TH and XFKBP12 interact and regulate proliferation and differentiation in neural ectoderm through the TOR signaling pathway

During development, embryos must carefully integrate the processes of cell proliferation and differentiation. TH has been identified in Xenopus laevis as a gene product that functions in regulating differentiation of the neural ectoderm through its effect on cell proliferation. However, the mechanism and molecular pathway through which TH functions are not known. We identified the Xenopus FK506 binding protein homolog (XFKBP12) as a protein that interacted with TH in a yeast two-hybrid screen with TH as the bait. The direct and specific interaction between TH and XFKBP12 was supported by several tests including CO-IP, drug competence assay and mutagenesis analysis. To investigate the function of XFKBP12 during embryogenesis, we created an XFKBP12 loss of function embryo using antisense morpholino oligonucleotides (MO). XFKBP12 MO injected embryos displayed similar phenotypes as TH depleted embryos. We also demonstrated that both TH and XFKBP12 functioned through the TOR signaling pathway which is a target for cancer therapies. The interaction between TH and XFKBP 12 was required to regulate the proliferation of neural cells. Therefore, our study indicates that TH represents the endogenous ligand of XFKBP12 and together they coordinate neural cell proliferation and differentiation through the conserved rapamycin sensitive TOR pathway. Thus, understanding how this pathway functions in development will not only provide us important insights into the relationship between proliferation and differentiation, but help design rational cancer therapies targeting this pathway. ^

Identification and characterization of Xenopus Kazrin, a nucleocytoplasmic shuttling protein that interacts with ARVCF catenin

In common with other members of the p120-catenin subclass of catenins, ARVCF-catenin appears to have multiple cellular and developmental functions. In Xenopus, our lab recently demonstrated that xARVCF- and Xp120-catenins are each essential for early vertebrate embryogenesis, being functionally linked to Rho-family GTPases (RhoA, Rac) and cadherin metabolic stability. For the project described here, the yeast two-hybrid system was employed to screen a Xenopus laevis neurula library for proteins that interact with xARVCF, resulting in the identification of the Xenopus homolog of Kazrin (xKazrin). Kazrin is a variably-spliced protein of unknown function that has been shown to interact with periplakin and envoplakin, components of desmosomal junctions. Kazrin's primary sequence is highly conserved across vertebrate species and is composed of an amino-terminal nuclear export sequence (NES), a carboxy-terminal nuclear localization sequence (NLS) and a central predicted coiled-coil domain. In vitro and in vivo authenticity tests demonstrated that xARVCF-catenin interacts directly with xKazrin via xARVCF's Armadillo and carboxy-terminal regions and xKazrin's coiled-coil domain. The interaction of xARVCF-catenin with xKazrin is specific and does not extend to the related Xp120-catenin. xKazrin co-localized with E-cadherin at sites of cell-cell contact and could be co-immunoprecipitated with components of the cadherin complex. xKazrin was also present in the cytoplasm and nucleus. Suggestive of a nuclear role, mutation of xKazrin's predicted NLS resulted in nuclear exclusion, while deletion of the predicted NES resulted in loss of sensitivity to nuclear export inhibitors. Within Xenopus embryos, xKazrin was expressed across all developmental stages and appeared at varying levels in adult tissues. Morpholino depletion of xKazrin from Xenopus embryos resulted in axial elongation abnormalities and loss of tissue integrity after neurulation. Over-expression of xKazrin had no effect, while over-expression of a NLS mutant resulted in a mild phenotype similar to that seen in xKazrin depleted embryos. Interestingly, the axial phenotype resulting from reduced xKazrin levels was largely rescuable by xARVCF over-expression. In conjunction with xARVCF-catenin, xKazrin has properties consistent with its function at cell-cell contact sites and in the nucleus. ^

The p120-catenin/Kaiso signaling pathway

The canonical and non-canonical Wnt signaling pathways appear to interact with one another as a network in development, or when hyper-activated, in the progression of disease. A much studied key mediator of the canonical Wnt pathway, β-catenin, is characterized by a central armadillo-repeat domain that engages in multiple protein-protein interactions, such as those with cadherins functioning at cell-cell contact regions. In the nucleus, β-catenin forms a complex with the repressor TCF/LEF, promoting the activation of genes participating in processes such as proliferation, differentiation and stem cell survival. Somewhat similarly, the p120-catenin binds the distinct transcriptional repressor Kaiso, relieving Kaiso-mediated repression to promote gene activation. Here, employing Xenopus laevis, I report upon both downstream and upstream aspects of the p120-catenin/Kaiso pathway which was previously poorly understood. I first show that Kaiso, a BTB/POZ zinc-finger family member, directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1 and c-Myc) in conjunction with TCF. Depletion or dominant-negative inhibition of xKaiso results in Siamois de-repression, while xKaiso over-expression induces additional Siamois repression through recruitment of N-CoR co-repressor and chromatin modifications. Functional interdependencies are further corroborated by the capacity of Kaiso to suppress β-catenin-induced axis duplication. Thus, my work inter-relates the p120-catenin/Kaiso and β-catenin/TCF pathways at the level of specific gene promoters important in development and cancer progression. Regarding upstream aspects of the p120-catenin/Kaiso pathway, I collaboratively identified p120 in association with Frodo, a protein previously identified as a component of the canonical (β-catenin dependent) Wnt pathway. I determined that canonical Wnt signals result in Frodo-mediated stabilization of p120-catenin, resulting in the sequestration of Kaiso to the cytoplasm and thereby the activation (relief of repression) of gene targets. Developmental evidence supporting this view included findings that Frodo has the capacity to partially rescue Kaiso over-expression phenotypes in early Xenopus embryos. Taken together, my studies point to the convergence of p120-catenin/Kaiso and β-catenin/TCF signaling pathways at the level of gene transcription as well as at more upstream points during vertebrate development. ^

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

Analysis of Wnt/beta-catenin signaling in kidney tubulogenesis

The β-catenin/Lef/Tcf-mediated Wnt pathway is central to the developmental of all animals, stem cell renewal, and cancer progression. Prior studies in frogs and mice have indicated that the ligand Wnt-4 is essential for the mesenchyme to epithelial transition that generates tubules in the context of kidney organogenesis. More recently, Wnt-9b in mice, was likewise found to be required. Yet despite the importance of Wnt signals in renal development, the corresponding Frizzled receptor(s) and downstream signaling mechanim(s) are unclear. My work addresses these knowledge gaps using in vitro (Madin-Darby Canine Kidney cells) and in vivo (Xenopus laevis and zebrafish pronephros) tubulogenic kidney model systems. Employing established reporter constructs of Wnt/β-catenin pathway activity, I have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions. I have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/β-catenin pathway using β-Engrailed and dnTCF-4, constructs that suppress this pathway. I have further found that MDCK cells express the Frizzled-6 receptor, and that Wnt-4 forms a biochemical complex with Frizzled-6, yet does not appear to transduce Wnt-4's canonical signal. Additionally, I demonstrate that standard Hepatocyte Growth Factor (HGF)-mediated (non-physiologic) induction of MDCK tubulogenesis in collagen matrices is not altered by activation or suppression of β-catenin signaling activity; however, β-catenin signaling maintains cell survival in this in vitro system. Using a Wnt/β-catenin signaling reporter in Xenopus laevis, I detect β-catenin signaling activity in the early pronephric epithelial kidney tubules. By inhibiting the Wnt/β-catenin signaling pathway in both zebrafish and Xenopus , a significant loss of kidney tubulogenesis is observed with little or no effect on adjoining axis or somite development. This inhibition also leads to the appearance of severe edema that phenocopies embryos depleted for Wnt-4. Tubulogenic loss does not appear to be caused by increased cell death in the Xenopus pronephric field, but rather by lessened expression of tubule epithelium genes associated with cellular differentiation. Together, my results show that Wnt/β-catenin signaling is required for renal tubule development and that Wnt-4 is a strong candidate for activating this pathway. ^

Modulation of high voltage -activated calcium channels by phosphorylation

High voltage-activated (HVA) calcium channels from rat brain and rabbit heart are expressed in Xenopus laevis oocytes and their modulation by protein kinases studied. A subtype of the HVA calcium current expressed by rat brain RNA is potentiated by the phospholipid- and calcium-dependent protein kinase (PKC). The calcium channel clone $\alpha\sb{\rm1C}$ from rabbit heart is modulated by the cAMP-dependent protein kinase (PKA), and another factor present in the cytoplasm.^ The HVA calcium channels from rat brain do not belong to the L-type subclass since they are insensensitive to dihydropyridine (DHP) agonists and antagonists. The expressed currents do contain a N-type fraction which is identified by inactivation at depolarized potentials, and a P-type fraction as defined by blockade by the venom of the funnel web spider Agelenopsis Aperta. A non N-type fraction of this current is potentiated, by using phorbol esters to activate PKC. This residual fraction of current resembles the newly described Q-type channel from cerebellar granule cells in its biophysical properties, and potentiation by activation of PKC.^ The $\alpha\sb{\rm1C}$ clone from rabbit heart is expressed in oocytes and single-channel currents are measured using the cell-attached and cell-excised patch clamp technique. The single-channel current runs down within two minutes after patch excision into normal saline bath solution. The catalytic subunit of PKA + MgATP is capable of reversing this rundown for over 15 minutes. There also appears to be an additional factor present in the cytoplasm necessary for channel activity as revealed in experiments where PKA failed to prevent rundown.^ These data are important in that these types of channels are involved in synaptic transmission at many different types of synapses. The mammalian synapse is not accessible for these types of studies, however, the oocyte expression system allows access to HVA calcium channels for the study of their modulation by phosphorylation. ^

Identification and characterization of the vegetally localized Hermes mRNA

One way developing embryos regulate the expression of their genes is by localizing mRNAs to specific subcellular regions. In the oocyte of the frog, Xenopus laevis, many RNAs are localized specifically to the animal or the vegetal halves of the oocyte. The localization of these RNAs contributes to the primary polarity of the oocyte, the asymmetry that is the basis for patterning and lineage specification in the embryo. I have screened a cDNA library for clones containing the Xlsirt repeat, an element known to target RNAs to the vegetal cortex of the oocyte. I have identified seventeen cDNA clones that contain this element. One of these cDNAs encodes the RNA binding protein Hermes. The Hermes mRNA is localized to the vegetal cortex of the oocyte. Additionally, Hermes protein is also vegetally localized in the oocyte and is found in subcellular structures known to contain localized mRNAs. This suggests that Hermes might interact with localized RNAs. While Hermes protein is present in oocytes, it disappears at germinal vesicle breakdown during maturation. We therefore believe that the time period during which Hermes functions is during oogenesis or maturation prior to the time of Hermes degradation. To determine Hermes function, an antisense depletion strategy was used that involved injecting morpholino oligos (HE-MO) into oocytes. Injection of these morpholinos causes the level of Hennes protein to drop prematurely during maturation. Embryos produced from these oocytes exhibit cleavage defects that are most prevalent in the vegetal blastomeres. The phenotype can be partially rescued by injection of a heterologous Hermes mRNA and is therefore specific to Hermes. The Hermes expression and depletion results are consistent with a model in which Hermes interacts with one or more vegetally localized mRNAs in the oocyte and during the early stages of maturation. The interaction is required for cleavage of the vegetal blastomeres. Therefore, it is likely that at least one mRNA that interacts with Hermes is a cell cycle regulator. ^

Isolation and characterization of a novel human Mix -like homeobox gene MIXL

Molecular events involved in specification of early hematopoietic system are not well known. In Xenopus, a paired-box homeodomain family (Mix.1–4) has been implicated in this process. Although Mix-like homeobox genes have been isolated from zebrafish (bon), chicken (CMIX) and mice (MmI/MIXL1), isolation of a human Mix-like gene has remained elusive. ^ We have recently isolated and characterized a novel human Mix-like homeobox gene with a predicted open reading frame of 232 amino acids designated the Mix.1 homeobox (Xenopus laevis)-like gene (MIXL). The overall identity of this novel protein to CMIX and MmI/MIXL1 is 41% and 69%, respectively. However, the identity in the homeodomain is 66% to that of Xenopus Mix.1, 79% to that of CMIX, and 94% to that of MmI/MIXL1. In normal hematopoiesis, MIXL expression appears to be restricted immature B and T lymphoid cells. Several acute leukemic cell lines of B, T and myeloid lineages express MIXL suggesting a survival/block in differentiation advantage. Furthermore, Xenopus animal cap assay revealed that MIXL could induce expression of the α-globin gene, suggesting a functional conservation of the homeodomain. ^ Biochemical analysis revealed that MIXL proteins are phosphorylated at multiple sites. Immunoprecipitation and immunoblotting confirmed that MIXL is tyrosine phosphorylated. Mutational analysis determined that Tyr20 appears to be the site for phosphorylation. However, deletion analysis preliminarily showed that the proline-rich domain appears not to be necessary for tyrosine phosphorylation. The novel finding will help us make a deeper understanding of the regulation on homeodomain proteins by rarely reported tyrosine phosphorylation. ^ Taken together, isolation of the MIXL gene is the first step toward understanding novel regulatory circuits in early hematopoietic differentiation and malignant transformation. ^

Diversity and faunal composition of benthic foraminifera in Hole 44-390A on the Blake Nose Plateau

Benthic foraminiferal assemblages are a widespread tool to understand changes in organic matter flux and bottom-water oxygenation and their relation to paleoceanographic changes in the Upper Cretaceous oceans. In this study, assemblage data (diversity, total number, and number per species and gram) from Deep Sea Drilling Project (DSDP) Site 390 (Blake Nose, western North Atlantic) were processed for the lower Maastrichtian (Globotruncana falsostuarti - Gansserina gansseri Planktic Foraminiferal Zone). These data document significant changes in nutrient flux to the sea floor as well as bottom-water oxygenation during this time interval. Parallel to the observed changes in the benthic foraminiferal assemblages the number of inoceramid shells decreases, reflecting also a significant increase in bottom-water oxygenation. We speculate, that these data could reflect the onset of a shift from warmer low-latitude to cooler high-latitude deep-water sources. This speculation will predate the major reorganization of the oceanic circulation resulting in a circulation mode similar to today at the Early/Late Maastrichtian boundary by ~1 Ma and therefore improves our understanding of Late Cretaceous paleoceanography.

Foraminifera and microfossil occurence in Late Jurassic sediments of the North and South Atlantic

Biostratigraphical, taxonomical, and palaeocological results were obtained from Oxfordian to Tithonian foraminifers of the Northern and Southern Atlantic Ocean boreholes of the DSDP Legs 1, 11, 36, 41, 44, 50, and 79. An oversight on the cored Jurassic sections of the DSDP Legs 79 and the corresponding foraminiferal descriptions are given. The reddish brown, clayey and carbonaceous Cat Gap Formation (Oxfordian to Tithonian) of the Northern Atlantic Ocean, rich in radiolarians, yields less or more uniform, in most cases allochthonous foraminiferal faunas of Central European shelf character. No Callovian and Upper Tithonian foraminiferaI zones can be established. The zone of Pseudomarssonella durnortieri covers the Oxfordian/Kimmeridgian, the zone of Neobulimina atlantica the Kimmeridgian/Lower Tithonian interval. Characteristic foraminiferal faunas are missing since the Upper Tithonian to Valanginian for reason of a widely distributed regression which caused hiatuses observed all over the Northern Atlantic Ocean and in parts of Europe. The Upper Jurassic cannot be subdivided into single stages by foraminiferal biostratigraphy alone. The fovaminiferal zones established by Moullad (1984) covering a Callovian-Tithonian interval may be of some local importance in the Tethyan realm: It has too long-ranging foraminiferal species to be used as index marker in the word-wide DSDP boreholes. Some taxonomical confusion is caused because in former publications some foraminiferal species have got different names both in the Jurassic and Cretaceous. The foraminiferal biostratigraphy of drilled sections from DSDP boreholes is restricted by the drilling technique and for palaeo-oceanographical, biological, and geological reasons. Foraminiferal faunas from the DSDP originally described as ,,bathyal, or ,,abyssal,, have to be derived from shallower water. This contrasts the palaeo-water depths of 3000-4000 m which result from sedimentological and palaeo-geographical investigations.

Benthic foraminifers from the Japan Sea

Five holes were drilled at two sites in the Sea of Japan during Ocean Drilling Program (ODP) Leg 128. Site 798 is located on Oki Ridge at a depth of about 900 m. Sediment age at Site 798 ranges from Pliocene to Holocene. Site 799 is located in the Kita-Yamato Trough at depth of 2000 m and below the present calcite compensation depth (CCD); the sediment ranges from Miocene to Holocene in age. Samples from all holes contain benthic foraminifers. Faunal evidence of downslope displacement is frequent in Holes 799A and 799B. The vertical frequency distribution of some dominant species shows that significant faunal changes occur in Holes 798A-C on Oki Ridge. Based on the faunal change and the thickness of sediments, it appears that the Oki Ridge was uplifted more than 1,000 m during last 4 m.y. Benthic foraminifers also demonstrate that the water depth of Site 799 rapidly changed from upper bathyal to lower bathyal during middle Miocene time. The appearance of benthic foraminifer species common to anaerobic environments suggests that the dysaerobic to anaerobic bottom conditions existed during the evolution of the Sea of Japan. Faunal distributions also suggest that the 'Tertiary-type' species recognized in the Neogene strata of the Japan Sea coastal regions disappeared sequentially from the Sea of Japan during Pliocene to late Pleistocene.

Occurrence and abundance of late Early Cretaceous radiolarians at DSDP Leg 62 Holes

Well-preserved Mesozoic radiolarian faunas have been recovered at four sites of Deep Sea Drilling Project Leg 62. Late Early Cretaceous assemblages, which occur always with foraminifers or calcareous nannoplankton, allow the description of 21 new species, the introduction of a new zone scheme, and calibration of the radiolarian zones with the geochronological scale.

Mollusc biostratigraphy and Bolboforma abundance in sediments obtained near Gross Pampau, north Germany

A thirty-six meter thick section of Miocene mica clay of Gross Pampau was studied for molluscs and bolboformas. The molluscs define the regional substages of late Reinbekian to late Langenfeldian. The bolboformas enable the cross-correlation with the nannoplankton subdivision and the geological time scales of BERGGREN et al. (1995). New species are Periploma ariei, Ringicula tiedemanni, Bolboforma robusta badenensis, and Bolboforma contorta.