886 (D.J.G.). Deposited in PMC for release after 12 months.

AUTHOR CONTRIBUTIONS

M.J.J. and D.J.G. conceived the study, analyzed data and wrote the manuscript. M.J.J. designed and performed the experiments with assistance from D.J.G.

REFERENCES

Affolter
M
,
Basler
K
(
2007
).
The Decapentaplegic morphogen gradient: from pattern formation to growth regulation
.
Nat Rev Genet
.
8
,
663
674
.
Aoki
K
,
Nakamura
T
,
Inoue
T
,
Meyer
T
,
Matsuda
M
(
2007
).
An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor-stimulated PC12 cells
.
J Cell Biol
.
177
,
817
827
.
Artemenko
Y
,
Gagnon
A
,
Sorisky
A
(
2009
).
Catalytically inactive SHIP2 inhibits proliferation by attenuating PDGF signaling in 3T3-L1 preadipocytes
.
J Cell Physiol
.
218
,
228
236
.
Backers
K
,
Blero
D
,
Paternotte
N
,
Zhang
J
,
Erneux
C
(
2003
).
The termination of PI3K signalling by SHIP1 and SHIP2 inositol 5-phosphatases
.
Adv Enzyme Regul
.
43
,
15
28
.
Beenken
A
,
Mohammadi
M
(
2009
).
The FGF family: biology, pathophysiology and therapy
.
Nat Rev Drug Discov
.
8
,
235
253
.
Birnbaum
MJ
(
2005
).
SHIPing news: a new way to keep your weight down
.
Cell Metab
.
1
,
90
92
.
Blero
D
,
De Smedt
F
,
Pesesse
X
,
Paternotte
N
,
Moreau
C
,
Payrastre
B
,
Erneux
C
(
2001
).
The SH2 domain containing inositol 5-phosphatase SHIP2 controls phosphatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin
.
Biochem Biophys Res Commun
.
282
,
839
843
.
Christian
JL
,
Moon
RT
(
1993
).
Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus
.
Genes Dev
.
7
,
13
28
.
Clement
S
,
Krause
U
,
Desmedt
F
,
Tanti
JF
,
Behrends
J
,
Pesesse
X
,
Sasaki
T
,
Penninger
J
,
Doherty
M
,
Malaisse
W
, et al. 
(
2001
).
The lipid phosphatase SHIP2 controls insulin sensitivity
.
Nature
409
,
92
97
.
Cohen
JMM
(
2004
).
FGFs/FGFRs and associated disorders
. In
Inborn Errors of Metabolism
(eds
Epstein
CJ
,
Erickson
JD
,
Wynshaw-Boris
A
), pp.
380
400
.
New York
:
Oxford University Press
.
Coskun
T
,
Bina
HA
,
Schneider
MA
,
Dunbar
JD
,
Hu
CC
,
Chen
Y
,
Moller
DE
,
Kharitonenkov
A
(
2008
).
Fibroblast growth factor 21 corrects obesity in mice
.
Endocrinology
149
,
6018
6027
.
De Robertis
EM
,
Larrain
J
,
Oelgeschlager
M
,
Wessely
O
(
2000
).
The establishment of Spemann’s organizer and patterning of the vertebrate embryo
.
Nat Rev Genet
.
1
,
171
181
.
Decker
SJ
,
Saltiel
AR
(
2005
).
Staying in SHIP shape
.
Nat Med
.
11
,
123
124
.
Doria
A
,
Patti
ME
,
Kahn
CR
(
2008
).
The emerging genetic architecture of type 2 diabetes
.
Cell Metab
.
8
,
186
200
.
Engelman
JA
,
Luo
J
,
Cantley
LC
(
2006
).
The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism
.
Nat Rev Genet
.
7
,
606
619
.
Fekany
K
,
Yamanaka
Y
,
Leung
T
,
Sirotkin
HI
,
Topczewski
J
,
Gates
MA
,
Hibi
M
,
Renucci
A
,
Stemple
D
,
Radbill
A
, et al. 
(
1999
).
The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures
.
Development
126
,
1427
1438
.
Fukui
K
,
Wada
T
,
Kagawa
S
,
Nagira
K
,
Ikubo
M
,
Ishihara
H
,
Kobayashi
M
,
Sasaoka
T
(
2005
).
Impact of the liver-specific expression of SHIP2 (SH2-containing inositol 5′-phosphatase 2) on insulin signaling and glucose metabolism in mice
.
Diabetes
54
,
1958
1967
.
Furthauer
M
,
Reifers
F
,
Brand
M
,
Thisse
B
,
Thisse
C
(
2001
).
sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish
.
Development
128
,
2175
2186
.
Furthauer
M
,
Van Celst
J
,
Thisse
C
,
Thisse
B
(
2004
).
Fgf signalling controls the dorsoventral patterning of the zebrafish embryo
.
Development
131
,
2853
2864
.
Hammerschmidt
M
,
Mullins
MC
(
2002
).
Dorsoventral patterning in the zebrafish: bone morphogenetic proteins and beyond
.
Results Probl Cell Differ
.
40
,
72
95
.
Helgason
CD
,
Damen
JE
,
Rosten
P
,
Grewal
R
,
Sorensen
P
,
Chappel
SM
,
Borowski
A
,
Jirik
F
,
Krystal
G
,
Humphries
RK
(
1998
).
Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span
.
Genes Dev
.
12
,
1610
1620
.
Hinsby
AM
,
Olsen
JV
,
Mann
M
(
2004
).
Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4
.
J Biol Chem
.
279
,
46438
46447
.
Imai
Y
,
Gates
MA
,
Melby
AE
,
Kimelman
D
,
Schier
AF
,
Talbot
WS
(
2001
).
The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish
.
Development
128
,
2407
2420
.
Joly
JS
,
Joly
C
,
Schulte-Merker
S
,
Boulekbache
H
,
Condamine
H
(
1993
).
The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos
.
Development
119
,
1261
1275
.
Jurynec
MJ
,
Xia
R
,
Mackrill
JJ
,
Gunther
D
,
Crawford
T
,
Flanigan
KM
,
Abramson
JJ
,
Howard
MT
,
Grunwald
DJ
(
2008
).
Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle
.
Proc Natl Acad Sci USA
105
,
12485
12490
.
Kagawa
S
,
Sasaoka
T
,
Yaguchi
S
,
Ishihara
H
,
Tsuneki
H
,
Murakami
S
,
Fukui
K
,
Wada
T
,
Kobayashi
S
,
Kimura
I
, et al. 
(
2005
).
Impact of SRC homology 2-containing inositol 5′-phosphatase 2 gene polymorphisms detected in a Japanese population on insulin signaling
.
J Clin Endocrinol Metab
.
90
,
2911
2919
.
Kagawa
S
,
Soeda
Y
,
Ishihara
H
,
Oya
T
,
Sasahara
M
,
Yaguchi
S
,
Oshita
R
,
Wada
T
,
Tsuneki
H
,
Sasaoka
T
(
2008
).
Impact of transgenic overexpression of SH2-containing inositol 5′-phosphatase 2 on glucose metabolism and insulin signaling in mice
.
Endocrinology
149
,
642
650
.
Kan
SH
,
Elanko
N
,
Johnson
D
,
Cornejo-Roldan
L
,
Cook
J
,
Reich
EW
,
Tomkins
S
,
Verloes
A
,
Twigg
SR
,
Rannan-Eliya
S
, et al. 
(
2002
).
Genomic screening of fibroblast growth-factor receptor 2 reveals a wide spectrum of mutations in patients with syndromic craniosynostosis
.
Am J Hum Genet
.
70
,
472
486
.
Kharitonenkov
A
,
Shiyanova
TL
,
Koester
A
,
Ford
AM
,
Micanovic
R
,
Galbreath
EJ
,
Sandusky
GE
,
Hammond
LJ
,
Moyers
JS
,
Owens
RA
, et al. 
(
2005
).
FGF-21 as a novel metabolic regulator
.
J Clin Invest
.
115
,
1627
1635
.
Koch
A
,
Mancini
A
,
El Bounkari
O
,
Tamura
T
(
2005
).
The SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocyte growth factor (HGF)-induced lamellipodium formation, cell scattering and cell spreading
.
Oncogene
24
,
3436
3447
.
Koos
DS
,
Ho
RK
(
1999
).
The nieuwkoid/dharma homeobox gene is essential for bmp2b repression in the zebrafish pregastrula
.
Dev Biol
.
215
,
190
207
.
Lazar
DF
,
Saltiel
AR
(
2006
).
Lipid phosphatases as drug discovery targets for type 2 diabetes
.
Nat Rev Drug Discov
.
5
,
333
342
.
Leslie
NR
,
Downes
CP
(
2004
).
PTEN function: how normal cells control it and tumour cells lose it
.
Biochem J
.
382
,
1
11
.
Liu
Q
,
Sasaki
T
,
Kozieradzki
I
,
Wakeham
A
,
Itie
A
,
Dumont
DJ
,
Penninger
JM
(
1999
).
SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival
.
Genes Dev
.
13
,
786
791
.
Logan
CY
,
Nusse
R
(
2004
).
The Wnt signaling pathway in development and disease
.
Annu Rev Cell Dev Biol
.
20
,
781
810
.
Luo
Y
,
Yang
C
,
Jin
C
,
Xie
R
,
Wang
F
,
McKeehan
WL
(
2009
).
Novel phosphotyrosine targets of FGFR2IIIb signaling
.
Cell Signal
.
21
,
1370
1378
.
Lusis
AJ
,
Attie
AD
,
Reue
K
(
2008
).
Metabolic syndrome: from epidemiology to systems biology
.
Nat Rev Genet
.
9
,
819
830
.
Manahan
CL
,
Iglesias
PA
,
Long
Y
,
Devreotes
PN
(
2004
).
Chemoattractant signaling in dictyostelium discoideum
.
Annu Rev Cell Dev Biol
.
20
,
223
253
.
Marion
E
,
Kaisaki
PJ
,
Pouillon
V
,
Gueydan
C
,
Levy
JC
,
Bodson
A
,
Krzentowski
G
,
Daubresse
JC
,
Mockel
J
,
Behrends
J
, et al. 
(
2002
).
The gene INPPL1, encoding the lipid phosphatase SHIP2, is a candidate for type 2 diabetes in rat and man
.
Diabetes
51
,
2012
2017
.
Mohammadi
M
,
McMahon
G
,
Sun
L
,
Tang
C
,
Hirth
P
,
Yeh
BK
,
Hubbard
SR
,
Schlessinger
J
(
1997
).
Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors
.
Science
276
,
955
960
.
Montero
JA
,
Kilian
B
,
Chan
J
,
Bayliss
PE
,
Heisenberg
CP
(
2003
).
Phosphoinositide 3-kinase is required for process outgrowth and cell polarization of gastrulating mesendodermal cells
.
Curr Biol
.
13
,
1279
1289
.
Muenke
M
,
Schell
U
,
Hehr
A
,
Robin
NH
,
Losken
HW
,
Schinzel
A
,
Pulleyn
LJ
,
Rutland
P
,
Reardon
W
,
Malcolm
S
, et al. 
(
1994
).
A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome
.
Nat Genet
.
8
,
269
274
.
Muoio
DM
,
Newgard
CB
(
2008
).
Mechanisms of disease: molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes
.
Nat Rev Mol Cell Biol
.
9
,
193
205
.
Muraille
E
,
Pesesse
X
,
Kuntz
C
,
Erneux
C
(
1999
).
Distribution of the src-homology-2-domain-containing inositol 5-phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells
.
Biochem J
.
342
,
697
705
.
Nguyen
VH
,
Schmid
B
,
Trout
J
,
Connors
SA
,
Ekker
M
,
Mullins
MC
(
1998
).
Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes
.
Dev Biol
.
199
,
93
110
.
Odenthal
J
,
Nusslein-Volhard
C
(
1998
).
fork head domain genes in zebrafish
.
Dev Genes Evol
.
208
,
245
258
.
Ooms
LM
,
Horan
KA
,
Rahman
P
,
Seaton
G
,
Gurung
R
,
Kethesparan
DS
,
Mitchell
CA
(
2009
).
The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease
.
Biochem J
.
419
,
29
49
.
Ornitz
DM
(
2000
).
FGFs, heparan sulfate and FGFRs: complex interactions essential for development
.
BioEssays
22
,
108
112
.
Pesesse
X
,
Moreau
C
,
Drayer
AL
,
Woscholski
R
,
Parker
P
,
Erneux
C
(
1998
).
The SH2 domain containing inositol 5-phosphatase SHIP2 displays phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity
.
FEBS Lett
.
437
,
301
303
.
Pesesse
X
,
Dewaste
V
,
De Smedt
F
,
Laffargue
M
,
Giuriato
S
,
Moreau
C
,
Payrastre
B
,
Erneux
C
(
2001
).
The Src homology 2 domain containing inositol 5-phosphatase SHIP2 is recruited to the epidermal growth factor (EGF) receptor and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF-stimulated COS-7 cells
.
J Biol Chem
.
276
,
28348
28355
.
Raible
F
,
Brand
M
(
2001
).
Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development
.
Mech Dev
.
107
,
105
117
.
Ramel
MC
,
Lekven
AC
(
2004
).
Repression of the vertebrate organizer by Wnt8 is mediated by Vent and Vox
.
Development
131
,
3991
4000
.
Roehl
H
,
Nusslein-Volhard
C
(
2001
).
Zebrafish pea3 and erm are general targets of FGF8 signaling
.
Curr Biol
.
11
,
503
507
.
Sasaoka
T
,
Hori
H
,
Wada
T
,
Ishiki
M
,
Haruta
T
,
Ishihara
H
,
Kobayashi
M
(
2001
).
SH2-containing inositol phosphatase 2 negatively regulates insulin-induced glycogen synthesis in L6 myotubes
.
Diabetologia
44
,
1258
1267
.
Sasaoka
T
,
Kikuchi
K
,
Wada
T
,
Sato
A
,
Hori
H
,
Murakami
S
,
Fukui
K
,
Ishihara
H
,
Aota
R
,
Kimura
I
, et al. 
(
2003
).
Dual role of SRC homology domain 2-containing inositol phosphatase 2 in the regulation of platelet-derived growth factor and insulin-like growth factor I signaling in rat vascular smooth muscle cells
.
Endocrinology
144
,
4204
4214
.
Sasaoka
T
,
Wada
T
,
Tsuneki
H
(
2006
).
Lipid phosphatases as a possible therapeutic target in cases of type 2 diabetes and obesity
.
Pharmacol Ther
.
112
,
799
809
.
Sharma
PM
,
Son
HS
,
Ugi
S
,
Ricketts
W
,
Olefsky
JM
(
2005
).
Mechanism of SHIP-mediated inhibition of insulin- and platelet-derived growth factor-stimulated mitogen-activated protein kinase activity in 3T3-L1 adipocytes
.
Mol Endocrinol
.
19
,
421
430
.
Shinya
M
,
Koshida
S
,
Sawada
A
,
Kuroiwa
A
,
Takeda
H
(
2001
).
Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos
.
Development
128
,
4153
4164
.
Sleeman
MW
,
Wortley
KE
,
Lai
KM
,
Gowen
LC
,
Kintner
J
,
Kline
WO
,
Garcia
K
,
Stitt
TN
,
Yancopoulos
GD
,
Wiegand
SJ
, et al. 
(
2005
).
Absence of the lipid phosphatase SHIP2 confers resistance to dietary obesity
.
Nat Med
.
11
,
199
205
.
Stachel
SE
,
Grunwald
DJ
,
Myers
PZ
(
1993
).
Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish
.
Development
117
,
1261
1274
.
Thisse
B
,
Thisse
C
(
2005
).
Functions and regulations of fibroblast growth factor signaling during embryonic development
.
Dev Biol
.
287
,
390
402
.
Tomlinson
E
,
Fu
L
,
John
L
,
Hultgren
B
,
Huang
X
,
Renz
M
,
Stephan
JP
,
Tsai
SP
,
Powell-Braxton
L
,
French
D
, et al. 
(
2002
).
Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity
.
Endocrinology
143
,
1741
1747
.
Tsang
M
,
Dawid
IB
(
2004
).
Promotion and attenuation of FGF signaling through the Ras-MAPK pathway
.
Sci STKE
2004
, p
e17
.
Tsang
M
,
Maegawa
S
,
Kiang
A
,
Habas
R
,
Weinberg
E
,
Dawid
IB
(
2004
).
A role for MKP3 in axial patterning of the zebrafish embryo
.
Development
131
,
2769
2779
.
Turner
N
,
Grose
R
(
2010
).
Fibroblast growth factor signalling: from development to cancer
.
Nat Rev Cancer
10
,
116
129
.
Wada
T
,
Sasaoka
T
,
Funaki
M
,
Hori
H
,
Murakami
S
,
Ishiki
M
,
Haruta
T
,
Asano
T
,
Ogawa
W
,
Ishihara
H
, et al. 
(
2001
).
Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5′-phosphatase catalytic activity
.
Mol Cell Biol
.
21
,
1633
1646
.
Wang
Y
,
Keogh
RJ
,
Hunter
MG
,
Mitchell
CA
,
Frey
RS
,
Javaid
K
,
Malik
AB
,
Schurmans
S
,
Tridandapani
S
,
Marsh
CB
(
2004
).
SHIP2 is recruited to the cell membrane upon macrophage colony-stimulating factor (M-CSF) stimulation and regulates M-CSF-induced signaling
.
J Immunol
.
173
,
6820
6830
.
Westerfield
M
(
2000
).
The Zebrafish Book
.
Eugene
:
University of Oregon Press
.
Wisniewski
D
,
Strife
A
,
Swendeman
S
,
Erdjument-Bromage
H
,
Geromanos
S
,
Kavanaugh
WM
,
Tempst
P
,
Clarkson
B
(
1999
).
A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells
.
Blood
93
,
2707
2720
.
Xu
J
,
Lloyd
DJ
,
Hale
C
,
Stanislaus
S
,
Chen
M
,
Sivits
G
,
Vonderfecht
S
,
Hecht
R
,
Li
YS
,
Lindberg
RA
, et al. 
(
2009
).
Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice
.
Diabetes
58
,
250
259
.

COMPETING INTERESTS

The authors declare no competing financial interests.

Supplementary information