SHIP2, a factor associated with diet-induced obesity and insulin sensitivity, attenuates FGF signaling in vivo

Abstract

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.

Fig. 1.

The zebrafish Ship2a protein and expression of maternal ship2a transcripts. (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. 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. 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. 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. 6.

Overexpression of bmp2b restores 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.