Lack of de novo Phosphatidylinositol Synthesis Leads to Endoplasmic Reticulum Stress and Hepatic Steatosis in cdipt-Deficient Zebrafish

Abstract

Hepatic steatosis is the initial stage of non-alcoholic fatty liver disease (NAFLD) and may predispose to more severe hepatic disease, including hepatocellular carcinoma. Endoplasmic reticulum (ER) stress has been recently implicated as a novel mechanism that may lead to NAFLD, although the genetic factors invoking ER stress are largely unknown. During a screen for liver defects from a zebrafish insertional mutant library, we isolated the mutant cdipt^hi559Tg/+ (hi559). CDIPT is known to play an indispensable role in phosphatidylinositol (PtdIns) synthesis. Here we show that cdipt is expressed in the developing liver and its disruption in hi559 mutants abrogates de novo PtdIns synthesis, resulting in hepatomegaly at 5-dpf. The hi559 hepatocytes display features of NAFLD, including macrovesicular steatosis, ballooning, and necroapoptosis. Gene set enrichment of microarray profiling revealed significant enrichment of ER stress response (ERSR) genes in hi559 mutants. ER stress markers, including atf6, hspa5, calr, xbp1, are selectively upregulated in the mutant liver. The hi559 expression profile showed significant overlap with that of mammalian hepatic ER stress and NAFLD. Ultrastructurally, the hi559 hepatocytes display marked disruption of ER architecture with hallmarks of chronic unresolved ER stress. Induction of ER stress by tunicamycin in wild-type larvae results in a fatty liver similar to hi559, suggesting that ER stress could be a fundamental mechanism contributing to hepatic steatosis. Conclusion: Cdipt-deficient zebrafish exhibit hepatic ER stress and NAFLD pathologies, implicating a novel link between PtdIns, ER stress, and steatosis. The tractability of hi559 mutant provides a valuable tool to dissect ERSR components, their contribution to molecular pathogenesis and evaluation of novel therapeutics of NAFLD.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD), one of the most common causes of chronic liver disease, represents a spectrum of liver disorders extending from simple hepatic steatosis to steatohepatitis, cirrhosis and fibrosis in the absence of significant alcohol abuse.^{1, 2} While this disease is highly prevalent, its molecular pathogenesis is poorly understood, hindering the development of effective therapeutics. Hepatic steatosis is believed to be the initial stage that progresses to a more severe form of NAFLD.

Currently, there is a lack of genetic models to investigate molecular mechanisms of hepatic steatosis. In this study, we present a zebrafish model to unravel potential mechanisms of hepatic steatosis.

Zebrafish are an elegant genetic model for identifying genes and elucidating molecular pathways critical to development and disease of the digestive system. Zebrafish gastrointestinal (GI) tissues share striking similarities in anatomy, cellular composition and function with their mammalian counterparts.^{3, 4} Gene expression profiles and active pathways during zebrafish GI development are also analogous to those observed in mammalian GI development and cancer.⁵ Furthermore, prominent histopathological similarities are seen between zebrafish and mammalian GI diseases, such as fatty liver, cholestasis and neoplasia.^{6, 7}

An insertional zebrafish mutant library has been established,⁸ allowing identification of genes with a role in liver development and establishment of novel models of liver diseases.⁷ Here, we provide molecular characterization of the insertional mutant cdipt^hi559Tg/+ (hi559), which displays striking liver defects at 5 days post fertilization (dpf) and subsequent death beginning at 6.5-dpf. The mutated gene responsible for the hepatic phenotype is CDIPT (CDP-diacylglycerol-inositol 3-phosphatidyltransferase), also known as phosphatidylinositol synthase (PIS). CDIPT is a highly conserved integral membrane protein found on the cytoplasmic side of the endoplasmic reticulum (ER) and has an indispensable role in the synthesis of a critical phospholipid, phosphatidylinositol (PtdIns).⁹ Phosphorylated derivatives of PtdIns, known as phosphoinositides (PIs), are crucial regulators of calcium homeostasis, membrane trafficking, secretory pathways, and signal transduction. Formation and turnover of PIs are catalyzed by evolutionarily conserved families of PI kinases and phosphatases.^{10, 11} Improper function of several of these metabolic enzymes is associated with both benign and malignant human diseases.^{12, 13} We recently reported that inositol metabolism and PI3-kinase (PI3-K) signaling pathways were enriched in the developing liver and inhibition of PI3-K pathway resulted in hepatic abnormalities.⁵

As an integral component of the ER, PtdIns and the PI signaling components are crucial for ER and its secretory functions.¹⁴ Transmembrane, organellar and secreted proteins are folded and modified in the ER and exit by vesicular transport. Perturbations of ER homeostasis, such as elevated secretory protein synthesis and accumulation, glucose deprivation, and ER calcium depletion can cause ER stress, triggering an evolutionarily conserved response, termed the ER stress response (ERSR) or unfolded protein response (UPR).¹⁵ ER stress has been associated with a wide range of diseases, including neurodegeneration, cardiac diseases, cancer and diabetes.^{16, 17} Secretory cells, such as hepatocytes, process large amounts of protein in their ER and hence are vulnerable to ER stress-associated pathology. Hepatocellular ER stress is believed to contribute to insulin resistance in diabetes and obesity, liver disorders such as α1-antitrypsin deficiency, and non-alcoholic fatty liver disease (NAFLD).¹⁸ Additionally, increased expression of ER stress-related genes was recently reported in hepatocellular carcinoma.¹⁹ Although the precise molecular pathways leading to ER stress in these diseases are largely unknown, components of PI signaling plays pivotal roles in vesicular trafficking at ER exit sites, suggesting that abnormal PI signaling may cause disruption of ER and subsequent pathologies.²⁰

Analyses of hi559 larvae reveal that a lack of de novo PtdIns synthesis causes severe disruption of the ER architecture in hepatocytes with ultrastructural pathology indicating excessive ER stress and hepatic steatosis. The ER stress and cytopathologies seen in the hi559 liver resemble those seen in human NAFLD. Furthermore, gene set enrichment analysis (GSEA) of microarray data identified selective enrichment of genes involved in ERSR pathway in hi559 larvae; several of these genes are selectively overexpressed in the mutant liver. Together, these data support a model in which disrupted PtdIns synthesis leads to ER stress-mediated intracellular damage resulting in hepatic pathology similar to that seen in NAFLD.

MATERIALS AND METHODS

Zebrafish Mutant

The zebrafish line hi559 was obtained from a large-scale insertional mutagenesis screen.⁸ All fish husbandry was carried out in accordance with local institutional animal care and use committee protocols. Heterozygous and homozygous fish were confirmed by genotyping using multiplex PCR.

Whole Mount Staining

CY3-streptavadin (CY3-SA) labeling was performed as illustrated previously.⁷ For whole-mount in situ hybridization (ISH), embryos were processed as described.⁵ See supplementary text for probes and corresponding accession numbers. Alkaline phosphatase staining for vasculature and whole-mount Oil-red-O (ORO) staining were carried out as described.^{21, 22}

RT-PCR, morpholino knockdown and mRNA rescue

Total RNA was extracted from 5-dpf wild-type and hi559 larvae using RNAeasy (Qiagen). Oligo dT primed cDNA was then synthesized using SuperScript II RT (Invitrogen) and probed by PCR. Cdipt mRNA was synthesized from a full-length linear DNA template using mMessage (Ambion) and purified by RNA clean™ (Zymo Research). Cdipt and gfp mRNAs were injected into 1-cell stage embryos. For knockdown analyses of Cdipt, two zebrafish cdipt splice-blocking morpholinos (see supplementary text for sequences) were co-injected with tp53 morpholino into wild-type embryos. tp53 morpholino alone was injected as a control morpholino. See supplementary text for primer and morpholino sequences.

Radioactive Phosphatidylinositol Synthesis (PIS) assay

The PIS assay was performed essentially as described previously.^{23, 24} The assay was conducted in 100 μl total volume containing 0.2 mM CDP-DAG, 0.5 mM myo-[³H]inositol (5000 cpm/nmol), 2 mM MnCl₂, 50 mM Tris-Hcl, pH8.0, 0.15% Triton X-100, and 50 μg of total protein isolated either from wild-type or hi559 larvae. After 1 h incubation at 37° C, the reaction was terminated by adding 0.35 ml chloroform and 0.5 ml 1 M MgCl₂. The organic phase was separated for lipid extraction. PIS activity was measured as amount of myo-[³H]inositol incorporation into PtdIns per mg of protein, as determined by scintillation counting.

Histology and Transmission Electron Microscopy (TEM)

Wild-type and hi559 larvae were fixed in 4% PFA/PBS at 4° C overnight, dehydrated with ethanol and embedded in JB-4 (Polysciences). Serial sagittal and transverse sections (4 μm) were stained with Hematoxylin and Eosin (H&E). For semi-thin sections, epoxy resin embedded embryos were sectioned (20 nm) and stained with Toluedene-blue. For lipid staining, freshly collected embryos were embedded in OCT (Tissue-Tek), frozen in liquid nitrogen, sectioned (5 μm) using a cryostat at −20° C and stained with ORO. Sectioning and TEM imaging was performed by the EM facility of the Renal Pathology Service at the University of Pittsburgh Medical Center (Pittsburgh, PA). See supplementary text for further details.

Microarray Analyses

Total RNA was extracted from three samples each of 5-dpf wild-type and mutant larvae (n=25) using RNAeasy (Qiagen). Hybridization of Affymetrix GeneChips, microarray data collection and analyses were performed as described previously, using Ingenuity’s pathway analysis (IPA; http://www.ingenuity.com) and Gene Set Enrichment Analysis (GSEA; http://www.broad.mit.edu/gsea/).^{5, 25} Microarray data are deposited with GEO (GSE17711).

RESULTS

hi559 Mutants Exhibit Defects in Liver Development

Heterozygous hi559 carriers are phenotypically indistinguishable from their wild-type siblings; the hi559 phenotype is completely penetrant in homozygotes. The hi559 embryos hatch and are phenotypically normal until 5-dpf when homozygous hi559 larvae become easily distinguishable from wild-type siblings by a globular (abnormally shaped), darkish liver, as seen by bright-field microscopy and CY3-SA labeling (Figure 1A-C). hi559 larvae also display a smaller intestine and slightly smaller eyes. The pancreas does not exhibit any noticeable defects (Figure S1). hi559 larvae begin to die around 6.5-dpf.

Figure 1. hi559 larvae exhibit defects in liver development.

(A) 5-dpf larval morphology, wild-type on top, mutant below. (B) hi559 larvae show globular liver (yellow) and smaller intestine (red). (C) Globular liver is apparent by Cy3-SA labeling. Lateral view of ISH at 5-dpf showing expression of liver-specific markers sepp1b (D), cp (E), and fabp10a (F). L, liver, ib; intestinal bulb; gb, gas-bladder; y, yolk.

To analyze developmental abnormalities in the liver, we characterized the 5-dpf hi559 larvae by ISH using RNA probes against three liver-specific transcripts, sepp1b, cp, and fabp10a (Figure 1D-F). Although their expression appears similarly intense in wild-type and hi559, the abnormal shape of the liver is apparent (Figure 1D). We did not notice any difference in expression of the liver markers in clutches of embryos between 2 and 4-dpf (data not shown), indicating that there are no overt defects in liver formation at early stages. We observed no noticeable differences in the expression of markers specific to exocrine (try) and endocrine (ins) pancreas (Figure S1A). The defects in hi559 liver at 5-dpf suggest an important role of the wild-type gene product in hepatic development and function.

Retroviral Insertion Disrupts Cdipt Expression

By inverse PCR, the retroviral insert was mapped to the first intron (35 nucleotides past the first exon) of cdipt (Figure 2A).⁸ RT-PCR results show lack of cdipt mRNA in hi559, but products of expected sizes in wild-type siblings (Figure 2B). Bioinformatic analyses of the zebrafish genome (Build 7, Ensembl) did not reveal a second copy of cdipt. To investigate if knockdown of Cdipt can replicate the hi559 phenotype, we injected two different splice-blocking morpholinos against cdipt into wild-type embryos. To reduce non-specific effects due to activation of tp53, we co-injected tp53 morpholino.²⁶ Approximately, 60% (53/90) of the injected embryos displayed hepatic abnormalities similar to hi559 (Figure S2A-D). Injection of 100 pg of cdipt mRNA resulted in significant reduction of larvae with hepatic phenotype from a clutch, rescuing presumably mutant embryos (Figure S2E). In a clutch (n=147) of cdipt mRNA injected embryos, only 5 (3.4%, expected 25%) displayed the typical hi559 phenotype at 5-dpf; all remaining larvae appeared normal. In the control group (injected with gfp mRNA), ~25% (19/70) showed the typical hi559 phenotype. These results strongly suggest that hi559 phenotype is due to loss of Cdipt function.

Figure 2. Disruption of cdipt expression and PtdIns synthesis in hi559 larvae.

(A) The retroviral insertion (triangle) was mapped to the first intron of the cdipt gene.⁸ White boxes indicate exons, grey boxes introns. (B) cdipt expression is disurpted in hi559 larvae (RT-PCR amplification of three different regions, indicated in A). (C-E) Developmental expression pattern of cdipt in wild-type embryos. Note the absence of cdipt expression in hi559 larvae (wild-type, F, left; mutant, F, right). Intestine (arrow), liver (arrowhead). (F) PtdIns synthesis is disrupted in hi559 larvae. The bar chart represents values from three biological replicates.

Cdipt Is Expressed in the Liver During Development

At 24-hpf, cdipt mRNA expression is ubiquitous, but becomes restricted to the developing liver and intestine by 48-hpf and remains high in these tissues through 5-dpf (Figure 2D-E). Cdipt is also expressed in the brain, retina and branchial arches throughout development. As expected, hi559 embryos lack cdipt expression (Figure 2F, right and Figure S3).

hi559 Larvae Are Deficient in de novo PtdIns Synthesis

Since CDIPT plays a critical role in PtdIns synthesis, we wanted to confirm alteration of PtdIns levels in the absence of cdipt expression. Surprisingly, comparative phopholipid profile by TLC shows that levels of PtdIns and other phospholipids of deyolked wild-type and hi559 larvae are similar at 5-dpf (Figure S4). However, we noticed that the embryonic yolk at 1-cell stage contains abundant PtdIns, suggesting that it is maternally deposited. We reasoned that de novo PtdIns synthesis might be disrupted in hi559 embryos and tested PIS activity in the 5-dpf wild-type and mutant larvae. PIS activity is negligible in hi559, but robust in wild-type (Figure 2F). To further confirm that chemical inhibition of PIS replicates hi559 phenotype, we treated wild-type larvae with δ-Hexachlorocyclohexane (δ-HCH), a drug with the same configuration as myo-inositol, known to inhibit myo-inositol incorporation into PtdIns.²⁷ Treatment with δ-HCH resulted in hepatomegaly and a darkish liver similar to hi559 (Figure S5). These results suggest that maternally deposited and de novo synthesized PtdIns are not functionally equivalent and that de novo synthesis of PtdIns is required for normal hepatic development.

hi559 Livers Display Features of NAFLD

In sagittal sections, the hi559 liver appears swollen and vacuolar with enlarged hepatocytes and increased internuclear distance between adjacent hepatocytes as compared to wild-type (Figure 3A). Toluedene-blue staining of semi-thin transverse sections reveals marginalization of nuclei with rarefied cytoplasm, and vesicles often filled with dense material, suggestive of fat accumulation (Figure 3B). Whole-mount ORO staining shows that hi559 larvae have fatty livers (Figure 3C). ORO staining of frozen histological sections reveals substantial fat accumulation in the form of small and large lipid droplets in hi559 hepatocytes (Figure 3D). At the onset of NAFLD at 5-dpf, the hi559 liver displays admixture of normal hepatocytes and foci of microvesicular and macrovesicular steatosis, without apparent necrosis (Figure 4B). However, with progression of NAFLD, most hepatocytes exhibit severe macrovesicular steatosis and some display fragmented nuclei (Figure 4C-D). In some cases of severely steatotic livers, hepatic sinusoids appear smaller (Figure S6). The distortion of the sinusoid architecture may be attributed to the grossly enlarged hepatocyte plates compressing the adjacent sinusoids. Hepatocellular injury in the form of ballooning degeneration, apoptosis and necrotic foci are prominent in hi559 liver by 6-dpf (Figure 4D). The ballooned hepatocytes often have rarefied cytoplasm containing perinuclear hyaline inclusion bodies. Thus, many of the characteristic histological features of NAFLD, such as enlarged hepatocytes, cytoplasmic clearing, accumulation of small and large membrane-bound lipid, and subsequent necrosis are observed in hi559 livers.^{1, 28} Despite the severe hepatic histopathology reminiscent of NAFLD, inflammation was not conspicuous in hi559 livers at histological level, although we noticed the presence of macrophages adjacent to the necrotic hepatocytes ultrastructurally, indicating mild inflammation (Figure 7H). The paucity of inflammation may be attributed to an incompletely matured zebrafish immune system at this stage of larval development.

Figure 3. Histopathological abnormalities of hi559 liver.

(A) H&E-stained sagittal sections of 5-dpf wild-type and hi559 liver. Mutant liver architecture is abnormal with vacuolar appearance and smaller sinuses (asterisk). (B) Toluedene staining of semi-thin transverse sections reveals large, vacuolated hepatocytes with marginalized nuclei (arrow) in hi559 liver. (C) Whole mount ORO staining of 5-dpf larvae shows fatty liver (dotted line) in the mutant. (D) ORO staining of frozen tissue sections of 5-dpf larvae reveals substantial steatosis in hi559 hepatocytes. Arrows indicate lipid droplets (red), arrowheads nuclei. ib, intestinal bulb; in, intestine; L, liver; y, yolk; wt, wild-type. Scale bars: A, 20 μM; B, 50 μM; D, 20 μm.

Figure 4. NAFLD progression in hi559 liver.

H&E sections of wild-type liver at 6-dpf (A), and stages of NAFLD progression in hi559 liver at 5-dpf (B), 5.5-dpf (C), and 6-dpf (D). At early stage of NAFLD at 5-dpf, foci of microvesicular (arrow) and macrovesicular (arrowhead) steatosis are evident with no apparent apoptosis. At 5.5-dpf, most of the hepatocytes exhibit macrovesicular steatosis with a few apoptotic hepatocytes (arrow, the dotted line area magnified in the inset). At 6-dpf, hepatocellular ballooning (arrow), often with perinuclear hyaline inclusions (arrowhead), and necrotic foci (dotted line) are apparent. (E) Bar-charts showing percentages of embryos with hepatic steatosis at 5-dpf. (F) Bar-charts showing percentages of apoptotic hepatocytes in 6-dpf liver. Data are representative of 5 biological replicates. Scale bars: 20 μM.

Figure 7. Ultrastructural pathology of hepatocytes.

(A-D) Electron-micrographic comparison of wild-type and hi559 hepatocytes. Cytoplasmic clearing (asterisk, B), ER luminal swelling (triangle, D), and abnormal mitochondria are frequently observed in hi559 hepatocytes. (E) hi559 hepatocyte shows mitochondrial damage and excessive ER luminal vacuolization. The ER lumens are filled with granular materials. (F) The hi559 hepatocytes often contain membrane-bound lipids (lipolysosomes, arrow) of variable electron density. (G) Autophagosome-like structures (arrow) containing reticular materials are noticed within hi559 hepatocytes. (H) Presence of a macrophage (arrow) adjacent to necrotic hepatocytes. mc, mitochondria; n, nucleus; er: endoplasmic reticulum. wt, wild-type; mut, hi559 mutant. Scale bars: A-B & F, 2 μm; C-E & G,-H, 500 nm.

Microarray Analyses Reveal Upregulation of ERSR in hi559 Larvae

We performed Affymetrix array analyses to decipher deregulated pathways and gene networks associated with hi559 phenotype. Our analyses reveal a set of 465 genes that are significantly differentially regulated (p<0.05) in hi559 compared to wild-type siblings at 5-dpf, 186 of which are upregulated. GSEA revealed enrichment of a set of genes involved in ERSR/UPR (Figure 5A-B). We noticed significant upregulation of critical ERSR indicators in the mutant. Many of these genes encode ER resident proteins that collectively take part in the UPR or in Ca²⁺ homeostasis, including calr, hspa5/bip/grp78, hsp90b1/grp94, caln, and atf6 (Figure 5). We subsequently compared our gene expression profile with a previously published gene set on hepatic ER stress in mice²⁹ and found a significant overlap between the two (Figure 5C-D). IPA identified acute phase response signaling as the top most upregulated canonical pathways, suggesting activated transcription of immune/inflammatory response factors in hi559 larvae (data not shown). Overlaying gene expression values onto the ERSR pathway generated by IPA show transcriptional upregulation of ERSR components and ER stress-induced apoptotic signals (Figure S7). To validate our microarray data, we analyzed the expression patterns of a set of ERSR markers by ISH. Interestingly, these genes are selectively overexpressed in hi559 liver (Figure 5A-E). Together, these data implicate lack of PtdIns synthesis in leading to hepatocellular ER stress, causing the hepatic pathology in hi559 larvae.

Figure 5. Deregulation of genes involved in ERSR.

GSEA enrichment plots and expression profile (shown by heat-map) of genes involved in the ERSR/UPR pathway (A-B) and integrated stress-response pathway²⁹ (C-D). Running enrichment score (ES, top) and signal-to-noise ratio (bottom) used for ranking genes (positive: upregulated in mutant; negative: downregulated in mutant) are shown in the GSEA plots.²⁵ Vertical bars (middle) indicate the position of genes in the ERSR/UPR within the sorted microarray data, showing enrichment among genes upregulated in hi559. Normalized ES (NES) and nominal p-value, below.

ER Architecture Is Severely Disrupted in hi559 Hepatocytes

We performed TEM to analyze the ultrastructural pathology of hi559 hepatocytes. Wild-type hepatocytes exhibit a homogeneous, grainy cytoplasm, generally without clearing areas (Figure 7A). By contrast, the hi559 hepatocytes have abnormal mitochondria, large cytoplasmic clearing areas with several membrane-bound structures containing granular materials (Figure 7B). Irregular shaped lipolysosomes containing lipid droplets of variable electron-density are frequently seen in hi559 hepatocytes (Figure 7F). Most strikingly, hi559 hepatocytes have large, excessively dilated (luminal swelling), abnormally distributed ER (Figure 7C-D). It appears that the prominent clearing areas in hi559 hepatocytes may be the sequelae of excessive ER luminal swelling and vacuolation. The lumens of the expanded ER in hi559 hepatocytes are often filled with aggregates of variable electron density, suggestive of accumulated proteins (Figure 7E). In some instances, the ER-membranes are selectively sequestered and tightly packaged into autophagosome-like structures (Figure 7G). These ultrastructural pathologies are consistent with chronic unresolved ER stress and resemble that seen in NAFLD.

Tunicamycin-induced ER Stress Results in Fatty Liver Similar to hi559

While analyzing the expression of ER stress markers, we noticed elevated expression of the crucial ER stress sensor hspa5 in hi559 livers at 4-dpf, prior to onset of the hepatic phenotype (Figure 8A). This implicates that hepatocellular ER stress may be a major contributor to the hepatic steatosis seen in hi559 larvae at 5-dpf. To test whether ER stress during this developmental stage could cause hepatic steatosis, we treated wild-type larvae with tunicamycin, an inhibitor of protein N-glycosylation that induces ER stress. Chronic treatment with 1 μM tunicamycin from 3.5-dpf through 5.5-dpf induced defects similar to those seen in hi559 larvae in ~90% of the treated embryos (Figure 8B-E). Embryos subsequently die at 6 to 7-dpf, similar to hi559, when tunicamycin treatment was continued. Induction of ER stress upon tunicamycin treatment was confirmed by ISH with the crucial ER stress marker hspa5. The ubiquitously elevated expression of hspa5 is apparent in tunicamycin-treated embryos (Figure 8C). Whole-mount ORO staining further confirmed the development of a fatty liver in tunicamycin-treated embryos (Figure 8D). These results implicate that ER stress triggers hepatic steatosis in zebrafish larvae and that chronic hepatocellular ER stress may be predisposing to NAFLD in hi559 embryos.

Figure 8. Tunicamycin-induced ER stress causes fatty liver in wild-type larvae.

(A) Elevated expression of hspa5 in hi559 liver at 4-dpf by ISH (arrow). (B) Chronic exposure of 1 μM tunicamycin from 3.5-dpf to 5.5-dpf causes globular darkish liver (yellow outline) similar to hi559. (C) hspa5 expression is ubiquitously elevated, including liver (arrow) in tunicamycin-treated larvae. (D) ORO staining shows presence of fatty liver (yellow outline) in tunicamycin-treated larvae. (E) Bar-charts indicating percentage of larvae with hepatic steatosis at 5.5-dpf in DMSO and tunicamycin (TM) treated groups. Data are representative of 3 clutches (n=55).

DISCUSSION

To our knowledge, hi559 is the first in vivo model linking PtdIns synthesis, ER stress, and NAFLD. Multiple lines of evidence support the conclusion that loss of Cdipt function eliminates PtdIns synthesis. First, Cdipt is specifically inactivated in hi559 zebrafish, evident by undetectable levels of cdipt mRNA by RT-PCR and ISH, rescue of the mutant phenotype with cdipt mRNA and phenocopy of the hi559 phenotype by injection of cdipt morpholinos. Secondly, we believe that cdipt is the sole enzyme responsible for de novo PtdIns synthesis in zebrafish: in extracts from mutant larvae, no PtdIns synthesis could be detected. Thirdly, zebrafish cdipt is highly homologus to mammalian CDIPTs (Figure S9) and no other potential orthologs could be detected in the zebrafish genome. Lastly, Drosophila embryos deficient in dPIS (the ortholog of CDIPT) were unable to synthesize PtdIns, and died during embryogenesis.²³ Taken together, we infer that Cdipt is essential for PtdIns synthesis and its disruption leads to the hi559 phenotype.

Although Cdipt is indispensable for PtdIns synthesis, widespread developmental abnormalities are not observed in hi559 embryos during early development, possibly due to maternally deposited PtdIns in the yolk (Figure S4). The later phenotypic abnormalities reflects a requirement of de novo PtdIns synthesis, since pools of PtdIns are locally made and used in intracellular PI signaling almost instantly after synthesis.³⁰ Thus, despite an abundant supply of maternal PtdIns, cells may still require de novo synthesis of PtdIns for appropriate PI signaling and PtdIns function. Hence, we surmise that lack of de novo PtdIns synthesis during development causes aberrant PtdIns function and PI signaling in secretory hepatocytes of hi559 larvae.

The dynamics and function of PtdIns and their patho-physiolological roles in various human diseases remain elusive. In this study, disruption of PtdIns de novo synthesis results in persistent hepatocellular ER stress, evident by robust activation of ER stress sensors and chaperones in the hi559 liver, and grossly expanded ER lumens. Aberrant PtdIns functions can affect ER homeostasis and cause subsequent ER stress-associated cytopathologies in several ways, such as calcium misregulation, alteration of secretory pathways and accumulation of proteins in the ER (Figure S8). First, intracellular Ca²⁺ signaling and Ca²⁺ homeostasis in the ER are dependent on the PtdIns breakdown products, IP₃. ^{31, 32} Aberrant Ca²⁺ results in dysfunction of ER chaperones, thus affecting proper folding of proteins in ER. Second, PKC signaling requires PtdIns and its breakdown products, inositol and diacylglycerol, and calcium. Altered PKC signaling can cause elevated transcription of secretory proteins.³³ Third, the continual exchange of proteins and lipids between the ER and Golgi apparatus through vesicular transport is also a Ca²⁺ and PtdIns-dependent process.³⁴ One of the transient PIs, PtdIns-4-phosphate is critical in vesicular trafficking and ERAD, and its lack may contribute to the accumulation of secretory proteins in the ER lumen, causing ER stress.^{20, 35} Therefore, we hypothesize that disrupted PtdIns synthesis alters one or more of these molecular processes, resulting in unresolved ER stress and consequent hepatic pathology. Consistent with this hypothesis, UPR is activated when yeast are cultured on inositol-deficient media and inactivated upon inositol supplementation, as a result of modulation of PtdIns levels.^{36, 37}

Concurrent with ER stress, the hi559 liver displays NAFLD pathologies, which we believe are a consequence of unresolved ER stress. Hepatocytes cope with ER-stress by the UPR, but chronic unresolved ER stress can unleash pathological consequences, including hepatic fat accumulation, cell death and inflammation, thus contributing to NAFLD.^{18, 38} XBP1, a critical mediator of ERSR, is reported to be involved in increased hepatic lipogenesis and we found selective upregulation of xbp1 in the hi559 liver. Upregulation of hspa5, the master ER stress sensor, is apparent in the hi559 liver at 4-dpf, before onset of the hepatic phenotype (Figure 8A). Additionally, pharmacological induction of ER stress by tunicamycin caused hepatic steatosis similar to hi559. These results suggest that chronic unresolved ER stress may predispose the secretory hepatocytes to hepatic steatosis in hi559 larvae.

Hyperlipidemia, obesity and diabetes may predispose to NAFLD, a disease with increasing prevalence in Western societies and currently without effective therapy.^{1, 28}The similarity of cytopathological features of hi559 liver to NAFLD emphasizes the potential of this mutant as an in vivo model for unraveling molecular pathogeneses of this disease. Here, we report a novel association between PtdIns, ER stress, and hepatic steatosis, suggesting that modulation of PtdIns may mitigate the contribution of ER stress to the pathology of NAFLD. With the increasing recognition of the role of ER stress in human disease, including hepatocellular carcinoma, several ER stress-modulating compounds are being explored for their therapeutic potential.^{16, 38} The hi559 mutant described in this manuscript is uniquely positioned to aid in the functional characterization of these compounds in a live animal model, and in the identification and analyses of potentially new treatment paradigms.

Figure 6. Preferential upregulation of ERSR/UPR genes in the liver of hi559 embryos.

ISH of ERSR markers in wild-type (top) and hi559 embryos (bottom) at 5-dpf. The respective gene symbols are indicated in each panel. Note the elevated expression in the hi559 liver (arrow).

Abbreviations Used in Manuscript

dpf

days post fertilization

ER

endoplasmic reticulum

ERSR

Endoplasmic Reticulum Stress Response

GFP

green fluorescent protein

GI

gastrointestinal

NAFLD

non-alcoholic fatty liver disease

PI

phosphoinositides

PtdIns

phosphatidylinositol