Research Article 4303
Crucial role of phosphatidylinositol 4-kinase III in development of zebrafish pectoral fin is linked to phosphoinositide 3-kinase and FGF signaling
Hui Ma1, Trevor Blake2, Ajay Chitnis3, Paul Liu2 and Tamas Balla1,* 1Section on Molecular Signal Transduction, National Institute of Child Health and Human Development, 2Oncogenesis and Development Section, National Human Genome Research Institute, and 3Section on Neural Developmental Dynamics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA *Author for correspondence ([email protected])
Accepted 15 September 2009 Journal of Cell Science 122, 4303-4310 Published by The Company of Biologists 2009 doi:10.1242/jcs.057646
Summary Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first establishment of the sharp signaling center formed by the apical committed step in the synthesis of phosphoinositides, important ectodermal ridge and the underlying mesenchyme. This, in turn lipid regulators of signaling and trafficking pathways. Here we caused a prominent loss of the induction of one of the mitogen- cloned Pik4a, one of the zebrafish PI4K enzymes, and studied activated protein kinase (MAPK) phosphatases, Mkp3, an its role(s) in vertebrate development using morpholino essential intermediate in vertebrate limb development. These oligonucleotide-based gene silencing in zebrafish. changes were associated with impaired proliferation in the Downregulation of Pik4a led to multiple developmental developing fin bud due to a loss of balance between the MAPK abnormalities, affecting the brain, heart, trunk and most and PI3K branch of FGF-initiated signals. Our results identify prominently causing loss of pectoral fins. Strikingly similar Pik4a as an upstream partner of PI3Ks in the signaling cascade defects were caused by treatment of the developing embryos orchestrated by FGF receptors with a prominent role in forelimb with the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. development. To investigate the cause of the pectoral fin developmental defect, we focused on fibroblast growth factor (FGF) signaling Supplementary material available online at pathways because vertebrate limb development requires the http://jcs.biologists.org/cgi/content/full/122/23/4303/DC1 concerted action of a series of FGF ligands. Using in situ hybridization, the pectoral fin defect was traced to disruption Key words: PI4K, PI3K, FGF signaling, Zebrafish, Limb of the early FGF signaling loops that are crucial for the development Journal of Cell Science
Introduction pectoral fin development in zebrafish. (The nomenclature of PI4Ks Embryonic development relies on the coordinated interplay between is somewhat confusing. The human PI4KIII gene was formerly several signaling pathways to ensure the right balance between cell designated as PIK4CA. Currently, the human gene is named PI4KA proliferation, differentiation and apoptosis. Signaling during early whereas the gene encoding PI4K type II alpha is termed PI4K2A. embryonic development is based on external cues and their gradients Based on this, a more logical name for the gene encoding PI4KIII that are derived from adjacent cells and differs in many ways from would be PI4K3A. However, we have followed the current the classical endocrine signals that act at a distance with the help of nomenclature and used pi4ka for the zebrafish gene encoding Pi4ka.) circulating fluids. Secreted fibroblast growth factor (FGF) ligands PI4Ks catalyze the first step in the synthesis of phosphatidylinositol have important roles in many developmental processes (Bottcher and 4,5-bisphosphate (PtdIns(4,5)P2), the major phosphoinositide of the Niehrs, 2005; Thisse and Thisse, 2005). These ligands act on four plasma membrane and a precursor of the PI3K lipid product, FGF receptor subtypes and activate several signal transduction PtdIns(3,4,5)P3. Hence, it is expected that PI4Ks are placed cascades including the mitogen-activated protein kinase (MAPK) and upstream from the PI3Ks in the cascade generating PtdIns(3,4,5)P3. phosphoinositide 3-kinase (PI3K) pathways (Eswarakumar et al., There are four distinct PI4K genes in vertebrates (Balla and Balla, 2005). Very limited information is available on how these signaling 2006) including zebrafish. In this study we chose to study the Pi4ka pathways are linked to the developmental program during FGF action. enzyme because we found recently that it contributes to the A recent study showed that the MAP kinase phosphatase 3 (MKP3) generation of the plasma membrane pool of phosphoinositides in is a crucial component in the FGF8 signaling cascade during mammalian cells (Balla et al., 2007). Our results show that PI3K vertebrate limb development (Kawakami et al., 2003). In that study inhibition as well as downregulation of Pi4ka causes a striking defect the induction of mkp3 by the apical ectodermal ridge (AER)-derived in zebrafish pectoral fin development. Detailed analysis of this FGF8 within the limb bud was found to occur through PI3Ks phenotype showed a defect in the establishment of the sharp (Kawakami et al., 2003). These studies were the first to establish the organizing center between the Fgf24-positive cells within the apical importance of the PI3K signaling pathway in FGF action during ectodermal ridge (AER) and the Fgf10- and Mkp3-producing cells vertebrate limb development. within the underlying mesenchyme leading to greatly reduced In the present study we investigated whether phosphatidylinositol induction of mkp3 within the fin bud. In addition, downregulation 4-kinase III alpha (Pi4ka) can be linked to PI3K signaling during of Pi4ka and inhibition of PI3K were both associated with increased 4304 Journal of Cell Science 122 (23)
generalized apoptosis. Decreased proliferation was also prominent in the fin bud as a result of an imbalance between the MAPK and PI3K-Akt signaling pathways.
Results Pi4ka deficiency and PI3K inhibition impairs pectoral fin development in zebrafish embryos We cloned the full-length zebrafish pi4ka and located the encoding gene on chromosome 10. (The sequences of all four fish PI4Ks have been deposited in GenBank under the following accession numbers: AY929293, PI4KIII; AY929292, PI4KIII; AY929291, PI4KII; AY929290, PI4KII.)The gene comprises 54 exons with an ORF of 6180 bp. No other copies of this gene were found in the zebrafish genome. Sequence comparisons indicated that the Pik4a protein shares a high degree of sequence homology with its mammalian orthologs, exhibiting 82.3% overall amino acid identity with the human sequence (AF012872). The zebrafish protein shares the same domain structure with the other type III mammalian PI4Ks; it contains a lipid kinase unique domain followed by a putative PH domain and the lipid kinase catalytic domain (Fig. 1A). In vitro translated Pik4a displayed strong phosphatidylinositol 4-kinase activity, that was sensitive to inhibition by wortmannin (Wm), confirming its identity as a type III PI4K (not shown). Whole-mount in situ hybridization analysis showed the pi4ka transcript detectable at 1.5 hpf, indicating its probable maternal origin. It was uniformly expressed throughout the embryos from 3 to 24 hpf, with distinct expression in the somites. At later stages (36, 48 and 60 hpf), the expression was primarily restricted to the brain, the branchial arches and the pectoral fin-bud mesenchyme (Fig. 1B). To downregulate pi4ka, two morpholinos, MO1 and MO2 Fig. 1. Expression and downregulation of zebrafish Pi4ka. (A)Pi4ka cloned were designed to target the exon-intron boundaries flanking intron from zebrafish shows high homology to other mammalian Pi4ka enzymes 49 to block splicing of exon 50, which contains the most conserved sharing the same domain organization and highest conservation within the C- part of the catalytic domain. RT-PCR analysis showed their terminal catalytic domain. (B)Expression pattern of pi4ka mRNA during effectiveness when injected into freshly fertilized eggs (Fig. 1C left). zebrafish embryogenesis. Expression is ubiquitous in early embryos (1.5-24 hpf) but is primarily restricted to the brain, branchial arches (ba), and fin buds
Journal of Cell Science Two additional non-overlapping antisense MOs (MO3 and MO4) (fb, indicated by arrowheads) at later stages (36 and 48 hpf). targeted the 5ЈUTR of pi4ka and their efficiency in blocking the (C)Downregulation of zebrafish Pi4ka by morpholino injection targeting the translation of the transcript was confirmed by an in vitro translation splicing of exon 50 that encodes a crucial region within the catalytic domain assay (Fig. 1C right). (MO1 and MO2). Left panels: RT-PCR analysis showed that antisense morpholinos could eliminate the transcript containing exon 50. Both MOs Injection of MO1 or MO2 produced a complex phenotype caused exon skipping in a dose-dependent manner, whereas control injections affecting several structures (supplementary material Table S1). At (FITC) were without effect. Right panel: in vitro translation assay showing the the 24 hpf stage, mutant brains were dark, non-transparent and ability of MO3 and MO4 targeting the translation initiation site to reduce in atrophic. At 48 hpf the most conspicuous defects were smaller heads vitro translation of Pi4ka (cMO, control morpholino). (D)Lateral view of and eyes, hooked tails, domed heads, shortened body axis and MO1 (9 ng)-injected morphants show a complex phenotype affecting several structures. Note the shorter trunk with curved tail, smaller head and eye, pericardial edema (Fig. 1D). However, the most prominent feature reduced and disorganized pigmentation, and pericardial edema. Dorsal view of of the mutant embryos was the abnormal development of pectoral the embryos at 72 hpf, demonstrate the larger yolk sac, smaller head and eyes, fins most obvious at 72 hpf stage (Fig. 1D). Injection of MO3 caused and the loss of pectoral fins (pf, arrowheads) in morphants. similar abnormalities but these were less severe than those of the MO1-injected embryos (supplementary material Table S1). We attributed the milder defects in the MO3-injected embryos to the more than half of pi4ka morphants injected with full length mRNA possibility that an alternative downstream start site exist that could together with MO1 showed normal brain size and eye development yield a limited amount of partially functional enzyme in the MO3- (Fig. 2A). Injection of pi4ka mRNA alone did not cause any gross treated embryos but this was not studied in further detail. Injection abnormalities (not shown). of controls did not cause any developmental defects (not shown). To determine whether inhibition of the PI3K-Akt signaling could These results suggested that Pi4ka plays an important role in limb reproduce some of the effects we observed in embryonic or fin initiation and/or differentiation. We attempted to rescue the development in Pi4ka-deficient embryos, we exposed developing phenotype by injecting one-cell stage embryos with wild-type pi4ka embryos to the PI3K inhibitor LY294002 (Vlahos et al., 1994). We mRNA. The translation of this mRNA is not expected to be sensitive treated embryos with 10 M of the inhibitor from the onset of to the MOs that interfere with mRNA splicing at the transcriptional blastulation and analyzed the phenotype. This is a lower level. As shown in Fig. 2A,B, co-injection of 1 ng (n60) or 3 ng concentration than used previously (30-100 M) to effectively block (n79) pi4ka full-length mRNA with MO1 significantly alleviated PI3K activity in Xenopus embryos (Carballada et al., 2001; Peng the pectoral fin phenotype (55% and 81%, respectively). In addition, et al., 2004), but 30 M LY294002 caused 76% of the embryos to Phosphatidylinositol 4-kinase in zebrafish 4305
Fig. 2. pi4ka mRNA co-injection can reverse, and PI3K inhibitors can mimic Fig. 3. Effect of pi4ka downregulation and LY294002 treatment expression of the MO1-induced phenotype. Fertilized zebrafish eggs were injected at the several genes on the early FGF signaling pathway. Fertilized zebrafish eggs one-cell stage with either a control morpholino (cMO) or MO1 together with were injected at the one-cell stage either with a control morpholino (cMO) or Journal of Cell Science in vitro transcribed and purified pi4ka mRNA. Embryos were allowed to with MO1 or treated with 10M LY294002 and analyzed by whole-mount in develop for 1-3 days (24-72 hpf). MO1 interferes with splicing of the situ hybridization for the presence of the indicated mRNA at different stages of endogenous mRNA causing exon skipping but does not affect the translation development. (A)At 24 hpf embryos mkp3 expression is reduced in the fin- of the injected full length mRNA. (A) Co-injection of 1 ng mRNA can largely bud mesenchyme in the morphants as well as in embryos treated with 10M alleviate the brain defect at 24 hpf and 48 hpf. (B)The pectoral fin develops to LY294002. This difference becomes very prominent by the 36 hpf stage and almost normal length, but it poorly separates from the yolk sac and does not also affects the branchial arches. (B)Expression of tbx5 is only slightly fully develop in the rescued embryos (left panels; arrowhead). Measurements reduced in the fin-bud mesenchyme of morphant embryos both at 24 and 32 of the length of the pectoral fin at the 72 hpf stage (right panel) show partial hpf. The expression of fgf24 is comparable to controls in the fin-bud rescue by the injected mRNA (values are means ± s.e.m. of 70-110 mesenchyme of pi4ka morphant embryos at 24 hpf. However, at 32 hpf, fgf24- observations). (C)The PI3K inhibitor, LY294002 (10M) mimics the effects positive cells move to the AER in control embryos but are still dispersed in the of pi4ka downregulation on zebrafish development. LY-treated embryos have a mesenchyme and fail to migrate to the AER in pi4ka morphant embryos. As a smaller brain and eyes, a hooked tail and pericardial edema at 48 hpf. At 72 result, expression of fgf10, which is controlled by fgf24 in the AER, is greatly hpf, LY-treated embryos have larger yolk sacs and also reduced pectoral fins. reduced in the fin-bud mesenchyme of Pi4ka morphant embryos. Note that These changes are very similar to those observed in pi4ka downregulated some signals in the control are saturated because identical exposures were embryos (see Fig. 1D). chosen to still see the weak signal in the morphant embryos.
die before the 24 hpf stage (not shown). In LY294002-treated al., 2003). Therefore, we next determined the expression of mkp3 embryos the size of brain and eyes was reduced and the body axis in the pi4ka morphants and in LY294002-treated embryos. In control severely shortened at 24 hpf stage. At 48 hpf, pericardial edema embryos, mkp3 is detectable in the pectoral fin bud, the mid-hind has developed. At 72 hpf, LY294002-treated embryos also had brain boundary, branchial arches (Fig. 3A) and the tip of the tail smaller pectoral fins. These changes were very reminiscent of those (not shown) at 24 hpf. However, mkp3 expression was almost observed in pi4ka morphants (Fig. 2C). completely lost at this stage in the pectoral fin bud and expressed at a greatly reduced level in the branchial arches of morphant MKP3 induction is grossly affected in pi4ka-deficient embryos embryos (Fig. 3A). Expression of mkp3 was also weaker in the and after pharmacological inhibition of PI3K mid-hindbrain boundary and in the tip of the tail (not shown). At As discussed above, the MKP3 MAPK phosphatase is induced in a later stage (36 hpf), the expression of mkp3 was substantially the mesenchyme by FGF ligands of the AER via the PI3K-Akt weaker in the branchial arches, pectoral fin bud (Fig. 3A) and mid- pathway during vertebrate limb or fin development (Kawakami et hindbrain boundary of mutant embryos, whereas its expression in 4306 Journal of Cell Science 122 (23)
of the earliest genes activated in the lateral plate mesenchyme (LPM) in fin-bud development is the Tbx5 transcription factor, a master regulator of many genes in the limb-developmental program (Capdevila and Izpisua Belmonte, 2001). The first member of the Fgf family that appears at this location is fgf24 followed by fgf10 (Fischer et al., 2003). As shown in Fig. 3B, there was only a slight reduction in the expression of tbx5 in the lateral plate mesenchyme at the 24 hpf stage in pi4ka morphants. At this point the expression of fgf24 was slightly reduced but the fgf24-positive cells appeared to be more scattered in the morphant embryos. Expression of Fgf10, however, was already more severely affected even at this early stage. Analysis of embryos at a slightly later stage (32 hpf) showed more prominent differences. Here, the migration of fgf24-positive cells toward the AER could be clearly observed in control embryos (shown by the arrows) together with high expression of fgf10. By contrast, fgf24-positive cells failed to concentrate in the AER and fgf10 induction had been almost abolished in the pi4ka morphant embryos (Fig. 3B). The same changes were observed after LY294002 treatment (not shown). This process was followed at higher spatial resolution using fluorescent in situ hybridization (Clay and Ramakrishnan, 2005) and confocal microscopy to better resolve the distribution of the fgf24 and mkp3-positive cells. Fig. 4A shows that in control embryos, the fgf24-positive cells that had already appeared at 24 hours gradually moved toward the ectoderm and populated the AER. By 36 hpf, these fgf24-positive cells were clearly separated from the mkp3-positive cells that were located in the mesenchyme below the AER (Fig. 4A,B). Both in pi4ka morpholino-treated embryos and after LY294002 treatment, the fgf24-positive cells appeared at the early stages but remained scattered at the later stages when the AER is supposed to form. These cells did not move toward the ectoderm and the AER did not develop properly (Fig. 4C, upper row). Since the AER is the signaling center that controls fin/limb outgrowth, the primordial limb area remains rudimentary. This is
Journal of Cell Science also reflected in the mkp3 distribution and expression, as the early mkp3-positive cells do not show proliferation and remain weak and Fig. 4. Fluorescent in situ hybridization (FISH) and confocal analysis of fgf24 scattered by 36 hours (Fig. 4C, lower row). By contrast, in control and mkp3 expression and localization. (A)Time sequence of the position of the embryos the mkp3-positive cells are present in great number lining fgf24-positive cells showing their migration to the AER that is very clear by 36 up against the AER within the mesenchyme (Fig. 4B). These results hpf. At the same time, the number of mkp3-positive cells and their signal progressively increases but remains beneath the AER. The DAPI staining is suggested that the early mkp3-positive cells do not undergo not shown for better clarity but the contours of the ectoderm based on the proliferation and are probably subject to apoptosis because of the DAPI staining are indicated by a dashed line (see panel B). (B)Representative lack of an appropriate AER. images showing the position of the developing fin bud in a lateral view of a control fish embryo at the 36 hpf stage analyzed by FISH for mkp3 (orange) and counterstained with DAPI (green). The boxed region of each image is Loss of Pi4ka leads to widespread cell death and inhibits cell enlarged in the panel to the right. (C) Left: schematic illustration of the proliferation in the fin bud interplay between the AER and the underlying mesenchyme showing the These data prompted us to test whether apoptosis and proliferation position of fgf24- and mkp3-positive cells at the 30 hpf stage. Right: at the 36 is affected by these manipulations. For this, injected embryos at hpf stage the fgf24-expressing cells do not approach the apical layer of the different stages were stained with Acridine orange (AO) or were budding fin and the AER is not developing. As a consequence the mkp3 expression remains weak and the cells are scattered in the pi4ka MO1-injected subjected to TUNEL analysis to detect apoptotic cells. Fig. 5A shows and LY294002-treated embryos. Note that these pictures do not reflect the true that only a few AO-staining or TUNEL-positive cells were observed relative signals in the control and morphant embryos, because the images were in control embryos injected with negative MOs at all development adjusted to make the position of the positive cells clear. LPM, lateral plate stages tested. However, embryos injected with pi4ka MOs had high mesenchyme. numbers of apoptotic cells throughout all tissues at 24 hpf stage, especially in the brain, eyes and the trunk. Increased cell death was not particularly apparent in the lateral plate mesoderm that later gives rise to the pectoral fin. LY294002 treatment caused similar the telencephalon and tail were less affected (not shown). Similar, increases in apoptotic cells at all stages examined (Fig. 5A). It is but even stronger, reductions in mkp3 induction were observed in important to note that parallel downregulation of p53 by morpholino the fin bud area after LY294002 treatment. injections failed to prevent the increased general apoptosis (or the The severe defect in mkp3 induction prompted us to investigate pectoral fin defect) evoked by Pi4ka downregulation (data not the upstream steps in the origination of the developing fin bud. One shown). Phosphatidylinositol 4-kinase in zebrafish 4307
decreased proliferation dominating the picture in the case of the developing fin bud.
Discussion The experiments described above were designed to clarify the link between PI4KIII and PI3K signaling using the zebrafish as a model. This model offered several advantages. First, the developmental processes are thoroughly explored and mapped in this species with several genes and signaling pathways characterized and their testing reagents available. Second, the morpholino- induced downregulation of genes is not an all or none process and, therefore, even essential genes can be studied when remaining small expression permits survival of the cells, and pinpoint the pathways that most depend on the expression of the gene. The recently identified role of PI3K signaling in vertebrate limb development (Kawakami et al., 2003) also offered a biological readout that could be studied in a whole organism. Downregulation of Pi4ka caused a number of developmental abnormalities consistent with a presumed pleiotropic role of the enzyme in trafficking and signaling (Balla and Balla, 2006). However, one of the most prominent defects was the lack of pectoral fin development. Notably, the brain and pectoral fin buds were also the areas showing highest expression of the transcript in normal fish embryos. The impaired fin development pointed to a defect in FGF signaling. There is ample literature suggesting that sequential activation of members of the FGF family are the key to the development of pectoral fin in zebrafish (Grandel and Schulte- Merker, 1998), a process analogous to forelimb development in birds and mammals. Vertebrate limb/fin development has two crucial phases: first is the establishment of a limb field at the lateral plate mesenchyme and second, the limb outgrowth that is controlled by Fig. 5. Loss of Pi4ka function leads to widespread cell death and reduced cell the interplay between the AER and the underlying mesenchyme. proliferation. (A)Cell death detected by TUNEL analysis showed few positive cells in control embryos (cMO) at 24 hpf (i, iii) and 36 hpf stage (v). Embryos In the fish, Fgf24, a member of the FGF8/17/18 family, is the earliest injected with pi4ka MO1 had large numbers of apoptotic cells throughout the Fgf expressed in the pectoral fin bud, and it promotes migration of
Journal of Cell Science embryo at both 24 and 36 hpf stages especially in the brain, eyes and the trunk. tbx5-positive cells towards the fin field and helps maintain tbx5 Notably, there was significant cell death in the lateral plate mesoderm region expression (Fischer et al., 2003) (Fig. 4C). In pi4ka morphant (iv, vi) the early precursor of the pectoral fin bud. Acridine orange (AO) staining (vii-x) showed similar pattern of apoptosis at 24 hpf in pi4ka MO1- embryos the establishment of the limb field appears normal, as injected (vii versus viii) or LY294002-treated (ix versus x) embryos. indicated by the early markers, tbx5 and fgf24. By contrast, limb (B)Proliferating cells were detected by immunohistochemistry using anti-H3P outgrowth does not occur and the AER does not form in the antibody. Proliferation was significantly lower in the brain of pi4ka MO1- morphant embryos. This is clearly manifested in the lack of fgf10 injected embryos (MO1) than in that of controls (cMO). Proliferation was also induction in the pi4ka morphant embryos. Disruption of the mouse greatly reduced at the somites and lateral plate mesenchyme at 24 hpf and almost completely disappeared in the pectoral fin buds region of pi4ka Fgf10 gene, which is expressed in the lateral plate mesoderm even morphants at 48 hpf. (C)H3P-positive cells in the mesenchyme of fin buds at before any visible outgrowth occurs (Ohuchi et al., 1997), also 48 hpf were quantified.Values are means ± s.d. of at least 20 measurements on results in complete loss of limbs (Min et al., 1998; Sekine et al., independent embryos. 1999). The AER-derived Fgf8 is the main signal to the underlying mesenchyme to direct outgrowth of the limb bud and maintain fgf10 expression as well as the further induction of Mkp3, which is also We also identified the proliferating cells using an anti-H3P an essential intermediate in limb development (Kawakami et al., antibody, widely used as a mitosis marker (Wei et al., 1998). H3P- 2003) (Fig. 6). The substantial reduction both in fgf10 and mkp3 positive cells were broadly detected in the brain region and trunk expression in the pectoral fin bud in the morpholino-injected of control MO-injected embryos at 24 hpf (Fig. 5B). By contrast, embryos suggested that the reduced Pi4ka activity impacts AER they were seldom observed in the same parts of pi4ka morphants. development and functions. This suggested that Pi4ka-depleted mutants underwent lower level Most of the effects of Pi4ka morpholino injections in fin of mitosis. H3P-positive cells were also detected in the mesenchyme development were phenocopied by the PI3K inhibitor, LY294002, of fin buds of control embryos at 48 hpf, but their number in the confirming previous claims that the PI3K branch of FGF signaling mesenchyme of fin buds of the MO1-injected embryos was greatly is important for limb development (Kawakami et al., 2003). reduced by 72% (Fig. 5C). These results showed that cell Although LY294002 inhibits mammalian casein kinase 2 (Davies proliferation in the mesenchyme of the Pi4ka-depleted fin buds was et al., 2000) and at higher concentration the type III PI4Ks (Balla considerably decreased. Together these data indicated that reduction et al., 2008), the Kawakami study together with the results of of Pi4ka function during early stages of zebrafish development leads another study in zebrafish identifying PI3Ks necessary for to a major imbalance between apoptosis and proliferation with a process formation and cell polarization and migration of 4308 Journal of Cell Science 122 (23)
An overall decrease in PI4K activity or changes in endogenous PtdIns4P, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 levels is not expected to be demonstrable after these manipulations. Such changes have been difficult or impossible to show after downregulation of type III PI4Ks even in cultured cells with well established lipid labeling or imaging methods. It is most likely that the lipid changes affect specific cellular compartments and only in certain cells and tissues during development. Generation of transgenic fish lines expressing phosphoinositide-specific fluorescent reporters might help us identify such localized and subtle changes. These efforts are in progress in several laboratories. The importance of specific PI4Ks at the level of the whole organism was shown by a recent study describing a late onset spino- cerebellar degeneration in a mouse line with gene-trapped PI4K type-II (Simons et al., 2009). Those studies showed that a complete elimination of the PI4KII caused no major developmental abnormalities and the mice started to develop the above pathology only at a later stage of life causing a reduced Fig. 6. Schematics of the proposed participation of Pi4ka and PI3Ks in the life span of the animals. This mouse study exemplifies the FGF signaling cascade in the developing fin bud mesenchyme. Pi4ka difficulty of linking a complex phenotype to a specific contributes to the generation of the PtdIns(4,5)P2 pool that is converted by the biochemical process at the level of the whole organism and the PI3Ks to PtdIns(3,4,5)P3 in the plasma membrane. Production of this latter lipid activates the Akt signaling cascade that regulates a host of downstream unpredictability of a phenotype based on our knowledge of the genes important for proliferation or protection of the cells from apoptosis. One cell biology of the particular enzyme. Nevertheless, such studies of the most important genes regulated by this pathway is the MAPK provide important pieces of information to advance our phosphatase, MKP3 that is a key intermediate in driving pectoral fin understanding of the role of the lipid kinases in vertebrate development (Kawakami et al., 2003). This phosphatase keeps a right balance physiology and development. between MAPK and the Akt pathways. The right panel shows the FGF signaling cascade in which the Fgf24-positive cell migration appears to be the More studies are required to substantiate the validity of the first step showing impairment after Pi4ka depletion. tentative model shown in Fig. 6. However, the complex involvement of the phosphoinositide cascade at many levels of zebrafish development might make this a rather difficult task, and we need to emphasize that even the partial inactivation of the fish mesendodermal cells during gastrulation (Montero et al., 2003) Pi4ka resulted in a complex set of developmental defects. The makes it likely that the observed defects are the results of PI3K importance of other elements of the phosphoinositide signaling inhibition. Importantly, higher concentrations of LY294002 used cascade such as phospholipase C-mediated PtdIns(4,5)P2
Journal of Cell Science in the latter study (30 M) almost completely stopped breakdown and InsP3 production was shown in the very early four- development, but at a reduced concentration (10 M) we could cell stage development (Ashworth et al., 2007) as well as in reproducibly observe the pectoral fin developmental defect. It is dorsoventral patterning and Wnt signaling (Westfall et al., 2003). possible that a migration defect preventing the fgf24-positive cells PLC1 was also found to be important in zebrafish primitive from migrating and forming the AER contributes to the observed hematopoiesis (Ma et al., 2007) and vasculogenesis (Lawson et limb phenotype. However, it is more likely that PI3K is required al., 2003). However, it will be important to determine whether for the proper sequence of FGF ligand induction, which sets up kinase-dead versions of Pi4ka can rescue the pectoral-fin defect the activator-inhibitor loops between the AER and underlying and whether alternative induction or activation of the putative mesenchyme sharpening an initially broader signaling domain to downstream signaling molecules such as Akt and MKP3 can form a tighter organizing center that controls limb outgrowth. rescue this phenotype. Another possible explanation for the lack of fin bud outgrowth In summary, the present studies identify a principal component is the increased apoptosis that is observed in the embryos treated of the PI3K branch of FGF signaling cascade, namely Pi4ka. The with LY294002 or injected with the pi4ka morpholinos. Increased probable importance of this enzyme is to supply the plasma apoptosis is not unexpected when the PI3K signaling pathway is membrane with PtdIns4P that is further converted to PtdIns(4,5)P2 inhibited. However, we did not observe a particularly high by a phosphatidylinositol 4-phosphate 5-kinase and, hence, supply apoptotic activity in the fin-bud area mesenchyme and it was the the PI3K with its lipid substrate. This role of the fish Pi4k enzyme decreased rate of proliferation that dominated in the morphant is consistent with its evolutionary conservation as its homologs and LY294002-treated embryos. Nonetheless, it is probable that are responsible for the maintenance of plasma membrane increased apoptosis also contributed to the defect in limb phosphoinositide pools in yeast (Audhya and Emr, 2002) and outgrowth. In this regards it is worth noting that a generalized probably in mammalian cells (Balla et al., 2007). Pi4ka as well as increased apoptosis reported in perp (a p53 downstream target PI3Ks are vital for the induction of fgf10 and maintenance of fgf24 gene) overexpression did not result in a pectoral fin in the AER and expression of the MKP3 MAPK phosphatase gene, developmental defect (Nowak et al., 2005). The strong functional a previously identified FGF-regulated gene that is essential for limb connection between Pi4ka and PI3K in FGF signaling at the development. Further studies are needed to identify additional pectoral fin suggests that this PI4K is important for the generation elements of this signaling pathway as well as their exact of the PtdIns(4,5)P2 pool of the plasma membrane that is used contribution to the complex roles of FGF in vertebrate as substrate by the PI3Ks (Fig. 6). development. Phosphatidylinositol 4-kinase in zebrafish 4309
Materials and Methods Their effectiveness was confirmed by an in vitro translation assay (Blasiole et al., Zebrafish maintenance 2005). Zebrafish (Danio rerio) were raised and handled at the Fish Core Facility of the National Human Genome Research Institute under an approved National Institutes Microinjections of Health animal use protocol as previously described (Blake et al., 2000). After Microinjections were performed with a glass needle and a Narishige micromanipulator breeding, embryos were maintained in egg water [0.006% Instant Ocean sea salt linked to a nitrogen gas pressure system. Working dilutions of MOs and mRNA for (Aquarium Systems) in distilled water] with 2 ppm methylene blue to prevent fungal zebrafish Pi4ka were prepared with Danieau solution and injected in the yolk of one- growth. Fish of the wild-type strain EKW was used for the production of wild-type stage embryos. embryos for in situ hybridization and RNA isolation. Embryos were staged according to morphology and hours post-fertilization (hpf). Rescue of Pi4ka phenotype The full-length PI4KIII coding region was cloned into the expression vector pCS2+. Isolation and sequence analysis of zebrafish PI4K cDNAs Capped sense mRNA was synthesized from this construct using the ribomax large- Pieces of the zebrafish PI4K III gene were identified by BLAST searches of the scale RNA production system (Promega). After purification, PI4KIII mRNA (1-3 zebrafish EST and genomic sequence databases using the bovine PI4KIII (U88532) ng) was co-injected with MO1 (9-10 ng) into one-cell stage zebrafish embryos. amino acid sequence. The full-length pi4ka cDNA was cloned from two fragments Embryos were collected at 3 dpf. Pectoral fin lengths were measured and compared obtained by two RT-PCR reactions using the following primer pairs: (5Ј-ATGTG - with those of controls or PI4KIII morphants. TCCTGTGGACATCCGTGG-3Ј and 5Ј-GTAGGTGTGATTTGGTGACCGATG-3 Ј; 5Ј-TGGACAGCATAGTGAAGGACTTTGC-3Ј and 5Ј-GTCTCAGTATGGGATT - Inhibitor treatment TGGTTCTGG-3Ј). The full-length pi4ka cDNA was then assembled by combining To block PI3K activity, we used LY294002 (Calbiochem), a specific inhibitor of these two overlapping sequences in the pGEM T-Easy vector (Promega). PI3K and treated embryos from 50% epiboly stage in 10 M LY294002 and fixed in 4% paraformaldehyde at different stages. Assay of phosphatidylinositol 4-kinase Full-length Pi4ka protein was obtained by in vitro translation using the TNT quick Whole-mount antibody staining coupled transcription/translation systems (Promega). The activity of the enzyme was Phosphohistone H3 was detected in fixed embryos by immunohistochemistry using then measured with the standard kinase reaction method described previously an anti-H3P rabbit polyclonal antibody (1:100) (Upstate Biotechnology, Lake Placid, (Downing et al., 1996) in the absence or presence of 1 M wortmannin, an inhibitor NY) and HRP-conjugated anti-rabbit IgG (1:500) (KPL). of PI3Ks and the type III PI4K isoforms. We would like to thank Gregory Palardy for his help with the Whole-mount in situ hybridization and other histochemical injections and Milton English for his technical suggestions. This methods research was supported by the Intramural Research Program of the In situ hybridization was performed as previously described (Lyons et al., 2001). Antisense RNA probes were generated with UTP-digoxigenin according to the National Institute of Child Health and Human Development and the manufacturer’s instructions (Roche Diagnostics). The antisense probe (from 371 bp National Human Genome Research Institute of the National Institutes to 823 bp of the coding region) was used to analyze zebrafish Pi4ka expression. The of Health. Deposited in PMC for release after 12 months. plasmid was digested with SacII, and SP6 RNA polymerase was used to synthesize antisense RNA. In addition, the following RNA probes were used: fgf8 (Reifers et al., 1998), mpk3 (Tsang et al., 2004) and sef (Tsang et al., 2002). Probes for tbx5, References Ashworth, R., Devogelaere, B., Fabes, J., Tunwell, R. E., Koh, K. R., De Smedt, H. fgf10, fgf16 and fgf24 were generated from the full-length coding sequences amplified and Patel, S. (2007). Molecular and functional characterization of inositol trisphosphate from 24 hpf stage embryos and cloned in the pGEM-T-Easy T/A cloning plasmids. Ј receptors during early zebrafish development. J. Biol. Chem. 282, 13984-13993. (The following primers were used: fgf10 Fw, 5 -CAATGTGCAAATGG - Audhya, A. and Emr, S. D. (2002). Stt4 PI 4-kinase localizes to the plasma membrane Ј Ј Ј AAAGTGAC-3 ; fgf10 Rev, 5 -CCAAGTCTTTCCTCAGTGCAG-3 ; fgf16 Fw, and functions in the Pkc1-mediated MAP kinase cascade. Dev. Cell 2, 593-605. 5Ј-ATGGCA GAGGTGGCTGGATTTC-3Ј; fgf16 Rev, 5Ј-TCACCTATGCT - Balla, A. and Balla, T. (2006). Phosphatidylinositol 4-kinases; old enzymes with emerging GGAACAATTCTC-3Ј; fgf24 Fw, 5Ј-GATGTCTGTTCTGCCGTCAAG-3Ј; fgf24 functions. Trends Cell Biol. 16, 351-361.
Journal of Cell Science Rev, 5Ј-GTCCTTTGTG AACTTGACTCAG-3 Ј; tbx5 Fw, 5Ј-CATGGCGGACAG - Balla, A., Kim, Y. J., Varnai, P., Szentpetery, Z., Knight, Z., Shokat, K. M. and Balla, TGAAGACAC-3Ј; tbx5 Rev, 5Ј-TCTGCATGTTAGCTGGCTTCG-3Ј. Fluorescent T. (2007). Maintenance of hormone-sensitive phosphoinositide pools in the plasma in situ hybridization was performed as previously described (Clay and Ramakrishnan, membrane requires phosphatidylinositol 4-kinase III{alpha}. Mol. Biol. Cell 19, 711- 2005). In brief, after digoxigenin (DIG)-probe binding, the embryos were hybridized 721. with anti-DIG-POD antibody (Roche) and co-stained for 1 hour with TSA-AlexaFluor Balla, A., Tuymetova, G., Toth, B., Szentpetery, Z., Zhao, X., Knight, Z. A., Shokat, 555 (1:200, from Invitrogen) and DAPI (1.5 nM). Embryos were then mounted in K., Steinbach, P. J. and Balla, T. (2008). Design of drug-resistant alleles of type-III 0.8% low melting point agarose for fluorescent analysis. 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Phosphatidylinositol-3 kinase acts in nucleotides and uppercase indicates exon nucleotides) was to target the exon49- parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm intron49 junction whereas MO2 (5Ј-AGTTATACCGAGCctagaaatgagc-3Ј) was to induction. Development 128, 35-44. target the intron49-exon50 boundary. MOs were resuspended in Danieau solution (5 Clay, H. and Ramakrishnan, L. (2005). Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification. Zebrafish 2, 105-111. mM HEPES, 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, and 0.6 mM Ca(NO3)2, pH 7.6) and stored at –20°C. Subsequent dilutions were made in distilled water. MOs Davies, S. P., Reddy, H., Caivano, M. and Cohen, P. (2000). Specificity and mechanism were injected at doses ranging from 2 to 10 mg/ml. To confirm the splicing defects of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95-105. Downing, G. J., Kim, S., Nakanishi, S., Catt, K. 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