ROS signaling-induced mitochondrial Sgk1 expression regulates epithelial cell renewal.

Many types of differentiated cells can reenter the cell cycle upon injury or stress. The underlying mechanisms are still poorly understood. Here, we investigated how quiescent cells are reactivated using a zebrafish model, in which a population of differentiated epithelial cells are reactivated under a physiological context. A robust and sustained increase in mitochondrial membrane potential was observed in the reactivated cells. Genetic and pharmacological perturbations show that elevated mitochondrial metabolism and ATP synthesis are critical for cell reactivation. Further analyses showed that elevated mitochondrial metabolism increases mitochondrial ROS levels, which induces Sgk1 expression in the mitochondria. Genetic deletion and inhibition of Sgk1 in zebrafish abolished epithelial cell reactivation. Similarly, ROS-dependent mitochondrial expression of SGK1 promotes S phase entry in human breast cancer cells. Mechanistically, SGK1 coordinates mitochondrial activity with ATP synthesis by phosphorylating F1Fo-ATP synthase. These findings suggest a conserved intramitochondrial signaling loop regulating epithelial cell renewal.

Regulation of cell quiescence-proliferation balance by Ca2+-CaMKK-Akt signaling.

Compared with our extensive understanding of the cell cycle, we have limited knowledge of how the cell quiescence-proliferation decision is regulated. Using a zebrafish epithelial model, we report a novel signaling mechanism governing the cell quiescence-proliferation decision. Zebrafish Ca2+-transporting epithelial cells, or ionocytes, maintain high cytoplasmic Ca2+ concentration ([Ca2+]c) due to the expression of Trpv6. Genetic deletion or pharmacological inhibition of Trpv6, or reduction of external Ca2+ concentration, lowered the [Ca2+]c and reactivated these cells. The ionocyte reactivation was attenuated by chelating intracellular Ca2+ and inhibiting calmodulin (CaM), suggesting involvement of a Ca2+ and CaM-dependent mechanism. Long-term imaging studies showed that after an initial decrease, [Ca2+]c gradually returned to the basal levels. There was a concomitant decease in endoplasmic reticulum (ER) Ca2+ levels. Lowering the ER Ca2+ store content or inhibiting ryanodine receptors impaired ionocyte reactivation. Further analyses suggest that CaM-dependent protein kinase kinase (CaMKK) is a key molecular link between Ca2+ and Akt signaling. Genetic deletion or inhibition of CaMKK abolished cell reactivation, which could be rescued by expression of a constitutively active Akt. These results suggest that the quiescence-proliferation decision in zebrafish ionocytes is regulated by Trpv6-mediated Ca2+ and CaMKK-Akt signaling.

Alteration of organ size and allometric scaling by organ-specific targeting of IGF signaling.

The size of an organ is proportional to the other body parts or the whole body. This relationship is known as allometry. Understanding how allometry is determined is a fundamental question in biology. Here we tested the hypothesis that local insulin-like growth factor (Igf) signaling is critical in regulating organ size and its allometric scaling by organ-specific expression of Igf binding protein (Igfbp). Overexpression of Igfbp2a or 5b in the developing zebrafish eye, heart, and inner ear resulted in a disproportional reduction in their growth relative to the body. Stable transgenic zebrafish with lens-specific Igfbp5b expression selectively reduced adult eye size. The action is Igf-dependent because an Igf-binding deficient Igfbp5b mutant had no effect. Targeted expression of a dominant-negative Igf1 receptor (dnIgf1r) in the lens caused a similar reduction in relative eye growth. Furthermore, co-expression of IGF-1 with an Igfbp restored the eye size. Finally, co-expression of a constitutively active form of Akt with Igfbp or dnIgf1r restored the relative eye growth. These data suggest that local Igf availability and Igf signaling activity are critical determinants of organ size and allometric scaling in zebrafish.

IGF-2 mRNA binding protein 2 regulates primordial germ cell development in zebrafish.

Insulin-like growth factor 2 mRNA binding protein-2 (IGF2BP2 or IMP2) is a member of a conserved family of RNA binding proteins. These proteins bind to and regulate target mRNA localization, stability, and translation. Their structure, expression and functions in bony fish are not well understood. Here, we characterized the zebrafish igf2bp2 gene and investigated its functional role in early development. Zebrafish igf2bp2 gives rise to 4 alternatively spliced transcripts. When expressed in cultured cells, all 4 proteins were detected in the cytoplasm. Igf2bp2-A, the longest isoform, has a domain structure similar to its mammalian counterpart. Igf2bp2-B lacks one of the C-terminal KH domains, while Igf2bp2-C lacks the two N-terminal RRM domains. Igf2bp2-D lacks both regions. In adult fish, these igf2bp2 isoforms were detected exclusively in the oocyte. After fertilization, they disappeared within 6 h post fertilization (hpf). At 20 ~ 24 hpf, igf2bp2-A mRNA, but not other mRNAs, was re-expressed in the embryos including in primordial germ cells. Targeted knockdown of Igf2bp2s reduced the numbers of primordial germ cells but did not affect global patterning or growth. The effect was rescued by overexpression of Igf2bp2-A. Likewise, dominant-negative inhibition of Igf2bp2 resulted in a similar reduction in primordial germ cell number. These results not only provide new information about the structure and expression of zebrafish Igf2bp2, but also reveal a critical role of this conserved RNA binding protein in primordial germ cell development.

Gonadotropin-releasing hormone neuron development in vertebrates.

Gonadotropin-releasing hormone (GnRH) neurons are master regulators of the reproductive axis in vertebrates. During early mammalian embryogenesis, GnRH1 neurons emerge in the nasal/olfactory placode. These neurons undertake a long-distance migration, moving from the nose to the preoptic area and hypothalamus. While significant advances have been made in understanding the functional importance of the GnRH1 neurons in reproduction, where GnRH1 neurons come from and how are they specified during early development is still under debate. In addition to the GnRH1 gene, most vertebrate species including humans have one or two additional GnRH genes. Compared to the GnRH1 neurons, much less is known about the development and regulation of GnRH2 neuron and GnRH3 neurons. The objective of this article is to review what is currently known about GnRH neuron development. We will survey various cell autonomous and non-autonomous factors implicated in the regulation of GnRH neuron development. Finally, we will discuss emerging tools and new approaches to resolve open questions pertaining to GnRH neuron development.

Hypoxia-inducible factor 3 biology: complexities and emerging themes.

The hypoxia-inducible factor (HIF) family has three distinct members in most vertebrates. All three HIFs consist of a unique and oxygen-labile α-subunit and a common and stable ß-subunit. While HIF-1 and HIF-2 function as master regulators of the transcriptional response to hypoxia, much less is known about HIF-3. The HIF-3α gene gives rise to multiple HIF-3α variants due to the utilization of different promoters, different transcription initiation sites, and alternative splicing. These HIF-3α variants are expressed in different tissues, at different developmental stages, and are differentially regulated by hypoxia and other factors. Recent studies suggest that different HIF-3α variants have different and even opposite functions. There is strong evidence that full-length HIF-3α protein functions as an oxygen-regulated transcription activator and that it activates a unique transcriptional program in response to hypoxia. Many HIF-3α target genes have been identified. While some short HIF-3α variants act as dominant-negative regulators of HIF-1/2α actions, other HIF-3α variants can inhibit HIF-1/2α actions by competing for the common HIF-ß. There are also a number of HIF-3α variants yet to be explored. Future studies of these naturally occurring HIF-3α variants will provide new and important insights into HIF biology and may lead to the development of new therapeutic strategies.

Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family.

Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to hypoxia. To gain insight into the structural and functional evolution of the HIF family, we characterized the HIFα gene from amphioxus, an invertebrate chordate, and identified several alternatively spliced HIFα isoforms. Whereas HIFα Ia, the full-length isoform, contained a complete oxygen-dependent degradation (ODD) domain, the isoforms Ib, Ic, and Id had 1 or 2 deletions in the ODD domain. When tagged with GFP and tested in mammalian cells, the amphioxus HIFα Ia protein level increased in response to hypoxia or CoCl2 treatment, whereas HIFα Ib, Ic, and Id showed reduced or no hypoxia regulation. Deletion of the ODD sequence in HIFα Ia up-regulated the HIFα Ia levels under normoxia. Gene expression analysis revealed HIFα Ic to be the predominant isoform in embryos and larvae, whereas isoform Ia was the most abundant form in the adult stage. The expression levels of Ib and Id were very low. Hypoxia treatment of adults had no effect on the mRNA levels of these HIFα isoforms. Functional analyses in mammalian cells showed all 4 HIFα isoforms capable of entering the nucleus and activating hypoxia response element-dependent reporter gene expression. The functional nuclear location signal (NLS) mapped to 3 clusters of basic residues. (775)KKARL functioned as the primary NLS, but (737)KRK and (754)KK also contributed to the nuclear localization. All amphioxus HIFα isoforms had 2 functional transactivation domains (TADs). Its C-terminal transactivation (C-TAD) shared high sequence identity with the human HIF-1α and HIF-2α C-TAD. This domain contained a conserved asparagine, and its mutation resulted in an increase in transcriptional activity. These findings reveal many ancient features of the HIFα family and provide novel insights into the evolution of the HIFα family.

Pregnancy-associated plasma protein A (PAPP-A) modulates the early developmental rate in zebrafish independently of its proteolytic activity.

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.

Role of IGF signaling in catch-up growth and accelerated temporal development in zebrafish embryos in response to oxygen availability.

Animals respond to adverse environments by slowing down or arresting growth and development. Upon returning to normal conditions, they often show compensatory acceleration in growth and developmental rate. This phenomenon, known as `catch-up' growth, is widely documented in the animal kingdom. The underlying molecular mechanisms, however, are poorly understood. Using the zebrafish embryo as an experimental model system, we tested the hypothesis that changes in IGF signaling activities play an important role in the accelerated growth and temporal development resulting from re-oxygenation following hypoxia. We show that chronic hypoxia reduced, and re-oxygenation accelerated, embryonic growth and developmental rate. Whereas hypoxia repressed the Igf1 receptor and its downstream Erk1/2 and Akt signaling activities, re-oxygenation restored their activities. Specific inhibition of Igf1 receptor signaling during re-oxygenation by genetic and pharmacological approaches attenuated catch-up growth. Further analysis showed that whereas PI3K-Akt is required in both normal and catch-up growth, Mek1/2-Erk1/2 activation induced by elevated IGF signaling during re-oxygenation is particularly crucial for catch-up growth. These results suggest that the evolutionarily conserved IGF signaling pathway coordinates growth and temporal development in zebrafish embryos in response to oxygen availability.

Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth.

IGF binding protein 3 exerts its ligand-independent action by antagonizing BMP in zebrafish embryos.

IGFBP3 is a multi-functional protein that has IGF-dependent and IGF-independent actions in cultured cells. Here we show that the IGF binding domain (IBD), nuclear localization signal (NLS) and transactivation domain (TA) are conserved and functional in zebrafish Igfbp3. The in vivo roles of these domains were investigated by expression of Igfbp3 and its mutants in zebrafish embryos. Igfbp3, and its NLS and TA mutants had equally strong dorsalizing effects. Human IGFBP3 had similar dorsalizing effects in zebrafish embryos. The activities of IBD and IBD+NLS mutants were lower, but they still caused dorsalization. Thus, the IGF-independent action of Igfbp3 is not related to NLS or TA in this in vivo model. We next tested the hypothesis that Igfbp3 exerts its IGF-independent action by affecting Bmp signaling. Co-expression of Igfbp3 with Bmp2b abolished Bmp2b-induced gene expression and inhibited its ventralizing activity. Biochemical assays and in vitro experiments revealed that IGFBP3 bound BMP2 and inhibited BMP2-induced Smad signaling in cultured human cells. In vivo expression of Igfbp3 increased chordin expression in zebrafish embryos by alleviating the negative regulation of Bmp2. The elevated level of Chordin acted together with Igfbp3 to inhibit the actions of Bmp2. Knockdown of Igfbp3 enhanced the ventralized phenotype caused by chordin knockdown. These results suggest that Igfbp3 exerts its IGF-independent actions by antagonizing Bmp signaling and that this mechanism is conserved.

Duplicated Kiss1 receptor genes in zebrafish: distinct gene expression patterns, different ligand selectivity, and a novel nuclear isoform with transactivating activity.

The kisspeptin (Kiss1) and Kiss1 receptor (Kiss1r) pathway plays a central role in the neuroendocrine control of reproduction. In contrast to humans and mammals that have a single Kiss1 gene and a single Kiss1r gene, multiple Kiss ligand and receptor genes are found in nonmammalian vertebrates. Their functional relationship, however, is poorly understood. Here, we report that the duplicated zebrafish kiss1r genes have evolved a distinct gene expression pattern, different ligand selectivity, and novel nuclear isoforms. While a single kiss1ra mRNA was detected exclusively in the brain, 5 kiss1rb transcripts were found in many peripheral tissues. Functional assays showed that kiss1ra encodes a receptor activated by both Kiss1 and Kiss2, while kiss1rb encodes a receptor that has a preference for Kiss1. The four alternatively spliced kiss1rb mRNAs encoded 4 truncated isoforms, denoted kiss1rb-derived protein (KRBDP)1-4. When their subcellular localization was examined, KRBDP3 and KRBDP4 were found in the nucleus in cultured mammalian cells and in zebrafish embryos. One-hybrid transcription activation assays revealed that KRBDP3, but not KRBDP4, possesses ligand-independent transactivation activity. These findings highlight how the duplication of Kiss1r genes may facilitate their adaptation of specialized functions. The discovery of a nuclear Kiss1r isoform raises the possibility of novel function of Kiss1r in the nucleus.

Differential regulation of IGF-I and IGF-II gene expression in skeletal muscle cells.

Insulin-like growth factor (IGF)-I and IGF-II play major roles in the regulation of skeletal muscle growth and differentiation, and both are locally expressed in muscle cells. Recent studies have demonstrated that IGF-II up-regulates its own gene expression during myogenesis and this auto-regulatory loop is critical for muscle differentiation. How local IGF-I is regulated in this process is unclear. Here, we report that while IGF-II up-regulated its own gene expression, it suppressed IGF-I gene expression during myogenesis. These opposite effects of IGF-II on IGF-I and IGF-II genes expression were time dependent and dose dependent. It has been shown that IGFs activate the PI3K-Akt-mTOR, p38 MAPK, and Erk1/2 MAPK pathways. In myoblasts, we examined their role(s) in mediating the opposite effects of IGF-II. Our results showed that both the PI3K-Akt-mTOR and p38 MAPK pathways played critical roles in increasing IGF-II mRNA expression. In contrast, mTOR was required for down-regulating the IGF-I gene expression by IGF-II. In addition, Akt, Erk1/2 MAPK, and p38 MAPK pathways were also involved in the regulation of basal levels of IGF-I and IGF-II genes during myogenesis. These findings reveal a previously unrecognized negative feedback mechanism and extend our knowledge of IGF-I and IGF-II gene expression and regulation during myogenesis.

Hypoxia converts the myogenic action of insulin-like growth factors into mitogenic action by differentially regulating multiple signaling pathways.

Insulin-like growth factors (IGFs) stimulate myoblast proliferation and differentiation. It remains elusive how these mutually exclusive cellular responses are elicited by the same growth factor. Here we report that whereas IGF promotes myoblast differentiation under normoxia, it stimulates proliferation under hypoxia. Hypoxia activates the HIF-1 transcriptional program and knockdown of HIF-1alpha changes the mitogenic action of IGF into myogenic action under hypoxia. Conversely, overexpression of HIF-1alpha abolishes the myogenic effect of IGF under normoxia. Under normoxia, IGF activates the Akt-mTOR, p38, and Erk1/2 MAPK pathways. Hypoxia suppresses basal and IGF-induced Akt-mTOR and p38 activity, whereas it enhances and prolongs IGF-induced Erk1/2 activation in a HIF-1-dependent fashion. Activation of Akt-mTOR and p38 promotes myogenesis, and p38 also inhibits proliferation. Activation of Erk stimulates myoblast proliferation but inhibits differentiation. These results suggest that hypoxia converts the myogenic action of IGFs into mitogenic action by differentially regulating multiple signaling pathways via HIF-1-dependent mechanisms. Our findings provide a mechanistic explanation for the paradoxical actions of IGFs during myogenesis and reveal a novel mechanism by which cells sense and integrate growth factor signals and oxygen availability in their microenvironments.

Stanniocalcin 1a regulates organismal calcium balance and survival by suppressing Trpv6 expression and inhibiting IGF signaling in zebrafish.

Stanniocalcin 1 (Stc1) is well known for its role in regulating calcium uptake in fish by acting on ionocytes or NaR cells. A hallmark of NaR cells is the expression of Trpv6, a constitutively open calcium channel. Recent studies in zebrafish suggest that genetical deletion of Stc1a and Trpv6 individually both increases IGF signaling and NaR cell proliferation. While trpv6-/- fish suffered from calcium deficiency and died prematurely, stc1a-/- fish had elevated body calcium levels but also died prematurely. The relationship between Stc1a, Trpv6, and IGF signaling in regulating calcium homeostasis and organismal survival is unclear. Here we report that loss of Stc1a increases Trpv6 expression in NaR cells in an IGF signaling-dependent manner. Treatment with CdCl2, a Trpv6 inhibitor, reduced NaR cell number in stc1a -/- fish to the sibling levels. Genetic and biochemical analysis results suggest that Stc1a and Trpv6 regulate NaR cell proliferation via the same IGF pathway. Alizarin red staining detected abnormal calcium deposits in the yolk sac region and kidney stone-like structures in stc1a -/- fish. Double knockout or pharmacological inhibition of Trpv6 alleviated these phenotypes, suggesting that Stc1a inhibit epithelial Ca2+ uptake by regulating Trpv6 expression and activity. stc1a-/- mutant fish developed cardiac edema, body swelling, and died prematurely. Treatment of stc1a-/- fish with CdCl2 or double knockout of Trpv6 alleviated these phenotypes. These results provide evidence that Stc1a regulates calcium homeostasis and organismal survival by suppressing Trpv6 expression and inhibiting IGF signaling in ionocytes.

FK506-binding protein 5 regulates cell quiescence-proliferation decision in zebrafish epithelium.

Using a zebrafish ionocyte model, transcriptomics and genetic analyses were performed to identify pathways and genes involved in cell quiescence-proliferation regulation. Gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses revealed that genes involved in transcription regulation, cell cycle, Foxo signalling and Wnt signalling pathway are enriched among the up-regulated genes while those involved in ion transport, cell adhesion and oxidation-reduction are enriched among the down-regulated genes. Among the top up-regulated genes is FK506-binding protein 5 (Fkbp5). Genetic deletion and pharmacological inhibition of Fkbp5 abolished ionocyte reactivation and impaired Akt signalling. Forced expression of a constitutively active form of Akt rescued the defects caused by Fkbp5 inhibition. These results uncover a key role of Fbkp5 in regulating the quiescence-proliferation decision via Akt signalling.

Regulation of temporal and spatial organization of newborn GnRH neurons by IGF signaling in zebrafish.

When and how newborn neurons are organized to form a functional network in the developing brain remains poorly understood. An attractive model is the gonadotropin-releasing hormone (GnRH) neuron system, master regulator of the reproductive axis. Here we show that blockage of IGF signaling, a central growth-promoting signaling pathway, by the induced expression of a dominant-negative form of IGF1 receptor (IGF1R) or specific IGF1R inhibitors delayed the emergence of GnRH2 neurons in the midbrain and GnRH3 neurons in the olfactory bulb region. Blockage of IGF signaling also resulted in an abnormal appearance of GnRH3 neurons outside of the olfactory bulb region, although it did not change the locations of other olfactory neurons, GnRH2 neurons, or brain patterning. This IGF action is developmental stage-dependent because the blockade of IGF signaling in advanced embryos had no such effect. An application of phosphatidylinositol 3-kinase (PI3K) inhibitors phenocopied the IGF signaling deficient embryos, whereas the MAPK inhibitors had no effect, suggesting that this IGF action is mediated through the PI3K pathway. Real-time in vivo imaging studies revealed that the ectopic GnRH3 neurons emerged at the same time as the normal GnRH3 neurons in IGF-deficient embryos. Further experiments suggest that IGF signaling affects the spatial distribution of newborn GnRH3 neurons by influencing neural crest cell migration and/or differentiation. These results suggest that the IGF-IGF1R-PI3K pathway regulates the precise temporal and spatial organization of GnRH neurons in zebrafish and provides new insights into the regulation of GnRH neuron development.

IGF binding protein-6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis.

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here, we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a hypoxia-inducible factor (HIF)-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and vascular endothelial growth factor (VEGF), a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ∼40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.

Molecular, functional, and gene expression analysis of zebrafish hypoxia-inducible factor-3α.

Hypoxia-inducible factors 1-3 (HIF1-3) are transcription factors that regulate gene expression in response to hypoxia. Compared with our extensive understanding of HIF-1 and HIF-2, our knowledge of HIF-3 is limited. In this study, we characterized the zebrafish hif-3α gene and determined its temporal and spatial expression, physiological regulation, and biological activity. We show that the chromosomal location, gene structure, and protein structure of zebrafish hif-3α are similar to its mammalian orthologs. When tagged with enhanced green fluorescent protein and transfected into cultured cells, zebrafish Hif-3α was localized in the nucleus and stimulated reporter gene expression in a hypoxia response element-dependent manner. During early development, hif-3α mRNA was detected in all tissues with higher levels in the head. This expression pattern became more apparent in larvae at the 72, 96, and 120 hours post fertilization stages. In the adult stage, hif-3α mRNA was detected in all examined tissues with the highest levels in the ovary. Hypoxia treatment increased Hif-3α protein levels in both embryos and adults. Hypoxia also increased hif-3α mRNA expression levels, and this regulation was tissue-specific. Expression of a stabilized form of Hif-1α in zebrafish embryos increased the expression of igfbp-1a, a Hif-1 target gene, whereas it did not change hif-3α mRNA levels, suggesting that hif-3α is not a Hif-1α target. These results provide new information about the structural and functional conservation, spatial and temporal expression, and physiological regulation of hif-3α in a teleost model organism.

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.