The coordinated regulation of gene expression must be tightly controlled for normal development to occur. In mammals, this issue is further complicated by the requirement of both the maternal and paternal genomes for normal development, reflecting the fact that a subset of genes are monoallelically expressed depending on parental inheritance, a phenomenon known as genomic imprinting. The imprinted H19 and Igf2 genes are often considered as paradigms of genomic imprinting, since their monoallelic expression patterns are coordinated via a short stretch of sequence upstream of H19, known as the imprinting control region (ICR). This region is differentially methylated, with specific CpG methylation on the paternal allele. It is shown here that the ICR harbours several maternal-specific hypersensitive sites, located in linker regions between positioned nucleosomes. Furthermore, this region functions as an orientation-dependent insulator, that binds the chromatin insulator factor CTCF. The hypothesis that the methylation status of the ICR dictates the activity of the Igf2 gene 90 kb further upstream was confirmed by the demonstration that the insulator function is lost when the ICR is CpG methylated. The ICR has previously been shown to act as a silencer when positioned in a promotor-proximal position. The cause of this silencing was shown to be distance-dependent, suggesting that the silencing features of the ICR depend on a chromatin conformation that renders adjacent sequences inaccessible to the RNA polymerase. These data issue a cautionary note with respect to the interpretation of silencer functions. In several forms of cancer, the normally silent maternal IGF2 gene is expressed, possibly as a result of loss of insulator function at the ICR. The utilisation of CTCF target-sites was analysed in different tumours, and was shown to be highly variable. Methylation analysis showed that potential loss of insulator function and gain of methylation at the maternal ICR did not always correlate with biallelic expression of IGF2. Further investigations uncovered a novel mechanism, in which the activation of the IGF2 promoter was independent of insulator function in some cancers. This thesis shows that the regulation of the imprinted state of Igf2 depends on the formation of an epigenetically regulated chromatin insulator, and that the loss of IGF2 imprinting in human cancer can be attributed to several mechanisms, including a novel mechanism that neutralises chromatin insulator function.

No description available.

Branched tubular organs, such as the lung and vascular system fulfill the respiratory needs of most animals. Optimal tissue function relies on the uniform sizes and shapes of the constituting branches in each organ. The Drosophila tracheal airways provide a recognized genetic model system for identification and characterization of tube size regulators. We found that the programmed secretion and assembly of the apical extracellular matrix (ECM) is required for the expansion of the trachea and salivary glands (SG) tubes. We have characterized Vermiform (Verm) and Serpentine (Serp), two chitin-binding proteins with predicted polysaccharide deacetylase domains (ChLDs). Verm and Serp mutants show overelongated tubes, suggesting that luminal ECM modification restricts tracheal tube elongation. The luminal deposition of ChLDs, but not other secreted components, depends on paracellular septate junction integrity (SJs) in the tracheal epithelium. Deletion of the deacetylase domain renders Serp-GFP intracellular, arguing that the deacetylase domain harbors uncharacterized secretion signals. To explore this possibility we transferred the deacetylase domain from Serp to Gasp, another tracheal luminal protein, which requires the Emp24 adaptor for ER exit. The Gasp-Deac-GFP chimera was normally secreted in emp24 mutants indicating that the deacetylase domain contains potential ER-exit signals. To identify such signals we characterized conserved sequence motifs in the Serp deacetylase domain. Mutations of the N-glycosylation sites gradually reduced Serp-GFP luminal deposition suggesting that increased glycosylation enhances apical Serp secretion. By contrast, substitutions in three conserved amino acid stretches completely blocked the ER-exit of Serp-GFP. The mutated proteins were N-glycosylated suggesting that the motifs may be involved in a subsequent protein-folding step or facilitate ER exit through interactions with unidentified specific adaptors. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

In recent times, major advances have been made towards understanding sexual dimorphism in the brain on a molecular basis. This thesis summarises my modest contributions to these endeavours. Sexual dimorphisms are manifested throughout the spectrum of biological complexity, and can be studied by numerous approaches. The approach of this thesis is to explore sex-biased gene expression in mammalian somatic tissues. Paper I describes an evolutionarily conserved sexual gene expression pattern in the primate brain. Conserved sex-biased genes may underlie important sex differences in neurobiology. In Paper II, Y-chromosome genes expressed across several regions of the human male brain during mid-gestation are identified. Such genes may play male-specific roles during brain development. The studies of Papers III and IV explore sex-biased gene expression in several somatic tissues from mouse. The amount of genes with sex-biased expression varied in different brain regions. The striatum was particularly interesting, with an order of magnitude increase in the number of sex-biased genes as compared to the other included brain regions. Of potentially wider significance are my observations regarding the transcriptional regulation of domains that escape X-chromosome inactivation (XCI). Specifically, I provide the first evidence that long non-coding RNAs (lncRNAs) transcribe together with protein-coding genes in XCI-escaping domains. This raises the possibility that lncRNAs mediate the transcriptional regulation of XCI-escaping domains. I also present evidence that the mouse X-chromosome has undergone both feminisation and de-masculinisation during evolution, as indicated by the sex-skewed regulation of genes on this chromosome. This finding is relevant for understanding the selective forces that shaped the mammalian X-chromosome. In the final chapter, Paper V, the generation of a novel transgenic mouse line, Gpr101-Cre, is described. Its progeny can be used for functional studies of striatum, a brain structure with major sexual dimorphism, as is further demonstrated in the Papers of this thesis.

No description available.

No description available.

<p>The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (<i>Ambystoma mexicanum</i>), is a basal tetrapod, whereas the other, the Australian lungfish (<i>Neoceratodus forsteri</i>), belongs to the Dipnoi, the extant sister group of the Tetrapoda. </p><p>The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii.</p><p>The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. </p><p>To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived.</p><p>In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.</p>

The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (Ambystoma mexicanum), is a basal tetrapod, whereas the other, the Australian lungfish (Neoceratodus forsteri), belongs to the Dipnoi, the extant sister group of the Tetrapoda. The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii. The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived. In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.

A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting. The imprinted state of the H19 and Igf2 genes is controlled by a short stretch of sequences upstream of H19 known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited Igf2 allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the H19 ICR, which plays an important role in maintaining the imprinting status of H19 and Igf2, was shown to lose the insulator property upon CpG methylation. Another ICR in the Kcnq1 locus regulates long-range repression of p57Kip2 and Kcnq1 on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing H19 and Igf2, on the distal end of mouse chromosome 7. Similarly to the H19 ICR, the Kcnq1 ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions. The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the H19 ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF in vivo occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.

<p>For a complete picture of the lake ecosystem, it is necessary to understand the mechanisms regulating biomass and nutrient status (nutrient limitation, optimal supply, or surplus) of benthic algae, which are important primary producers and a food resource for grazers. This thesis gives an overview of the natural variation of benthic algae at different scales of space and time and on different substrates, and unravels some of the underlying factors. Algal nutrient status was assessed using the C:N:P (carbon:nitrogen:phosphorus) ratio of the entire natural benthic community. A review, observations, and experiments confirmed that a C:N:P ratio of about 158:18:1 (molar basis) represented an optimal nutrient supply, and that substantially higher C:N, N:P, or C:P ratios reflected algal growth limitation caused by an N or P nutrient deficiency. </p><p>Horizontal variation of benthic algal biomass and nutrient status was patchy, of similar amount for all investigated distances, substrates, and lakes, and constituted a dominant proportion of the total variation. For example, patches of nutrient limited algae were found within only 10 m distance from patches with a nutrient surplus. Thus, horizontal variation should not be neglected when sampling benthic algae in lakes. Field observations suggested an impact of wind, nutrients, and grazers on the horizontal variation. Light and nutrients might have caused the observed vertical and temporal variation. Field experiments confirmed a simultaneous control of benthic algal biomass by nutrients and grazing, mediated by light and temperature. Grazing effects were larger than nutrient effects, but the comparison of natural communities in lakes of different trophy suggested that benthic algal biomass was controlled by nutrients in the long run. </p><p>An important nutrient supply was animal excretions, causing a low C:N:P ratio of epizoon on zebra mussels, and algal communities associated with macrograzers. A field experiment revealed that 15N circulated one week longer in epizoon associated with a sessile caddisfly than in surrounding epilithon. In conclusion, the regulation of benthic algal biomass and nutrient status in lakes is complex, and benthic animals should be looked at not only as grazers, but also as a nutrient source.</p>