No description available.

Abstract Lysyl hydroxylase (LH, EC 1.14.11.4) catalyzes the post-translational hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. The hydroxylysyl residues participate in the formation of collagen cross-links, and some of the hydroxylysyl residues are further glycosylated. Three lysyl hydroxylase isoforms LH1, LH2 and LH3, encoded by three individual genes have been characterized and one isoform, LH3 is a multifunctional enzyme containing lysyl hydroxylase, collagen galactosyltransferase (GT, E.C. 2.4.1.50) and glucosyltransferase (GGT, E.C. 2.4.1.66) activities in vitro. In this thesis the genes for the mouse lysyl hydroxylases were each mapped to a different chromosome. In addition, the roles of the lysyl hydroxylase isoforms were characterized in mice by studying their expression during development and the distribution of LH2 and LH3 in adult mice. The results revealed a widespread expression of the mouse lysyl hydroxylases during embryonic development whereas LH2 and LH3 showed tissue- or cell-specific expression patterns in the adult. Alternative splicing of the gene for LH2 also showed developmental and tissue-specific regulation. The different functions of LH3 were studied in vivo by generating three different LH3 manipulated mouse lines. Analysis of the mouse lines revealed that LH3 has lysyl hydroxylase and glucosyltransferase activities in vivo, and that, in particular, the glucosyltransferase activity of LH3 is essential for normal development. The loss of glucosyltransferase activity caused disruption of basement membranes leading to embryonic lethality while the absence of lysyl hydroxylase activity led to ultrastructural alterations in muscle and basement membranes and disorganization of collagen fibrils. The disruption of basement membrane was due to an intracellular accumulation of unglycosylated type IV collagen, whereas the ultrastructural alterations were related to the abnormal aggregation and distribution of underglycosylated type VI collagen. The results demonstrate that hydroxylysine-linked glycosylations are critical for the secretion of type IV collagen and its assembly into basement membranes, and for the assembly and distribution of type VI collagen.

Abstract Arthropod photoreceptors are versatile sense organs. Any investigation of these organs has to consider that their structure and functional limitations at the moment of fixation depend on many factors: species, sex, developmental and nutritional state of the animal, time of day and ambient light. The microscopic image of an arthropod photoreceptor is always a sample frozen in time and space. Quite often publications on arthropod photoreceptors only provide the name of the species studied, but nothing beyond that. At least the developmental status of the study animals ought to be noted, possibly even the sex and body size. Forty publications on insect and 54 on crustacean photoreceptors were checked for the information that was given about the investigated animals: Out of these papers 40% provide only information on the name of the studied species and nothing else. The aim of this thesis, thus, was to investigate, to what extent the developmental state and the sex of the animal as well as the ambient light conditions affect the structure of the eye of a given species. Five species of arthropods were chosen: (a) the semi-terrestrial isopod Ligia exotica and two aquatic Branchiuran fishlice, Argulus foliaceus and A. coregoni, to represent the Crustacea, and (b) the stick insect Carausius morosus and the spittle bug Philaenus spumarius, both terrestrial, to represent the Insecta. The addition of new ommatidia was studied in a paper on L. exotica, which also dealt with the site of newly added ommatidia. It was found that all of these species had two sessile, large compound eyes firmly positioned on their heads (but fishlouse compound eyes were bathed in haemocoelic liquid). In all species, the compound eye was found to be of the apposition type. The gross structural organization of the ommatidia stayed approximately the same during the whole post-embryonic development. Lateral ocelli of the A. coregoni nauplius eye changed from elongated to spherical between the metanauplius and the 8th stage pre-adult. The sex of the specimens was not found to affect the structure of the eye. In all species, it turned out that the larger the animal and hence the eye, the better its sensitivity. The addition of new ommatidia in the L. exotica compound eye was concluded to take place in the anterior and ventral marginal areas of the eye.

Roles for the Cdx transcription factors during anteroposterior patterning and development of the posterior embryo have been well described, yet little is known about Cdx functions during mesoderm specification. In the studies presented here, novel roles for Cdx during gastrulation are presented. In the first of two studies, the role of Cdx factors during cardiac mesoderm specification is investigated. We demonstrate that Cdx factors epigenetically restrict cardiac progenitor specification, preventing the ectopic expression of cardiogenic genes within progenitor mesodermal populations. We provide evidence to suggest that this occurs through interaction with Brg1, a subunit of the SWI/SNF chromatin remodeling complex, and propose a molecular mechanism by which Cdx-mediated recruitment of the SWI/SNF complex is required to maintain repression of cardiac targets at developmental stages where Cdx transcription factors are no longer expressed. Following this, Cdx-dependent gene expression programs were identified in the extra-embryonic yolk sac. RNA-sequencing of Cdx-mutant yolk sacs revealed novel Cdx-dependent gene targets involved in ion and nutrient transport, functions analogous to those previously described for Cdx in the adult intestinal epithelium. Subsequent experimentation revealed that, for a subset of these targets, regulation correlates with the maintenance of H3K27me3 marks. Finally, we provide evidence to suggest Cdx-dependent H3K27me3 marks are established at early developmental stages, when yolk sac progenitors are specified from the streak. Together, these studies describe novel roles for Cdx factors in mesoderm specification during gastrulation, and evoke molecular mechanisms by which Cdx might program early mesodermal populations for gene expression at later developmental stages through interactions with epigenetic regulatory complexes.

No description available.

Cilia are cellular organelles involved in processing components of the hedgehog (hh) signalling pathway and determining left-right (L-R) axis formation in the embryo. An embryonic lethal mouse mutant, called gasping 6 (gpg6), was identified that demonstrated morphological and molecular defects associated with L-R development and hh signalling. gpg6 mutant embryos also demonstrate abnormally short cilia, which was hypothesised to be the primary morphological defect in gpg6 mutants. The underlying genetic lesion in gpg6 is a mutation in the DNA repair gene Atmin. The base pair change results in an amino acid substitution in a critical residue in the third zinc finger of Atmin. The consequence of this change is the failure to activate transcriptional targets of Atmin. This raised the possibility that previously unidentified Atmin target genes are important for ciliogenesis. Consistent with this hypothesis, Dynein light chain-LC8 (Dynll1) is downregulated in gpg6 mutants. LC8 (a homolog of mouse Dynll1) is required, in the single cell eukaryotic organism Chlamydomonas, for retrograde intraflagellar transport (IFT), a process crucial for ciliogenesis. These data led to the following hypothesis: Atmin activates expression of Dynll1, which functions in retrograde IFT to enable normal ciliogenesis. Knockdown of Atmin in a ciliated kidney cell line resulted in abnormally short cilia. Thus, Atmin functions in ciliogenesis. Investigation of gpg6 has therefore identified a novel role for Atmin in ciliogenesis and has added to the growing knowledge of genes that control cilia formation and embryonic development.

Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood. In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment. These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z. / Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt. In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen. Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen.

The ubiquitin-proteasomal system which is the major pathway for intracellular protein degradation is also involved in sperm quality control in the mammalian epididymis. Defective sperm become surface- ubiquitinated during epididymal passage. The level of sperm surface ubiquitination negatively correlates with their quality. Hypothetically it is possible that after fertilization, highly ubiquitinated sperm, naturally present in mammalian ejaculates, would be actively recognized by oocyte (probably via 26S proteasomal complex). Subsequent partial or total sperm degradation should negatively affect the development of the potentially defective embryo. In this study, we examined the effect of sperm ubiquitination on the early embryonic development in pig (Sus scrofa f. domestica) using the method of intracytoplasmic sperm injection (ICSI). In vitro embryonic development to the blastocyst stage after ICSI was comparable with other laboratories. In this study, no significant difference was observed in the formation of pronuclei between oocytes fertilized by lower and highly ubiquitinated sperm cells. On the other hand, significantly better embryonic development to the blastocyst stage was observed in oocytes fertilized by sperm with lower surface ubiquitination (17 %) compared with oocytes fertilized by highly...

No description available.

DNA methylation is a key epigenetic mark which undergoes global reprogramming during early mammalian embryonic development, resulting in almost complete erasure of the mark after fertilisation of the zygote. Genome-wide patterns of DNA methylation are subsequently re-established in the implanting blastocyst by de novo DNA methyltransferases Dnmt3a and Dnmt3b along with their catalytically inactive co-factor Dnmt3l, while these DNA methylation patterns are maintained through cell divisions by maintenance methyltransferase Dnmt1. The exact mechanisms by which these DNA methyltransferase enzymes are targeted to specific genomic regions remain unclear, but may involve interaction with modified histones and/or the participation of co-factors. Lsh (lymphoid specific helicase), a putative chromatin remodelling helicase, has been implicated in facilitating de novo methylation, as Lsh knockout embryos and derived somatic cell lines display substantial but specific DNA methylation losses at repetitive elements and single copy genes. This study aims to define the requirement for Lsh in establishing de novo DNA methylation and gene expression patterns during the early stages of mouse embryonic development. The '2i' culture system using two small molecule kinase inhibitors was harnessed to convert lsh-/- mouse embryonic stem cells (mESCs) to a hypomethylated 'ground state' of pluripotency. Culture conditions were then altered to transition these ground state mESCs to cells representing later, more methylated stages of development ('serum' mESCs, epiblast stem cells and embryoid bodies). Implementation of this model system suggests that Lsh does not contribute to DNA methylation establishment in a pluripotent context, but rather is important for facilitating de novo DNA methylation during differentiation to culture models representing later developmental stages. These investigations also reveal that Lsh differentially regulates DNA methylation at major and minor satellite repeats depending on cellular context, and that this regulation may involve a role for Lsh in maintenance of DNA methylation.