Boveri's research at the Zoological Station Naples: Rediscovery of his original microscope slides at the University of Würzburg.

Eric Davidson once wrote about Theodor Boveri: "From his own researches, and perhaps most important, his generalized interpretations, derive the paradigms that underlie modern inquiries into the genomic basis of embryogenesis" (Davidson, 1985). As luck would have it, the "primary data" of Boveri's experimental work, namely the microscope slides prepared by him and his wife Marcella during several stays at the Zoological Station in Naples (1901/02, 1911/12 and 1914), have survived at the University of Würzburg. More than 600 slides exist and despite their age they are in a surprisingly good condition. The slides are labelled and dated in Boveri's handwriting and thus can be assigned to his published experimental work on sea urchin development. The results allowed Boveri to unravel the role of the cell nucleus and its chromosomes in development and inheritance. Here, I present an overview of the slides in the context of Boveri's work along with photographic images of selected specimens taken from the original slides. It is planned to examine the slides in more detail, take high-resolution focal image series of significant specimens and make them online available.

Historical roots of centrosome research: discovery of Boveri's microscope slides in Würzburg.

Boveri's visionary monograph 'Ueber die Natur der Centrosomen' (On the nature of centrosomes) in 1900 was founded primarily on microscopic observations of cleaving eggs of sea urchins and the roundworm parasite Ascaris. As Boveri wrote in the introductory paragraph, his interests were less about morphological aspects of centrosomes, but rather aimed at an understanding of their physiological role during cell division. The remarkable transition from observations of tiny dot-like structures in fixed and sectioned material to a unified theory of centrosome function (which in essence still holds true today) cannot be fully appreciated without examining Boveri's starting material, the histological specimens. It was generally assumed that the microscope slides were lost during the bombing of the Zoological Institute in Würzburg at the end of WWII. Here, I describe the discovery of a number of Boveri's original microscope slides with serial sections of early sea urchin and Ascaris embryos, stained by Heidenhain's iron haematoxylin method. Some slides bear handwritten notes and sketches by Boveri. Evidence is presented that the newly discovered slides are part of the original material used by Boveri for his seminal centrosome monograph.

Visualizing protein interactions involved in the formation of the 42S RNP storage particle of Xenopus oocytes.

BACKGROUND INFORMATION: During early phases of Xenopus oogenesis, 5S rRNA and tRNAs are stored in the cytoplasm of young oocytes in the form of a common RNA-protein complex termed the 42S particle. These storage particles comprise two kinds of proteins with different RNA binding specificities. The tRNA-binding protein 42Sp50 belongs to the EF1A (eukaryotic translation elongation factor 1A) family of translation elongation factors, while 42Sp43 is a diverged form of the transcription factor TFIIIA (transcription factor IIIA) and binds 5S rRNA. Little is known about the mode of protein-protein interactions that stabilize the 42S particle. RESULTS: We have determined the intracellular localization of the protein components of the 42S particle by expressing fluorescent protein-tagged fusions in transparent previtellogenic oocytes. 42Sp50 and its isoforms (EF1A-S and EF1A-O) were excluded from the nuclei and distributed uniformly throughout the cytoplasm with no enrichment in the Balbiani bodies, as described earlier by immunocytochemistry. In contrast, 42Sp43 accumulated in the amplified nucleoli. However, when both proteins were simultaneously expressed, 42Sp43 was no longer present in the nucleoli but was retained, together with 42Sp50, in the cytoplasm, the most likely site of 42S particle assembly. In contrast, the somatic-type EF1A isoforms were unable to redirect 42Sp43 from the nucleolar to the cytoplasmic compartment. We also tested for in vivo interactions using transiently transfected mammalian cells (COS-7 cell line). In this heterologous cell system 42Sp43 remained bound to the nucleoli but, on co-expression, induced the redistribution of 42Sp50 from the cytoplasm to the nucleoli. CONCLUSIONS: The microscopic approach described allows visualization of protein-protein interactions involved in the assembly of 42S storage particles. In particular, the transfection assay using COS-7 cells provides a rapid screening test that should facilitate identification of critical residues and structural determinants that enable the proteins of the 42S storage particle to interact with each other and to establish distinct higher-order RNP (ribonucleoprotein) complexes.

p53 localizes to intranucleolar regions distinct from the ribosome production compartments.

The tumor suppressor p53 has been implicated in the regulation of ribosome biogenesis based on its inhibitory effect on RNA polymerase I (pol I)-dependent transcription. Consistent with this, p53 has been described in nucleoli, albeit under specific experimental conditions. Since data on the intranucleolar localization of p53 are controversial, we have analyzed in detail its subnucleolar distribution. Our results show that p53 does not localize to one of the well-known structural components of the nucleolus involved in ribosome biogenesis, but rather occupies distinct intranucleolar regions that constitute nucleolar cavities. When cells were treated with the proteasome inhibitor MG132, the size and frequency of p53-containing nucleolar cavities increased, and the protein partially colocalized with inactivated proteasomes. Importantly, p53 did not colocalize with pol I at the transcription sites in fibrillar centers (FCs) as has previously been reported. The observed intranucleolar distribution and accumulation of p53 raises the question of how the protein influences rDNA transcription in vivo.

Assembly of nuclear pore complexes mediated by major vault protein.

During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells.

Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins.

Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC-GC interface.

Expression of HMGA2 variants during oogenesis and early embryogenesis of Xenopus laevis.

The high mobility group proteins A2 (HMGA2) have been implicated in the control of cell proliferation and differentiation, in particular during embryogenesis. Here, we used Xenopus laevis to analyze HMGA2 gene expression patterns during oogenesis and early embryogenesis. We found two functional XlHMGA2 isoforms, which we named XlHMGA2alpha and XlHMGA2beta. As revealed by RT-PCR, real-time PCR and whole-mount in situ hybridization both mRNAs are maternally produced and stored in eggs. Whole-mount in situ hybridizations revealed a conspicuous redistribution of the XlHMGA2 transcripts during early embryogenesis. Initially, during oogenesis and in eggs, the transcripts are uniformly distributed in the cytoplasm. With activation of the eggs the transcripts accumulate near the animal pole and remain in the juxtanuclear regions of animal pole blastomeres until midblastula transition. According to real-time PCR data, XlHMGA2alpha appears to be preferentially expressed during oogenesis and after midblastula transition, whereas XlHMGA2beta expression predominates after neurulation, suggesting an individual transcriptional regulation.

The chromatin remodelling complex WSTF-SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription.

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

Dynamic interaction of HMGA1a proteins with chromatin.

High-mobility-group proteins A1 (HMGA1; previously named HMGI/Y) function as architectural chromatin-binding proteins and are involved in the transcriptional regulation of several genes. We have used cells expressing proteins fused to green fluorescent protein (GFP) and fluorescence recovery after photobleaching (FRAP) to analyze the distribution and dynamics of HMGA1a in vivo. HMGA1-GFP proteins localize preferentially to heterochromatin and remain bound to chromosomes during mitosis. FRAP experiments showed that they are highly mobile components of euchromatin, heterochromatin and of mitotic chromosomes, although with different resident times. For a more-detailed investigation on the interaction of HMGA1a with chromatin, the contribution of the AT-hook DNA-binding motifs was analyzed using point-mutated HMGA1a-GFP proteins. Furthermore, by inhibiting kinase or histone deacetylase activities, and with the help of fusion proteins lacking specific phosphorylation sites, we analyzed the effect of reversible modifications of HMGA1a on chromatin binding. Collectively our data show that the kinetic properties of HMGA1a proteins are governed by the number of functional AT-hooks and are regulated by specific phosphorylation patterns. The higher residence time in heterochromatin and chromosomes, compared with euchromatic regions, correlates with an increased phosphorylation level of HMGA1a. The regulated dynamic properties of HMGA1a fusion proteins indicate that HMGA1 proteins are mechanistically involved in local and global changes in chromatin structure.

Developmental role of HMGN proteins in Xenopus laevis.

HMGN proteins are architectural chromatin proteins that reduce the compaction of the chromatin fiber, facilitate access to nucleosomes and modulate replication and transcription processes. Here we demonstrate that in Xenopus laevis, the expression and cellular location of the HMGN proteins are developmentally regulated and that their misexpression leads to gross developmental defects in post-blastula embryos. HMGN transcripts and proteins are present throughout oogenesis; however, the proteins stored in the cytoplasm are not associated with lampbrush chromosomes, and are rapidly degraded when oocytes mature into eggs. During embryogenesis, HMGN expression is first detected in blastula stages and progresses to a tissue-specific expression reaching relative high levels in the mesodermal and neuroectodermal regions of tadpoles. Only after midblastula transition (MBT), alterations in the HMGN levels by either microinjection of recombinant proteins or by morpholino-antisense oligo treatments produced embryos with imperfectly closed blastopore, distorted body axis and showed abnormal head structures. Analyses of animal cap explants indicated that HMGN proteins are involved in the regulation of mesoderm specific genes. In addition, HMGN misexpression caused altered expression of specific genes at MBT rather than global changes of transcription rates. Our results demonstrate that proper embryonic development of Xenopus laevis requires precisely regulated levels of HMGN proteins and suggest that these nucleosomal binding proteins modulate the expression of specific genes.

On the formation of amplified nucleoli during early Xenopus oogenesis.

In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF's role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.

Autoantibodies to NOR 90/hUBF: longterm clinical and serological followup in a patient with limited systemic sclerosis suggests an antigen driven immune response.

We describe the clinical and serological followup of a 9-year-old girl with anti-nucleolar organizing region 90/human upstream-binding factor (anti-NOR 90/hUBF) who had features of systemic sclerosis over a period of 17 years, from childhood into adulthood. We review the associations of anti-UBF autoantibodies, and provide evidence that anti-NOR 90/UBF immune response is antigen driven.

A bisexually reproducing all-triploid vertebrate.

Green toads are common in the Palaearctic region, where they have differentiated into several taxa. The toads exist with variable amounts of ploidy, similar to other anuran species or reptiles. In vertebrate biology, the very rare occurrence of triploidy is coupled with infertility or unisexuality, or requires the coexistence of individuals of different ploidy in a reproductive community. The reproduction of naturally occurring triploids has been reported to occur only through parthenogenesis, gynogenesis or hybridogenesis. The bisexual reproduction of pure triploids has been considered to be impossible because of the problem of equally distributing three chromosome sets in meiosis. Here we report geographically isolated populations of green toads (Bufo viridis complex) that are all-triploid and reproduce bisexually.

[Development of gametogonia in ektopically transplanted gonads of Triturus]. / Entwicklung der Gametogonien in ektopisch transplantierten Gonaden beiTriturus.

Homografts of gonads including fat-bodies show fusion of the fat-body with liver tissue. Thus, contact between gonad and liver is only indirect. The differentiation of the gametogonia follows the normal way of development. In case of ovary homografts auxocytes appear. Not later than 27 days after transplantation vascularization is reestablished.Homo- and autografts of gonads without fat-body show a quite different development of the gametogonia. When the gonads are broadly fused with liver tissue one notices karyolysis of the gametogonia nuclei within the gonad liver contact region already 7 days after transplantation. After 3-4 weeks the graft represents a cyst formed by connective tissue without any germ cells. Erythrocytes indicate vascularization. In the gonad partly fused with liver normal structure and mitosis in the gametogonia appear in that part of the transplant not attached to liver tissue. The present experiments suggest that the degeneration of germ cells is depending on their position to extragonadal tissue.