Browsing by Subject "kehitysbiologia"

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  • Närhi, Katja (Helsingin yliopisto, 2012)
    Although mammary glands and hair are morphologically and functionally different organs, they share similar early developmental features and arise from ectoderm like other skin appendages. Their development begins by the formation of an epithelial placode and a mesenchymal dermal condensate and crosstalk between these tissue compartments directs the subsequent developmental steps resulting in epithelial morphogenesis and the generation of specific organ shapes. Different types of hair filaments are observed in various anatomical regions and are produced by hair follicles consisting of several epithelial cell layers and a dermal papilla. The mammary gland is constructed of a nipple rising above the skin and the glandular mammary tree producing milk. Both organs continue development postnatally; new hair is produced by repeated hair cycles lasting throughout the lifetime and postpubertal mammary ductal tree is remodelled upon pregnancy and following lactation and involution. Handful of conserved signaling pathways guides both the embryonic and postnatal developmental steps of skin appendages. Hair and mammary gland development are especially known to depend on signals from the -catenin-mediated Wnt pathway. The Wnt pathway is highly complex with multiple ligands, receptors and signaling modulators, and cross-talk with other signaling pathways is apparent. Here, I have examined the role of Wnt signaling in hair and mammary gland patterning and development and its relationship to other signaling molecules within this context. The study has involved three different mouse models in which Wnt signaling is modulated either by continuous activation of -catenin, inactivation of the Wnt and Bmp pathway regulator Sostdc1, or ablation of a Wnt target gene, Fgf20. Continuous Wnt/-catenin signaling in embryonic ectoderm in Catnbex3K14/+ mice caused precocious hair development and, the formation of ectopic and mispatterned hair placodes showing disturbed morphogenesis and hair filament formation. Fgf20-null mice showed a surprisingly early hair phenotype with a loss of expression of several dermal condensate markers but presence of grossly normal morphological patterning of placodes with altered placode marker expression patterns. Loss of Sostdc1 had very mild effects on pelage hair development but interestingly, Sostdc1 appears to play a role in determing correct vibrissal hair and nipple number and the regulation of mammary bud size/form, plausibly through inhibiting Wnt signaling.
  • Raatikainen-Ahokas, Anne (Helsingin yliopisto, 2014)
    Embryonic cells undergo sequential specification processes to generate multiple cell types of mature organs. Some cells retain pluripotency. They serve as stem or progenitor cells, and provide both new stem cells (self-renewal) and offspring for differentiation. The fate of some cells is to die by programmed cell death. In this thesis, the cell fates in nephrogenesis and spermatogenesis were studied. During kidney organogenesis, an outgrowth of the Wolffian duct, the ureteric bud, induces condensation of the metanephric mesenchyme into a cap condensate, the progenitor cell population that forms the epithelium of all future nephrons. The cap condensate is surrounded by stromal cells. The developmental fates of these cells that also surround the ureter and nascent nephrons, i.e. the kidney stroma, are poorly understood. Bone morphogenetic protein 4 (BMP4) inhibited the outgrowth of the ureteric bud from the Wolffian duct in organ culture. It also had an inhibitory effect on subsequent ureteric branching. The branching defect primarily reflected the effect of BMP4 on the mesenchymal components of the kidney. BMP4 promotes the recruitment of mesenchymal cells around the ureter and their differentiation into smooth muscle. This periureteric cell population likely has a regulatory function in subsequent ureteric growth and differentiation. The exogenous BMP4 also disrupted the cap condensates in kidney explants and large amounts of mesenchymal cells underwent apoptosis. BMP4 maintained the isolated metanephric mesenchymes while suppressing the nephrogenic potential, suggesting that BMP4 acts as a survival/differentiation factor for the stromal progenitors. The stromal cells are apparently essential for the formation and maintenance of the cap condensate. In some organs, such as the testis, the maintenance of stem cells throughout the life span is essential to the normal function, e.g. the formation of sperm cells. Spermatogonia with stem cell activity (SSCs) are among the undifferentiated spermatogonia located at the basement membrane of the seminiferous tubule. Daughters of SSCs both replenish the stem cell pool and enter the differentiation pathway into spermatozoa. Glial cell line-derived neurotrophic factor (GDNF), essential for ureteric branching morphogenesis, is also crucial to the self-renewal of the SSCs. Haploinsufficiency of the Gdnf gene in Gdnf+/- mice caused segmental exhaustion of stem cells, resulting in germ cell loss in old mice. In mice overexpressing GDNF in the testis, spermatogenesis was arrested and large clusters of spermatogonia accumulated in prepubertal animals. Thus, high GDNF concentration promotes the propagation of undifferentiated spermatogonia, whereas low GDNF levels allow SCCs to differentiate in excess and make them prone to depletion. In conclusion, signalling molecules, such as BMP4 and GDNF, affect the cell fates both in nephrogenesis and spermatogenesis by maintaining the precursor cells and promoting their differentiation.
  • Jukkola, Tomi (Helsingin yliopisto, 2007)
    The neuroectodermal tissue close to the midbrain hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) regulates cellular survival, patterning and proliferation in the midbrain (Mb) and rhombomere 1 (R1) of the hindbrain. Signaling molecules of the IsO, such as fibroblast growth factor 8 (FGF8) and WNT1 are expressed in distinct bands of cells around the MHB. It has been previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. In the present study, we have compared the gene expression profiles of wild-type and Fgfr1 mutant embryos. We show that the loss of Fgfr1 results in the downregulation of several genes expressed close to the MHB and in the disappearance of gene expression gradients in the midbrain and R1. Our microarray screen identified several previously uncharacterized genes which may participate in the development of midbrain R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1. As Wnt1 expression at the MHB region requires the FGF signaling pathway, WNT target genes, including Drapc1, were also identified in our screen. The microarray data analysis also suggested that the cells next to the midbrain hindbrain boundary express distinct cell cycle regulators. We showed that the cells close to the border appeared to have unique features. These cells proliferate less rapidly than the surrounding cells. Unlike the cells further away from the boundary, these cells express Fgfr1 but not the other FGF receptors. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.
  • Ollonen, Joni (Helsingin yliopisto, 2020)
    The skull represents the most highly diversified and evolutionarily adapted anatomical aspect of metazoans, and its development and evolution have been a major driving force in the expansion of vertebrates. The evolution of skull and lower jaw bones have led to the adaptive radiation of jawed vertebrates, and skull tissues have changed rapidly over time and were finely tuned to meet functional and ecological demands with tremendous precision. Because of the long-lasting interest in conventional animal models, there is no general genetic or developmental model of skull evolution and diversity in vertebrates. Squamate reptiles represent the best model to study those aspects because of their key basal phylogenetic position within amniotes (i.e., mammals, birds, reptiles) and their exceptionally high levels of morphological variation (including their kinetic skulls). In particular, their lower jaw bones display tremendous variation. In order to assess this variation and the ecological and developmental factors connected to it, several methods from different fields of biology have to be used. In this study, morphometric, embryology and developmental approaches are used to investigate the ecological and developmental factors associated with the diversification of lower jaw bones in snakes and lizards. The shape diversity of squamate lower jaw bones was approached in a systematic way, using geometric morphometrics. Embryological methods were used to compare the embryonic stage of available squamate model animals at oviposition and to assess the order of ossification of embryo with earliest developmental stage at oviposition (bearded dragon, Pogona vitticeps). In addition, expression of major conserved candidate genes at different stages of lower jaw development (pharyngeal arches, mesenchyme patterning, ossification) were assessed in this species. The results indicate that the lower jaw bones of snakes versus lizards but also of fossorial squamates versus other habitats are significantly different. Heterochrony was also detected at both early stages (pharyngeal arche development at oviposition) and at the onset of ossification in lizards and snakes. Coherent with that, alterations in the expression pattern of Dlx genes in pharyngeal arches were observed in bearded dragon in comparison to earlier studies with mice, while other conserved markers of skeletogenesis were rather conserved. This analysis of the genotype and phenotype map of the reptilian skull provides some new insights into the development, origin and divergence of vertebrate tissues. The results will establish a good basis for future studies involving comparative developmental biology of bearded dragon. Future studies will offer excellent new opportunities to link craniofacial morphology, genetics/genomics and development to both ecological adaptation and evolutionary biology.
  • Järvinen, Elina (Helsingin yliopisto, 2008)
    In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.
  • Nieminen, Pekka (Helsingin yliopisto, 2007)
    Congenital missing of teeth, tooth agenesis or hypodontia, is one of the most common developmental anomalies in man. The common forms in which one or a few teeth are absent, may cause occlusal or cosmetic harm, while severe forms which are relatively rare always require clinical attention to support and maintain the dental function. Observation of tooth agenesis is also important for diagnosis of malformation syndromes. Some external factors may cause developmental defects and agenesis in dentition. However, the role of inheritance in the etiology of tooth agenesis is well established by twin and family studies. Studies on familial tooth agenesis as well as mouse null mutants have also identified several genetic factors. However, these explain syndromic or rare dominant forms of tooth agenesis, whereas the genes and defects responsible for the majority of cases of tooth agenesis, especially the common and less severe forms, are largely unknown. In this study it was shown, that a dominant nonsense mutation in PAX9 was responsible for severe tooth agenesis (oligodontia) in a Finnish family. In a study of tooth agenesis associated with Wolf-Hirschhorn syndrome, it was shown that severe tooth agenesis was present if the causative deletion in 4p spanned the MSX1 locus. It was concluded that severe tooth agenesis was caused by haploinsufficiency of these transcription factors. A summary of the phenotypes associated with known defects in MSX1 and PAX9 showed that, despite similarities, they were significantly different, suggesting that the genes, in addition to known interactions, also have independent roles during the development of human dentition. The original aim of this work was to identify gene defects that underlie the common incisor and premolar hypodontia. After excluding several candidate genes, a genome-wide search was conducted in seven Finnish families in which this phenotype was inherited in an autosomal dominant manner. A promising locus for second premolar agenesis was identified in chromosome 18 in one family and this finding was supported by results from other families. The results also implied the existence of other loci both for second premolar agenesis and for incisor agenesis. On the other hand the results did not lend support for comprehensive involvement of the most obvious candidate genes in the etiology of incisor and premolar hypodontia. Rather, they suggest remarkable genetic heterogeneity of tooth agenesis. The available evidence suggests that quantitative defects during tooth development predispose to a failure to overcome a developmental threshold and to agenesis. The results of the study increase the understanding of the etiology and heredity of tooth agenesis. Further studies may lead to identification of novel genes that affect the development of teeth.
  • Voutilainen, Maria (Helsingin yliopisto, 2015)
    Mammary gland development begins during embryogenesis with the formation of species-typical number of mammary placodes that emerge along the flanks of the embryo at conserved positions. By birth, the mammary primordium has undergone branching morphogenesis and displays a small ductal tree with several branches. The organ development and growth continues throughout postnatal life and the mammary gland matures to functional form only during pregnancy and following lactation. Ectodysplasin (Eda), a member of the tumour necrosis factor family, is one of the key regulators of epithelial appendage development in all vertebrates. In humans, mutations in the Eda gene, or in other components of the signalling pathway, cause hypohidrotic ectodermal dysplasia (HED), a disorder characterized by sparse hair, missing teeth, and defects in several exocrine glands including the breast. Previous studies have shown that transgenic overexpression of Eda (K14-Eda mice) in the developing ectoderm leads to formation of ectopic mammary placodes, which give rise to supernumerary glands in the adult mice. Otherwise, effects of Eda signalling in the mammary gland have been fairly unknown. Here I have analysed the role of Eda in prepubertal mammary gland development. Characterization of the mammary glands of Eda gain- (K14-Eda) and loss-of-function (Eda−/−) mice revealed that the branching morphogenesis of the organs correlated with Eda levels. Overexpression of Eda induced precocious and accelerated branching whereas lack of Eda reduced number of ductal tips. Furthermore, Eda induced supernumerary mammary placode formation not only on the flank but also in the neck region. Analysis of the mouse line with suppressed NF-kappaB signaling (IκBαΔN mice) revealed that the transcription factor is a major mediator of Eda in the mammary gland. NF-kappaB activity was shown to be necessary for the ability of Eda to induce supernumerary mammary primordia and to accelerate branching morphogenesis. With a candidate gene approach and genome wide-profiling several potent Eda target genes were identified in the mammary gland. Among them were members of the Wnt/beta-cat pathway. The obtained results suggest that Eda promotes mammary cell fate by enhancing canonical Wnt pathway activity and other effects of Eda are cooperatively mediated by certain Wnt family members in addition to other factors. To study mammary placode formation and branching morphogenesis and to assess roles of individual downstream factors or pathways, ex vivo culture systems were developed and utilized in this thesis work.
  • Hakanen, Janne (Helsingin yliopisto, 2014)
    The development of the forebrain is dependent on controlled regulation of neural stem/progenitor cells, since the vast majority of all cells in the forebrain are generated by them. The cellular processes influencing forebrain development include cell proliferation, migration, differentiation, and apoptosis, which are regulated in a spatiotemporal manner by genetic programs and interactive protein networks in a given environment. In this thesis, the influence of Netrin1 (NTN1), Human papilloma virus (HPV) E6/E7, and actin-bundling protein with BAIAP2 homology (ABBA)proteins on neural stem/progenitor cells cellular processes was studied and evaluated. In vitro methods were used to uncover the cellular processes regulated and affected by these proteins. In addition, in vivo methods were used to assess their possible impact on forebrain development in mice. NTN1 belongs to a conserved family of laminin-related molecules and it is present in the developing and adult mouse brain. NTN1 has been thought to act as a diffusible long or short-range guidance cue, which influences growing axons and migrating cells in either a chemoattractive or repulsive manner, and NTN1 deficiency in the brain causes defects in axon guidance and cell migration. The main issue of this thesis was to determine the developmental impact of NTN1 in the formation of two forebrain structures, the olfactory bulb and the corpus callosum (CC). Olfactory bulb processes arriving odour information from the nasal cavity and transmit this information forward to various brain regions. During development, the different cell types of olfactory bulb are generated in distinct germinal regions of the brain. Projection neurons are mainly generated by the olfactory bulb stem/progenitor cells whereas majority of the interneurons are produced by the progenitors, which migrate from the forebrain germinal zones into the olfactory bulb via rostral migratory stream (RMS). The origin of non-neural cells, astrocytes and oligodendrocytes, is not as well understood as neurons. We observed that NTN1 has a significant impact on the beginning of stem/progenitor cells migration from the forebrain germinal zones into the olfactory bulb. In more detail, the migration of Ntn1 expressing stem/progenitor cells was delayed, which led to an accumulation of these cells in RMS, and to a substantial reduction of GABAergic interneurons and oligodendrocytes in the olfactory bulb. Thus, the results suggest that NTN1 acts mainly as a detachment/release factor for the Ntn1 expressing stem/progenitor cells in the beginning of their migration and this function is mediated in either cell-autonomous or paracrine manner. CC is the largest axon tract in the forebrain and it integrates motor, sensory and cognitive performances between cerebral hemispheres. In mice, the cerebral hemispheres have to fuse in the midline before the commissural axons can cross the midline and form the CC. This interhemispheric fusion normally includes the removal of leptomeningeal cells found between the hemispheres and disruption of pial basal lamina, which is produced and maintained by the leptomeningeal cells. In Ntn1 deficient mice the leptomeningeal cells were not removed and the pial basal lamina remained intact in the hemispheric midline. Thus, the results suggest that NTN1 is required for the interhemispheric fusion, which precedes midline crossing of commissural axons and is a prerequisite for the formation of the CC. ABBA belongs to the Bin amphiphysin Rvs167 (BAR) protein superfamily, which regulates plasma membrane morphology by directly influencing plasma membrane assembly or through rearrangement of the actin cytoskeleton. The functional and expression studies revealed that ABBA participates in the regulation of cell protrusions by enhancing actin dynamics and connecting plasma membrane deformation to actin cytoskeleton. ABBA was present in radial glia-like cells and radial glial cell extensions near meningeal pial basal lamina during CNS development. During early development of brain, interaction between radial glial end-feet and pial basal lamina is required to sustain radial migration and integrity of pial basal lamina. Thus, ABBA may have an important role in formation of radial glial extensions and their connections in CNS. Exquisite balance between proliferation, self-renew and differentiation of stem/progenitor cells is necessary to maintain appropriate tissue homeostasis. HPV16 E6/E7 oncoprotein mediated degradation of p53 and pRb family of proteins seemed to facilitate a defective coupling of proliferation, self-renewal, and differentiation processes inside neural stem/progenitor cells. In addition, these processes also seemed to be susceptible to the environmental signals. Hence, a defective coupling of proliferation, self-renewal, and differentiation processes in neural stem/progenitor cells may lead to an imbalance in normal tissue homeostasis and abnormal growth of tissue in the brain. In summary, even though NTN1, HPV E6/E7, and ABBA proteins affected different cellular processes of neural stem/progenitor cells, all these cellular processes participate in the development of forebrain conformation and, therefore, the future functionality of it. In addition, defects in regulation of neural stem/progenitor cells cellular processes may lead to congenital neural disorders in mice and humans. Thus, these results also increase our overall comprehension of the developmental cellular processes behind the different neural congenital disorders and diseases.
  • Ola, Roxana (Helsingin yliopisto, 2012)
    Glial-cell-line-derived neurotrophic factor (GDNF) signaling through the receptor tyrosine kinase Ret and its co-receptor Gfrα1 is indispensable for the ureteric bud (UB) outgrowth from Wolffian ducts during kidney differentiation. To search for novel genes regulated by GDNF in Wolffian ducts and early UB epithelium, we performed a genome wide analysis of Wolffian ducts exposed by GDNF in vitro, and identified 69 up-regulated genes. Plakophilin2 gene encoding for the armadillo protein- Pkp2 and Visinin like 1 gene, encoding for a neuronal calcium sensor protein- VSNL1 were among the highly up-regulated genes and their expression was missing in the Gdnf- null kidneys. Analysis of the Pkp2- null allele kidneys revealed a defect in ureteric branching. The Pkp2-/- kidney explants did not undergo UB formation and branching in vitro, but responded to GDNF by producing supernumerary buds, suggesting that Pkp2 is not essential for UB formation from the Wolffian duct. VSNL1 characterized the tip domain of the UB epithelium regardless of the inducer. In the UB tips, VSNL1 displayed a unique mosaic expression pattern that demonstrated the heterogeneity of cells in the tip. VSNL1 expression was mutually exclusive with β-catenin activity as shown in Bat-gal Wnt-reporter mice. VSNL1 was downregulated in both β-catenin stabilized and β-catenin deficient kidneys. Moreover, VSNL1 transduction compromised β-catenin stability in cultured cells. The results suggest an antagonistic effect between VSNL1 and β-catenin. The spatial and temporal expression of VSNL1 and its mRNA was further addressed during mouse embryogenesis. Vsnl1 was specifically confined to the cardiac anlagen. All myocardial cells that contributed to the remodeling of the venous pole of the heart were positive for Vsnl1. An expansion of the VSNL1 expression domain to the ventricles was seen soon after the first week of postnatal life, and this pattern was maintained to adulthood. The function of PlexinB2 during renal development was finally addressed by analyzing the kidney phenotype in Plxnb2- null mice. These embryos displayed hypoplastic kidneys, and occasional unilateral double kidneys and ureters. The defect in branching was intrinsic to the ureteric epithelium. By co-immunoprecipitation experiments, PlexinB2 was shown to interact with Ret suggesting that PlexinB2, activated by Sema4C ligand, may modulate ureteric branching by a direct regulation of Ret activity.
  • Perälä, Nina (Helsingin yliopisto, 2011)
    Plexins (plxn) are receptors of semaphorins (sema), which were originally characterized as axon guidance cues. Semaphorin-plexin signalling has now been implicated in many other developmental and pathological processes. In this thesis, my first aim was to study the expression of plexins during mouse development. My second aim was to study the function of Plexin B2 in the development of the kidney. Thirdly, my objective was to elucidate the evolutionary conservation of Plexin B2 by investigating its sequence, expression and function in developing zebrafish. I show by in situ hybridisation that plexins are widely expressed also in the non-neuronal tissues during mouse development. Plxnb1 and Plxnb2, for example, are expressed also in the ureteric epithelium, developing glomeruli and undifferentiated metanephric mesenchyme of the developing kidney. Plexin B2-deficient (Plxnb2-/-) mice die before birth and have severe defects in the nervous system. I demonstrate that they develop morphologically normal but hypoplastic kidneys. The ureteric epithelium of Plxnb2-/- kidneys has fewer branches and a lower rate of proliferating cells. 10% of the embryos show unilateral double ureters and kidneys. The defect in the branching is intrinsic to the epithelium as the isolated ureteric epithelium grown in vitro fails to respond to Glial-cell-line-derived neurotrophic factor (Gdnf). We prove by co-immunoprecipitation that Plexin B2 interacts with the Gdnf-receptor Ret. Sema4C, the Plexin B2 ligand, increases branching of the ureteric epithelium in controls but not in Plxnb2-/- kidney explants. These results suggest that Sema4C-Plexin B2 signalling modulates ureteric branching in a positive manner, possibly through directly regulating the activation of Ret. I cloned the zebrafish orthologs of Plexin B2, Plexin B2a and B2b. The corresponding proteins contain the conserved domains the B-subfamily plexins. Especially the expression pattern of plxnb2b recapitulates many aspects of the expression pattern of Plxnb2 in mouse. Plxnb2a and plxnb2b are expressed, for example, in the pectoral fins and at the midbrain-hindbrain region during zebrafish development. The nearly complete knockdown of Plexin B2a alone or together with the 45% knockdown of Plexin B2b did not interfere with the normal development of the zebrafish. In conclusion, my thesis reveals that plexins are broadly expressed during mouse embryogenesis. It also shows that Sema4C-Plexin B2 signalling modulates the branching of the ureteric epithelium during kidney development, perhaps through a direct interaction with Ret. Finally, I show that the sequence and expression of Plexin B2a and B2b are conserved in zebrafish. Their knockdown does not, however, result in the exencephaly phenotype of Plxnb2-/- mice.
  • Vulli, Jaana (Helsingin yliopisto, 2013)
    A small set of highly conserved signaling molecules performs a great number of tasks in different animals and developmental contexts. Among them, the bone morphogenetic proteins (BMPs) constitute a group of growth and differentiation factors that are involved in numerous developmental processes affecting cell proliferation, apoptosis and differentiation. In the fruit fly, Drosophila melanogaster, three BMP type proteins have been identified, each of which has a homolog in mammals. Decapentaplegic (Dpp) is a BMP2/4 type protein which plays a major role in dorsal-ventral patterning of the early embryo. It participates in midgut development, patterning and growth of imaginal tissues, wing vein formation and maintenance of germline stem cells in the germarium. Dpp is a morphogen which requires a second BMP type protein, Screw (Scw) or Glass bottom boat (Gbb) to be able to form proper concentration gradients in developing tissues. Scw and Gbb belong to the BMP5/6/7/8 subfamily and their expression domains are different; Scw is specifically expressed during the early events of embryogenesis, while Gbb has more functional roles during later stages of fly development, like wing morphogenesis. BMP type proteins are produced as large proproteins that require proteolytic cleavage prior to secretion and extracellular gradient formation. This study concentrated on the cleavage of Dpp and Scw to reveal the meaning of post-translational modifications in concentration gradient formation and BMP signaling. Three furin recognition sites were identified in the Dpp proprotein. Mutational analyses indicate that the upstream optimal furin site of the prodomain (furin site (FS) II) is critical for producing ligands and creating a long range concentration gradient in a wing imaginal disc. Cleavage of the other two FSs produce the differently sized Dpp ligands that contribute to BMP gradient formation in the early embryo and wing imaginal disc. It was noted that the cleavage requirements of BMP2/4 type proteins in different species vary to establish species-specific regulation of BMP signaling. Discovery of the scwE1 allele, that causes dominant negative effect in embryos heterozygous for a hypomorphic dpp allele, gave more information about how the cleavage patterns of prodomains can contribute to creating diversity in the regulation of signaling. The mutation responsible for the dominant negative function in scwE1 was located in the cleavage site that is in the prodomain of Scw. Mutational analyses showed that the mature ligand of ScwE1 is produced in lower amounts and in complex with an N-terminal prodomain peptide. ScwE1 preferentially binds Dpp and disrupts normal gradient formation possibly through interactions with molecules within the extracellular matrix. Phylogenetic analyses and functional studies of BMP cleavage mutants propose a mechanism by which post-translational regulation of proproteins modulates BMP signaling.
  • Koskenranta, Anna (Helsingin yliopisto, 2020)
    Hammaspuutokset ovat yleisin kraniofakiaalinen anomalia. Ne voivat esiintyä sekä erillisinä anomalioina että oireyhtymien yhteydessä. Niiden vaikutus elämänlaatuun on negatiivinen muun muassa heikentyneen suunterveyden ja sosiaalisten suhteiden vaikeutumisen kautta. Pekka Niemisen tutkimusryhmä Helsingin yliopistosta löysi hammaspuutospotilaiden perimätutkimuksissa kaksi uutta mahdollisesti hammaspuutoksia aiheuttavaa geenimutaatiota geenissä RNF43. RNF43-proteiini on normaalisti toimiessaan osa hampaiden kehitykseen ja kasvuun vaikuttavan WNT-viestinnän negatiivista takaisinkytkentää. Mutaatioiden arvellaan häiritsevän hampaiden kehitystä WNT-viestinnän aktiivisuuden muuntelemisen kautta. Tutkimuksessamme tutkimme mutaatioiden vaikutusta RNF43-proteiinin määrään ja sijoittumiseen solussa ja pyrimme näin todistamaan mutaatioiden patogeenisyyden. Käytimme tutkimuksessa HEK293-soluja. Transfektoimme niitä villityypin ja kahden eri mutaation plasmideilla, jotka sisälsivät myös GFP-proteiinin. Värjäsimme niitä membraani- ja tumaväriaineilla, jotta pystyimme hahmottamaan GFP:n ja siten proteiinin sijaintia solussa fluoresenssi- ja konfokaalimikroskooppien avustuksella. Mutaatioilla ei havaittu olevan vaikutusta RNF43:n tuotannon määrään tai sijoittumiseen soluissa, emmekä näin ollen voineet tässä tutkimuksessa todentaa niiden patogeenisyyttä. Veimme tutkimuksellamme kuitenkin kyseisten mutaatioiden ja hammaspuutosten genetiikan tutkintaa eteenpäin. Tästä tutkimuksesta saatujen havaintojen pohjalta voidaan RNF43-geenin mutaatioiden tutkimusta jatkaa muilla menetelmillä.
  • Pummila, Marja (Helsingin yliopisto, 2009)
    Several organs of the embryo develop as appendages of the ectoderm, the outermost layer of the embryo. These organs include hair follicles, teeth and mammary glands, which all develop as a result of reciprocal tissue interactions between the surface epithelium and the underlying mesenchyme. Several signalling molecules regulate ectodermal organogenesis the most important ones being Wnts, fi broblast growth factors (Fgfs), transforming growth factor -βs (Tgf-βs) including bone morphogenetic proteins (Bmps), hedgehogs (Hhs), and tumour necrosis factors (Tnfs). This study focuses on ectodysplasin (EDA), a signalling molecule of the TNF superfamily. The effects of EDA are mediated by its receptor EDAR, an intracellular adapter protein EDARADD, and downstream activation of the transcription factor nuclear factor kappa-B (NF-кB). Mice deficient in Eda (Tabby mice), its receptor Edar (downless mice) or Edaradd (crinkled mice) show identical phenotypes characterised by defective ectodermal organ development. These mouse mutants serve as models for the human syndrome named hypohidrotic ectodermal dysplasia (HED) that is caused by mutations either in Eda, Edar or Edaradd. The purpose of this study was to characterize the ectodermal organ phenotype of transgenic mice overexpressing of Eda (K14-Eda mice), to study the role of Eda in ectodermal organogenesis using both in vivo and in vitro approaches, and to analyze the potential redundancy between the Eda pathway and other Tnf pathways. The results suggest that Eda plays a role during several stages of ectodermal organ development from initiation to differentiation. Eda signalling was shown to regulate the initiation of skin appendage development by promoting appendageal cell fate at the expense of epidermal cell fate. These effects of Eda were shown to be mediated, at least in part, through the transcriptional regulation of genes that antagonized Bmp signalling and stimulated Shh signalling. It was also shown that Eda/Edar signalling functions redundantly with Troy, which encodes a related TNF receptor, during hair development. This work has revealed several novel aspects of the function of the Eda pathway in hair and tooth development, and also suggests a previously unrecognized role for Eda in mammary gland development.
  • Harjunmaa, Enni (Helsingin yliopisto, 2012)
    Teeth display considerable morphological variability, which mammals have been able to use to their advantage. Consequently, mammal teeth provide a bountiful research subject that combines information on development, functional proper-ties, and thanks to their durable substance, evolutionary history. This thesis work is focused on the patterning of cusps, the peaks that form the shape of the tooth crown, in the mouse. Mouse tooth development has been studied extensively and offers a wide variety of established methods, including culture of embryonic teeth, which allows their observation and manipulation, and the mapping of gene expression patterns and protein distributions on histological sections. It has been established that teeth develop through a series of inductive interactions between the epithelium and the mesenchyme. The interactions are mediated by signalling molecules mostly belonging to the Wnt, Bmp, Fgf, and Shh families and are used similarly in the development of other organs. The growth of a tooth is controlled by epithelial signalling centres called enamel knots, each of which gives rise to a cusp. The patterning of enamel knots, and thus of cusps, can be modelled with reaction-diffusion dynamics, which suggests the patterning to be robust against interference yet capable of propagating change. As a semi-independent developmental module, teeth can vary without affecting the rest of the organism, an assumed prerequisite for evolvability. However, the use of tooth development in evolutionary studies has been hampered by a lack of mutations and manipulations causing small-scale variation. We have explored the dynamics of cusp patterning by studying mouse mutants with altered cusp patterns and by producing cusp pattern variation in cultured molars. In addition to taking advantage of established methods, we have shown Shhwt/GFPcre reporter molars to allow real-time observation of cusp patterning in culture, derived quantified data from developing molars, and imaged their three-dimensional structure at cellular resolution with X-ray scanning. Our results indicate that cusp patterning is controlled by feedback inhibition of enamel knot differentiation, and we identify Bmp, Activin, Eda, and Fgf20 as activators, and ectodin and Shh as inhibitors of differentiation. Each of these has slightly different functions and the correct regulation of all of these is required for normal cusp patterning. Bmp and ectodin, and Eda and Shh, seem to form feedback loops providing developmental stability. The manipulation of Eda signalling provided an opportunity to quantify development, revealing that variation increased in a linear fashion the further one deviated from the wild type level of signalling. Our results support the use of reaction-diffusion dynamics in modelling cusp patterning, but they also show that growth dynamics play an equally important role. Consequently, the evolution of crown shape can be followed cusp by cusp, and the developmental order of enamel knot induction closely corresponds to the evolutionary order of cusp appearance. Thus the mechanisms of molar development can be assumed to restrict, or channel, variation available to selection. In agreement with this, most of the molar features we generated have counterparts in extinct or extant rodent species. A general trend in evolution, evident also in molars, has been an increase in complexity. In the absence of experimental ways to repeat this phenomenon, its dynamics have remained elusive. In tuning Eda, Activin, and Shh signalling we found that an increase in cusp number correlated with the number of signalling pathways tuned simultaneously. Though intuitively obvious, the result had not previously been reported. Should an increase in complexity require multiple simultaneous changes in development as a rule, the overwhelming majority of reports on decreasing complexity, typically studying the effects of a single change at a time, would be explained. In conclusion, our results provide new information on the developmental genetic mechanisms of cusp patterning, how they provide developmental stability, and what kind of evolutionary constraints they cause.
  • Lefebvre, Sylvie (Helsingin yliopisto, 2012)
    Several embryonic organs, such as the hair follicle, develop as appendages of the ectoderm, the outermost layer of the embryo. These organs develop as a result of reciprocal tissue interactions between the surface epithelium and the underlying mesenchyme. The fi rst morphological sign of a developing hair follicle is a thickening of the epithelium called a placode. Several major signaling pathways are important for the development of hair and other ectodermal organs such as Wnts, fi broblast growth factors (Fgfs), Transforming growth factor-beta (TGF-beta), Hedgehogs (Hh) and tumor necrosis factors (TNFs). This thesis focuses on the role of TNFs in hair development and more particularly on one member of the TNF superfamily: Ectodysplasin (Eda). Mutations in Eda pathway components including the TNF ligand Eda, its receptor (Edar), and downstream effectors essential for activation of transcription factor NFbappaB in mouse or human give rise to a disease called hypohidrotic ectodermal dysplasia (HED). HED is an inherited disorder characterized by impaired development of ectodermal organs such as hair, teeth and several exocrine glands. A hallmark of mouse HED (Eda null mouse) is the absence of primary hair placodes that form at embryonic day 14 (E14). In order to identify the direct target genes of Eda, we have performed a microarray analysis on genes differentially expressed upon short exposure to recombinant Eda protein on Eda null skin at E14. Several of the genes identifi ed belong to the major signaling pathways mentioned above and interestingly, include also six chemokines that have not previously been associated with hair follicle morphogenesis. The purpose of this study was to validate whether the upregulated genes were truly transcriptional target genes of Eda/NFkappaB and to study their functional relevance in ectodermal organogenesis, in particular in hair follicle development. Based on these studies, we were able to confirm some Wnt pathway members, such as Dkk4 and Lrp4,TNF family member A20, and two chemokines, cxcl10 and cxcl11, as likely direct target genes of Eda. It is shown that whereas Dkk4 and Lrp4 are expressed in all ectodermal organs, A20 and the two chemokines seem to be hair follicle specific. Further study of Dkk4 and Lrp4 during development led us to conclude that Wnt and Eda pathways interact closely to fine tune the development of hair and other ectodermal organs. The role of A20 seems to be restricted to the termination of NFkappaB signaling induced by the Eda pathway in hair follicles. The lack of cxcl10 and cxcl11 signaling during hair follicle formation leads to more widely spaced hair placodes. This work has revealed an important role of Eda during hair placode induction as a modulator of inhibitors and activators of the major pathways in order to direct the patterning of hair placodes.