Lipid-independent secretion of a Drosophila Wnt protein

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.     

Hypoxia impairs primordial germ cell migration in zebrafish (Danio rerio) embryos

Background

As a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear.

Methodology/Principal Findings

In the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration.

Conclusions/Significance

This study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1.     

Genetic interactions between Cdk1-CyclinB and the Separase complex in Drosophila

Cdk1-CycB plays a key role in regulating many aspects of cell-cycle events, such as cytoskeletal dynamics and chromosome behavior during mitosis. To investigate how Cdk1-CycB controls the coordination of these events, we performed a dosage-sensitive genetic screen, which is based on the observations that increased maternal CycB (four extra gene copies) leads to higher Cdk1-CycB activity in early Drosophila embryos, delays anaphase onset, and generates a sensitized non-lethal phenotype at the blastoderm stage (defined as six cycB phenotype). Here, we report that mutations in the gene three rows (thr) enhance, while mutations in pimples (pim, encoding Drosophila Securin) or separase (Sse) suppress, the sensitized phenotype. In Drosophila, both Pim and Thr are known to regulate Sse activity, and activated Sse cleaves a Cohesin subunit to initiate anaphase. Compared with the six cycB embryos, reducing Thr in embryos with more CycB further delays the initiation of anaphase, whereas reducing either Pim or Sse has the opposite effect. Furthermore, nuclei move slower during cortical migration in embryos with higher Cdk1-CycB activity, whereas reducing either Pim or Sse suppresses this phenotype by causing a novel nuclear migration pattern. Therefore, our genetic screen has identified all three components of the complex that regulates sister chromatid separation, and our observations indicate that interactions between Cdk1-CycB and the Pim-Thr-Sse complex are dosage sensitive.     

The mechanism for primordial germ-cell migration is conserved between Japanese eel and zebrafish

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).     

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.     

Isoprenoids control germ cell migration downstream of HMGCoA reductase

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAr) provides attractive cues to Drosophila germ cells, guiding them toward the embryonic gonad. However, it remains unclear how HMGCoAr mediates this attraction. In a genomic analysis of the HMGCoAr pathway, we found that the fly genome lacks several enzymes required for cholesterol biosynthesis, ruling out cholesterol and cholesterol-derived proteins as mediators of PGC migration. Genetic analysis of the pathway revealed that two enzymes, farnesyl-diphosphate synthase and geranylgeranyl-diphosphate synthase, required for the production of isoprenoids, act downstream of HMGCoAr in germ cell migration. Consistent with a role in geranylgeranylation, embryos deficient in geranylgeranyl transferase type I show germ cell migration defects. Our data, together with similar findings in zebrafish, implicate an isoprenylated protein in germ cell attraction. The specificity and evolutionary conservation of the HMGCoAr pathway for germ cells suggest that an attractant common to invertebrates and vertebrates guides germ cells in early embryos.     

A Novel Piggybac Transposon Inducible Expression System Identifies a Role for Akt Signalling in Primordial Germ Cell Migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.     

The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation

The ter (teratoma) mutation causes primordial germ cell (PGC) deficiency in ter/ter embryos at 9.5–12.5 days of post-coitum (dpc) in mouse strains 129/Sv- ter and LTXBJ- ter . To study the effects of the ter mutation on the PGC development more precisely, we examined the PGC number and distribution in 7.5–12.5 dpc embryo of ter congenic C57BL/6J- ter strain using their complete serial sections. The ter genotypes of embryos were identified by the polymerase chain reaction (PCR) polymorphisms of the microsatellite DNA of the Grl -1 locus mapped near the ter locus. Results showed that: (i) the PGC number in ter/ter embryos was similar to those of + / ter and + / + embryos at 7.5 dpc, and did not increase at 8.0–12.5 dpc, although those of normal littermates did usually; (ii) the PGC migration to genital ridges was never affected in all embryos; and (iii) the ter genotype difference in the PGC numbers was not recognized between + / ter and + / + embryos. We concluded that the ter mutation does not affect the PGC appearance around 7.5 dpc, but first causes PGC deficiency around 8.0 dpc at the beginning of their migration and proliferation, suggesting that the normal function of the ter gene may be essential for the proliferation or survival mechanisms of PGC.     

Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival

Insulin-like growth factor (IGF) signaling is a critical regulator of somatic growth during fetal and adult development, primarily through its stimulatory effects on cell proliferation and survival. IGF signaling is also required for development of the reproductive system, although its precise role in this regard remains unclear. We have hypothesized that IGF signaling is required for embryonic germline development, which requires the specification and proliferation of primordial germ cells (PGCs) in an extragonadal location, followed by directed migration to the genital ridges. We tested this hypothesis using loss-of-function studies in the zebrafish embryo, which possesses two functional copies of the Type-1 IGF receptor gene (igf1ra, igf1rb). Knockdown of IGF1Rb by morpholino oligonucleotides (MO) results in mismigration and elimination of primordial germ cells (PGCs), resulting in fewer PGCs colonizing the genital ridges. In contrast, knockdown of IGF1Ra has no effect on PGC migration or number despite inducing widespread somatic cell apoptosis. Ablation of both receptors, using combined MO injections or overexpression of a dominant-negative IGF1R, yields embryos with a PGC-deficient phenotype similar to IGF1Rb knockdown. TUNEL analyses revealed that mismigrated PGCs in IGF1Rb-deficient embryos are eliminated by apoptosis; overexpression of an antiapoptotic gene (Bcl2l) rescues ectopic PGCs from apoptosis but fails to rescue migration defects. Lastly, we show that suppression of IGF signaling leads to quantitative changes in the expression of genes encoding CXCL-family chemokine ligands and receptors involved in PGC migration. Collectively, these data suggest a novel role for IGF signaling in early germline development, potentially via cross-talk with chemokine signaling pathways.     

split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells

Signaling by DER, the Drosophila epidermal growth factor receptor tyrosine kinase (RTK), is essential for proper migration and survival of midline glial cells (MGCs) in the embryonic central nervous system (CNS) [1-4]. We recently isolated a gene called split ends (spen) in a screen designed to identify new components of the RTK/Ras pathway [5]. Drosophila Spen and its orthologs are characterized by a distinct set of RNA recognition motifs (RRMs) and a SPOC domain, a highly conserved carboxy-terminal domain of unknown function [5-7]. To investigate spen function in the context of RTK signaling, we examined the consequences of spen loss-of-function mutations on embryonic CNS development. We found that spen was required for normal migration and survival of MGCs and that embryos lacking spen had CNS defects strikingly reminiscent of those seen in mutants of several known components of the DER signaling pathway. In addition, spen interacted synergistically with the RTK effector pointed. Using MGC-targeted expression, we found that increased Ras signaling rescued the lethality associated with expression of a dominant-negative spen transgene. Therefore, spen encodes a positively acting component of the DER/Ras signaling pathway.     

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.     

Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints

Background

Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors.

Results

We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally.

Conclusion

In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.     

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.     

Porcupine-mediated lipidation is required for Wnt recognition by Wls

Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.