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The cell proliferation-quiescence decision plays fundamental roles in tissue formation and regeneration, and its dysregulation can lead to human diseases. In this study, we performed transcriptomics and genetic analyses using a zebrafish model to identify pathways and genes involved in epithelial cell quiescence-proliferation regulation. In thisin vivomodel, a population of GFP-labeled epithelial cells known as ionocytes were induced to reenter the cell cycle by a physiological stress. Transcriptomics analysis identified 1168 genes up-regulated and 996 genes down-regulated in the reactivated cells. GO and KEGG pathway analyses revealed that genes involved in transcription regulation, cell cycle, Foxo signaling, and Wnt signaling 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), a member of the conserved immunophilin family. CRISPR/Cas9-mediated Fkbp5 deletion abolished ionocyte reactivation and proliferation.Pharmacological inhibition of Fkbp5 had similar effects. Further analyses showed that genetic deletion and inhibition of Fkbp5 impaired Akt signaling. Forced expression of a constitutively active form of Akt rescued the defects caused by Fkbp5 inhibition. These results uncover a previously unrecognized role of Fbkp5 in regulating the quiescence-proliferation decision via Akt signaling.Impact StatementTranscriptomic and genetic deletion studies unravel a new role of Fkbp5 in promoting cell reactivation via Akt signaling.HighlightsTranscriptomic analysis reveals several molecular pathways altered during epithelial cell quiescence-proliferation transition.Fkbp5 is highly up-regulated in reactivated and dividing cells.Fkbp5 promotes epithelial cell reactivation and proliferation via 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.

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.

The mechanisms governing cell quiescence-proliferation balance are poorly defined. Using a zebrafish model, here we report that Stc1a, a glycoprotein known as a hypocalcemic hormone, not only inhibits epithelial calcium uptake but also functions as a Ca2+-regulated switch controlling epithelial cell quiescence-proliferation balance. Among the 4stcgenes, only thestc1aexpression is [Ca2+]-dependent. Genetic deletion ofstc1a, but notstc2b, resulted in elevated body Ca2+contents, ectopic Ca2+deposit, body swelling, and premature death. Reducing epithelial calcium channel Trpv6-mediated Ca2+uptake alleviated these phenotypes. Loss of Stc1a also promoted quiescent epithelial cells to re-enter the cell cycle. This action was accompanied by local IGF signaling activation and increased expression inpapp-aa, a zinc metalloproteinase degrading Igfbp5a. Genetic deletion ofpapp-aaorigfbp5aabolished the elevated epithelial cell reactivation instc1a-/-mutants. Likewise, inhibition of IGF1 receptor, PI3 kinase, Akt, and Tor signaling abolished epithelial cell reactivation. These results reveal that Stc1a plays dual roles in regulating epithelial calcium uptake and cell quiescence-proliferation balance and implicate Trpv6 and Papp-aa-Igfbp5a-IGF signaling in these functions.

Hif-3α, a member of the hypoxia-inducible factor (HIF) family, enters the nucleus and regulates gene expression in response to hypoxia. The molecular basis of its nuclear localization is not clear. HIF-1α and HIF-2α use a bipartite nuclear localization signal (NLS) to enter the nucleus. This motif is not conserved in Hif-3α. Although there is a conserved Arg/Lys rich motif in the Hif-3α N-terminal region, deletion of this region has minimal effect on Hif-3α nuclear localization. Here, we mapped the functional NLS to the unique C-terminal region of Hif-3α and identified two clusters of basic residues critical for its nuclear localization. The two NLS motifs are functionally redundant. Our results, thus, suggest that Hif-3α nuclear localization is mediated through two redundant NLS motifs located in its unique C-terminal region.

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.

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.

Human patients carrying inactivating mutations in the pregnancy-associated plasma protein-a2 (PAPP-A2) gene display short status and lower bone mineral density. The underlying mechanisms are not well understood. Using a zebrafish model, here we report a [Ca2+]-dependent mechanism by which Papp-aa regulates bone calcification via promoting Ca2+-transporting epithelial cell (ionocyte) reactivation. Ionocyte, normally quiescent, re-enter the cell cycle in response to low [Ca2+] stress. Deletion of Papp-aa abolished ionocyte reactivation and resulted in a complete lack of calcified bone. Re-expression of Papp-aa, but not its active site mutant, rescued ionocyte reactivation. Inhibition of Papp-aa activity pharmacologically or by overexpressing STC1 or STC2 impaired ionocyte reactivation. Loss of Papp-aa expression or activity resulted in diminished IGF1 receptor-mediated Akt-Tor signaling activity in ionocytes and expression of a constitutively active Akt rescued ionocyte reactivation. Biochemically, Papp-aa cleaved Igfbp5a, a high-affinity IGF binding protein specifically expressed in ionocytes. Under normal [Ca2+] conditions, the Papp-aa-mediated Igfbp5a proteolysis was suppressed and IGFs sequestered in the IGF/Igfbp5a complex. Forced release of IGFs from the complex was sufficient to activate the IGF-Akt-Tor signaling and promote ionocyte reactivation. These findings suggest that Papp-aa functions as a [Ca2+]-regulated molecular switch linking IGF signaling to adaptive epithelial growth and bone calcification.

Epithelial homeostasis and regeneration require a pool of quiescent cells. How the quiescent cells are established and maintained is poorly understood. Here we report that Trpv6, a cation channel responsible for epithelial Ca2+ absorption, functions as a key regulator of cellular quiescence. Genetic deletion and pharmacological blockade of Trpv6 promoted zebrafish epithelial cells to exit from quiescence and re-enter the cell cycle. Reintroducing Trpv6, but not its channel dead mutant, restored the quiescent state. Ca2+ imaging showed that Trpv6 is constitutively open in vivo. Mechanistically, Trpv6-mediated Ca2+ influx maintained the quiescent state by suppressing insulin-like growth factor (IGF)-mediated Akt and Tor signaling. In zebrafish epithelia and human colon cancer cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, which down-regulated IGF signaling and promoted the quiescent state. Our findings suggest that Trpv6 mediates constitutive Ca2+ influx into epithelial cells to continuously suppress growth factor signaling and maintain the quiescent state.

The phenotype gap is a challenge for genetically dissecting redundant endocrine signaling pathways, such as the six isoforms in the insulin-like growth factor binding protein (IGFBP) family. Although overexpressed IGFBPs can inhibit or potentiate IGF actions or have IGF-independent actions, mutant mice lacking IGFBP-encoding genes do not exhibit major phenotypes. We found that although zebrafish deficient in

The phenotype gap is a challenge for genetically dissecting redundant endocrine signaling pathways, such as the six isoforms in the insulin-like growth factor binding protein (IGFBP) family. Although overexpressed IGFBPs can inhibit or potentiate IGF actions or have IGF-independent actions, mutant mice lacking IGFBP-encoding genes do not exhibit major phenotypes. We found that although zebrafish deficient in igfbp5a did not show overt phenotypes when raised in Ca2+-rich solutions, they died prematurely in low Ca2+ conditions. A group of epithelial cells expressing igfbp5a take up Ca2+ and proliferate under low Ca2+ conditions because of activation of IGF signaling. Deletion of igfbp5a blunted low Ca2+ stress-induced IGF signaling and impaired adaptive proliferation. Reintroducing zebrafish Igfbp5a, but not its ligand binding-deficient mutant, restored adaptive proliferation. Similarly, adaptive proliferation was restored in zebrafish lacking igfbp5a by expression of human IGFBP5, but not two cancer-associated IGFBP5 mutants. Knockdown of IGFBP5 in human colon carcinoma cells resulted in reduced IGF-stimulated cell proliferation. These results reveal a conserved mechanism by which a locally expressed Igfbp regulates organismal Ca2+ homeostasis and survival by activating IGF signaling in epithelial cells and promoting their proliferation in Ca2+-deficient states. These findings underscore the importance of physiological context when analyzing loss-of-function phenotypes of endocrine factors.

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 β-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-β. 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.

Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia. Here we describe a simple method to induce hypoxia in zebrafish embryos and larvae. This protocol is easy and reproducible and does not require expensive equipment or specialized devices. It can be adapted in large, medium, and small scales. This protocol is also well-suited for experiments requiring chemical drug treatment and can be applied to other fish and amphibian species.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia. Here we describe a simple method to induce hypoxia in zebrafish embryos and larvae. This protocol is easy and reproducible and does not require expensive equipment or specialized devices. It can be adapted in large, medium, and small scales. This protocol is also well-suited for experiments requiring chemical drug treatment and can be applied to other fish and amphibian species.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as "catch-up" growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1-mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia.

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.

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. 775KKARL functioned as the primary NLS, but 737KRK and 754KK 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.—Gao, S., Lu, L., Bai, Y., Zhang, P., Song, W., Duan, C. Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family.

IGFs play key roles in regulating vertebrate development, growth, reproduction, and aging. In extracellular fluids, IGFs are bound and regulated by a family of IGF-binding proteins (IGFBPs). Although all known IGFBPs are secreted proteins, some are also found in the nucleus and possess IGF-independent activities. When and how these distinct modes of biological actions have evolved is unknown. In this study, we identified and analyzed an IGFBP gene from amphioxus. Amphioxus shares a common ancestor with the modern vertebrate lineage that dates back to more than 520 million years ago. The amphioxus IGFBP shares all major structural characteristics of vertebrate IGFBPs. Phylogenetic analyses place it in a basal position in the IGFBP lineage. Ligand blot analysis reveals that amphioxus IGFBP does not bind to IGF-I or -II. Changing its Phe70 into Leu, however, is sufficient to convert it into a functional IGF binder. When tested in cultured cells, amphioxus IGFBP is localized in the nucleus, and this is attributed to 2 redundant nuclear localization sequences in its L domain. Furthermore, the amphioxus IGFBP N-terminal domain has strong transcriptional activation activity. Forced expression of amphioxus IGFBP in zebrafish embryos results in dorsalized phenotypes. This action requires nuclear localization. These results suggest that the nuclear localization and transcription activation activity of IGFBPs are ancient functions and the IGF-binding function may have been acquired by opportunistic gain-of-functional mutations later in evolution.

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.

This study demonstrates inducible transgenic expression in the exceptionally short-lived turquoise killifish Nothobranchius furzeri, which is a useful vertebrate model for ageing research. Transgenic N. furzeri bearing a green fluorescent protein (Gfp) containing construct under the control of a heat shock protein 70 promoter were generated, heat shock-induced and reversible Gfp expression was demonstrated and germline transmission of the transgene to the F1 and F2 generations was achieved. The availability of this inducible transgenic expression system will make the study of ageing-related antagonistically pleiotropic genes possible using this unique vertebrate model organism.

Insulin-like growth factor-binding proteins (IGFBPs) are multifunctional proteins that possess IGF-dependent and -independent actions. Recent studies suggest that its IGF-independent action appeared early and that the IGF-binding function may have been acquired later in evolution. The timing of the emergence of IGF-dependent actions is unclear. Here, we identified and characterized an igfbp gene from sea lamprey, an agnathan, which was separated from the jawed vertebrates 450 million years ago. Phylogenetic and structural analyses suggested that the encoded protein belongs to the IGFBP-3 clade in the IGFBP family. Lamprey IGFBP-3 contains an IGF-binding domain (IBD), nuclear localization signal, and transactivation (TA) domain. Biochemical and functional analyses showed that these domains are all functional. Lamprey IGFBP-3 can bind IGFs and modulate IGF signaling when tested in mammalian cells. Lamprey IGFBP-3 also has the capacity to enter the nucleus and has strong TA activity. Forced expression of lamprey IGFBP-3, but not its IBD mutant, in zebrafish embryos decreased body growth and developmental speed. Lamprey IGFBP-3 inhibited BMP2 signaling in cultured cells and in zebrafish embryos, and this action is independent of its IGF-binding function. These results suggest that lamprey IGFBP-3 has both IGF-dependent and -independent actions and provide new insights into the functional evolution of the IGFBP family.

Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great in vivo complexities inherited in this pathway, there is an unmet need for whole organism based assays. Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca2+] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.

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.

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.

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-timein vivoimaging 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.

The insulin-like growth factor (IGF) signaling pathway consists of multiple IGF ligands, IGF receptors, and IGF-binding proteins (IGFBPs). Studies in a variety of animal and cellular systems suggest that the IGF signaling pathway plays a key role in regulating skeletal muscle growth, differentiation, and in maintaining homeostasis of the adult muscle tissues. Intriguingly, IGFs stimulate both myoblast proliferation and differentiation, which are two mutually exclusive biological events during myogenesis. Both of these actions are mediated through the same IGF-1 receptor. Recent studies have shed new insights into the molecular mechanisms underlying these paradoxical actions of IGFs in muscle cells. In this article, we provide a brief review of our current understanding of the IGF signaling system and discuss recent findings on how local oxygen availability and IGFBPs act to specify IGF actions in muscle cells.