Cell Biology

The ankyrin family of proteins form specialized membrane domains in various cell types, including neurons and cardiomyocytes but little is known about how this process occurs. The majority of ankyrins localize to the plasma membrane in these cells while ankyrins in fibroblasts are largely intracellular. This property of fibroblasts allows us to study intracellular ankyrin and potentially how ankyrin forms membrane domains in other cell types. In this thesis, we use western blots, immunofluorescence, RT-PCR to characterize the expression pattern of ankyrin in fibroblasts and find that both ankyrin-B and-G localize to both nuclear and intracellular compartments. The size of the compartments of both ankyrin-B and-G is affected by the genetic deletion of the large isoforms of ankyrin-B and-G. The ankyrin-B compartment has a weak association with recycling endosomes suggesting that ankyrin-B may be involved in membrane protein trafficking.

The ankyrin family of proteins form specialized membrane domains in various cell types, including neurons and cardiomyocytes but little is known about how this process occurs. The majority of ankyrins localize to the plasma membrane in these cells while ankyrins in fibroblasts are largely intracellular. This property of fibroblasts allows us to study intracellular ankyrin and potentially how ankyrin forms membrane domains in other cell types. In this thesis, we use western blots, immunofluorescence, RT-PCR to characterize the expression pattern of ankyrin in fibroblasts and find that both ankyrin-B and-G localize to both nuclear and intracellular compartments. The size of the compartments of both ankyrin-B and-G is affected by the genetic deletion of the large isoforms of ankyrin-B and-G. The ankyrin-B compartment has a weak association with recycling endosomes suggesting that ankyrin-B may be involved in membrane protein trafficking.

The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise approximately 30% of the transcriptome, the localization of mRNAs to the ER represents an extraordinarily prominent, ubiquitous, and yet poorly understood RNA localization phenomenon.

In current views, the mRNA transcriptome is partitioned between the cytosol and endoplasmic reticulum (ER) compartments by binary selection; mRNAs encoding cytosolic/nucleoplasmic proteins are translated on free ribosomes and mRNAs encoding topogenic signal-bearing proteins are translated on ER-bound ribosomes, with ER localization being conferred by the signal recognition particle pathway. In sub-genomic and genomic analyses of subcellular mRNA partitioning, we report an overlapping subcellular distribution of cytosolic/nucleoplasmic and topogenic signal-encoding mRNAs, with mRNAs of both cohorts displaying non-canonical subcellular partitioning patterns. Unexpectedly, the topogenic signal-encoding mRNA transcriptome was observed to partition in a hierarchical, cohort-specific manner. mRNAs encoding resident proteins of the endomembrane system were clustered at high ER enrichment values whereas mRNAs encoding secretory pathway cargo were broadly represented on free and ER-bound ribosomes. Two distinct modes of mRNA association with the ER were identified. mRNAs encoding endomembrane resident proteins were bound via direct, ribosome-independent interactions whereas mRNAs encoding secretory cargo displayed predominately ribosomedependent modes of ER association. These data indicate that mRNAs are partitioned between the cytosol and ER compartments via a hierarchical system of intrinsic and encoded topogenic signals and identify mRNA cohort-restricted modes of mRNA association with the ER.

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1(Ter/Ter)) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1(Ter/Ter) embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1(Ter/Ter) XX germ cells developed in a testi...

Testes and ovaries undergo sex-specific morphogenetic changes and adopt strikingly different morphologies, despite the fact that both arise from a common precursor, the bipotential gonad. Previous studies showed that recruitment of vasculature is critical for testis patterning. However, vasculature is not recruited into the early ovary. Peripheral innervation is involved in patterning development of many organs but has been given little attention in gonad development. In this study, we show that while innervation in the male reproductive complex is restricted to the epididymis and vas deferens and never invades the interior of the testis, neural crest-derived innervation invades the interior of the ovary around E16.5. Individual neural crest cells colonize the ovary, differentiate into neurons and glia, and form a dense neural network within the ovarian medulla. Using a sex-reversing mutant mouse line, we show that innervation is specific to ovary development, is not dependent on th...

STUDY QUESTION Can host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy? SUMMARY ANSWER We found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter. WHAT IS KNOWN ALREADY In women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary. STUDY DESIGN, SIZE, DURATION We first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of t...

After their arrival in the fetal gonad, mammalian germ cells express E-cadherin and are found in large clusters, similar to germ cell cysts in Drosophila. In Drosophila, germ cells in cysts are connected by ring canals. Several molecular components of intercellular bridges in mammalian cells have been identified, including TEX14, a protein required for the stabilization of intercellular bridges, and several associated proteins that are components of the cytokinesis complex. This has led to the hypothesis that germ cell clusters in the mammalian gonad arise through incomplete cell divisions. We tested this hypothesis by generating chimeras between GFP-positive and GFP-negative mice. We show that germ cell clusters in the fetal gonad arise through aggregation as well as cell division. Intercellular bridges, however, are likely restricted to cells of the same genotype.

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and perform...

Husbandry staff noticed a research-naïve, young-adult, female finch tossing its head back intermittently. A second finch exhibiting similar signs was reported a few days later. Postmortem necropsy and histopathology with hematoxylin and eosin and acid-fast staining on the first finch revealed the presence of acid-fast organisms in several organs. After presumptive diagnosis of mycobacteriosis, all remaining finches housed in the same room as the first underwent necropsy and histology. Three additional finches were positive for Mycobacterium-like acid-fast organisms. Incidental findings of megabacteriosis were noted histopathologically on 2 other finches.

ABSTRACTCryptococcus neoformansis an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense againstC. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective againstC. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected withC. neoformanshave a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility toC. neoformansby dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D−/−mice afterC. neoformansinfection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleu...