GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

While the basic cellular contributions to bone differentiation and mineralization are widely accepted, the regulation of these processes at the intracellular level remains inadequately understood. Our laboratory recently identified annexin 2 as a protein involved in osteoblastic mineralization. Annexin 2 was overexpressed twofold in SaOSLM2 osteoblastic cells as a fusion protein with green fluorescent protein. The overexpression of annexin 2 led to an increase in alkaline phosphatase activity as well as an increase in mineralization. Our data suggest that the increase in alkaline phosphatase activity does not result from increased alkaline phosphatase transcript or protein levels; therefore we evaluated mechanism of action. We determined that both annexin 2 and alkaline phosphatase activity were localized to membrane microdomains called lipid rafts in osteoblastic cells. Annexin 2 overexpression resulted in an increase in alkaline phosphatase activity that was associated with lipid microdomains in a cholesterol-dependent manner. Furthermore, disruption of lipid rafts with a cholesterol sequestering agent or reduction of annexin 2 expression by specific antisense oligonucleotides each resulted in diminished mineralization. Therefore, intact lipid rafts containing annexin 2 appear to be important for alkaline phosphatase activity and may facilitate the osteoblastic mineralization process.

Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adults. The development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. We used representational difference analysis to isolate genes differentially expressed between primary human osteosarcoma tumors and subsequent metastatic lung lesions to identify genes potentially involved in metastatic potential. Several genes were differentially expressed between the two tumor populations, including annexin2. The levels of annexin2 mRNA and protein inversely correlated with metastatic potential in a subset of human osteosarcoma tumor specimens, as well as in a human osteosarcoma cell line selected for increased metastatic potential. Annexin2 has been described in several cellular localizations with various functional implications, many of which may be relevant to metastatic potential. Therefore, the subcellular localization of endogenous annexin2 protein was evaluated biochemically by subcellular fractionation and immunologically by flow cytometry and immunofluorescence in osteoblastic cells. Annexin2 was localized to the cytoplasm and intracellular aspect of the plasma membrane, excluded from the nucleus and undetectable on the cell surface or in the conditioned medium. Overexpression of annexin2 in osteosarcoma cells did not alter several in vitro phenotypes often used to assess metastatic potential including motility, adhesion, and proliferation. However, our previous data have implicated annexin2 in the mineralization process of osteoblastic cells in vitro. Consistent with an increase in differentiation-induced mineralization, there was diminished tumorigenicity and experimental metastatic potential of osteosarcoma cells overexpressing annexin2. These data suggest that annexin2 may downregulate osteosarcoma aggressiveness by inducing a more differentiated state in osteoblastic cells.