The past two decades have highlighted the utility in using the fruit fly Drosophila as a model organism for unravelling the molecular and functional complexities of two important fields of research: the systems that guide retinal determination (RD) and circadian rhythms (the daily body clock or oscillator). RD and clock factors are of great interest as they are: (1) highly conserved in vertebrates; (2) also essential for other physiological systems; (3) implicated in several congenital disorders and other diseases. The RD factors operate within a network in which several of their interactions have been described. Two such factors, eyes absent (eya) and sine oculis (so), are known to function as a unit to direct transcriptional regulation during photoreceptor (PR) differentiation. The regulation of eya and so by a transcriptional repressor at the heart of the clock mechanism, vrille (vri), is here investigated. Two distinct observations advocated exploration of a link between vri and eya/ so is of interest. (1) vri is a core component of the clock and interacts with RD but the RD function is unknown. (2) Recent evidence suggests that an RD factor directly upstream of eya and so, twin-of-eyeless (toy), interacts with the oscillator mechanism through direct and indirect pathways. It is possible that the indirect influences of toy on the oscillator are mediated via eya and/ or so. Interactions between eya, so and vri during RD and within oscillator cells are investigated here.Eye development function was studied using immunohistochemistry and transgenic manipulation. VRI is not expressed within the developed PRs; rather, expression of VRI is down-regulated prior to differentiation. In addition, conversely to the hypothesised role, VRI is co-expressed with EYA in some regions. Together with data from transgenic manipulation of VRI regional expression, I propose that VRI is predominantly part of a developmental pathway but can attenuate eya and so expression.The VRI binding site has been described previously and several sites were identified within eya/ so loci, some of which were tested in an in vitro binding assay. Two such sites were located adjacent to a known enhancer of so. I generated two transgenic fly lines containing: 1) an extension of the original enhancer to contain the VRI sites; and 2) a similar construct with the VRI sites ablated. Comparison of the original enhancer to those from the current study confirmed that the VRI sites attenuate expression and that intervening regions must contain binding sites for other transcription factors.In adult brains over a circadian light-dark cycle, EYA protein was expressed in three of the central brain clock neurones. Furthermore, expression of eya and so transcripts in adult heads, PRs and the brain, changed over the light/ dark cycle independently of the clock - indicating that their expression is modulated over the light-dark cycle but not by the oscillator mechanism. These data suggest interactions between eye development factors eya/ so and oscillator components, or, the light/ dark cycle exist. These interactions may be important for tissue-specific circadian physiology as well as the overall oscillator mechanism and offer an intriguing route for future investigation.

Cryopreservation is now a common practice in the fields of aquaculture, conservation and biomedicine. The success of cryopreservation is usually analysed in terms of cell survival, although there are other potential adverse effects that don’t necessarily result in cell death. These include DNA damage, which could result in altered gene expression. The aim of this study is to discover if cryopreservation has an impact on gene expression using zebrafish (Danio rerio) as the model organism. As the whole embryo cannot yet be successfully cryopreserved, isolated blastomeres from the embryos were used to investigate the impact of cryo-treatment on gene expression. This study sets out firstly to determine an optimum cryopreservation protocol for 50% epiboly blastomeres (Epiboly displaces the blastoderm margin to 50% of the distance between the animal and vegetal pole). Blastomeres had the highest survival level (70.2 ± 3.2%) when a mixture of 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose were used as cryoprotectants. As quantitative analysis of gene expression using real-time PCR requires the use of a housekeeping gene as an internal control to normalize date, the second study aimed to identify and validate housekeeping genes for use in cryopreservation studies of zebrafish blastomeres. Seven potential housekeeping genes were analysed across a range of embryo stages and isolated blastomeres using the GeNorm and NormFinder software packages. Results indicated β-actin and EF1α housekeeping genes to be the most appropriate for cryopreservation studies on zebrafish embryos and blastomeres, and these housekeeping genes were used in the third study, the effect of cryopreservation on Pax gene expression. The results indicated that the trends (profile changes) in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryos which could have a detrimental effect on embryo development. This is the first study on the stability of housekeeping and transcription factor genes in chilled and cryopreserved embryonic cells of the zebrafish. This work will significantly enhance future studies investigating the impact of cryopreservation on gene expression in zebrafish embryos.

In this study, full coding sequences of Atlantic salmon (Salmo salar L.) muscle genes were cloned, including myogenic regulatory factors (MRFs) (myod1c, myog, mrf4, myf5), inhibitors of Myostatin (fst, decorin), markers of myogenic progenitor cell (MPC) proliferation (sox8) and fusion (calpastatin), a marker of slow muscle fibre differentiation (smlc1) and a novel eukaryotic gene involved in regulating growth (cee). Several of these genes were then characterised using a range of experimental and computational analyses with the aim to better understand their role in myogenesis and their evolution in teleosts. A series of experiments supported previous findings that teleosts have extra copies of many genes relative to tetrapods as a result of a whole genome duplication (WGD) event that occurred some 320-350 Mya. For example, it was shown that genes for myod and fst have duplicated in a common teleost ancestor, but were then specifically lost or retained in different lineages. Furthermore, several characterised Atlantic salmon genes were conserved as paralogues, likely from a later WGD event specific to the salmonid lineage. Phylogenetic reconstruction and comparative genomic approaches were used to characterise the evolution of teleost paralogues within a framework of vertebrate evolution. As a consequence of one experiment, a revised nomenclature for myod genes was proposed that is relevant to all diploid and polyploid vertebrates. The expression patterns of multiple myogenic genes were also established in Atlantic salmon embryos using specific complementary RNA probes and in situ hybridization. For example, co-ordinated embryonic expression patterns were revealed for six salmon MRFs (myod1a, myod1b, myod1c, myog, mrf4, myf5), as well as markers of distinct MPC populations (pax7, smlc1), providing insight into the regulatory networks governing myogenesis in a tetraploid teleost. Furthermore, it was shown that Atlantic salmon fst1 was expressed concurrently to pax7 in a recently characterised MPC population originating from the anterior domain of the epithelial somite, which is functionally analogous to the amniote dermomyotome. In another experiment, the individual expression domains of three Atlantic salmon myod1 paralogues were shown to together recapitulate the expression of the single myod1 gene in zebrafish, consistent with the partitioning of ancestral cis-acting regulatory elements among salmonid myod1 duplicates. Additionally, the in situ expression of cee a novel and highly conserved eukaryotic gene was revealed for the first time in a vertebrate and was consistent with an important role in development including myogenesis. Additionally, Atlantic salmon were reared at 2, 5, 8 or 10 ºC solely to a defined embryonic stage, which was just subsequent to the complete pigmentation of the eye. After this time, animals were provided an equal growth opportunity. Remarkably, changing temperature during this short developmental window programmed the growth trajectory throughout larval and adult stages. While 10 and 8 ºC fish were larger than those reared at 2 and 5 ºC at the point of smoltification, strong compensatory growth was subsequently observed. Consequently, after 18 months of on growing, size differences among 5, 8 and 10 ºC fish were not significant, although each group was heavier than 2 ºC fish. Furthermore, significant embryonic-temperature induced differences were observed in the final muscle fibre phenotype, including the number, size distribution and myonuclear density of muscle fibres. A clear optimum for the final muscle fibre number was observed in 5 ºC fish, which was up to 17% greater than other treatments. In a sub-sample of embryos, temperature induced heterochonies were recorded in the expression of some MRFs (myf5, mrf4) but not others (myod1a, myog). These results allowed the proposition of a potential mechanism explaining how temperature can program the muscle phenotype of adult teleosts through modification of the somitic external cell layer, a source of MPCs throughout teleost ontogeny.

Interferon (IFN)τ is the luteotrophic signal in ruminants and is secreted by bovine blastocysts both in vivo and in vitro. IFNτ secretion is highly variable and its control is only partly understood. Most studies on the effects of environmental factors on IFNτ production have evaluated IFNτ production during the time of embryo elongation and attachment. There is less knowledge of how IFNτ production at the blastocyst stage is modulated. Therefore, the hypothesis of this thesis was that the amounts of IFNτ expressed and/or secreted by bovine blastocysts produced in vitro were modulated by environmental factors. In the first set of experiments, bovine embryos were incubated with a cytokine (granulocyte macrophage colony stimulating factor, GM-CSF). GM-CSF had been shown previously to promote embryo viability in a range of species and to modulate IFNτ secretion by ovine blastocysts and thus was classified as a beneficial environmental factor. Three experiments were conducted to test whether GM-CSF stimulated bovine blastocyst development and IFNτ secretion. Embryos were incubated with a range of different concentrations of GM-CSF (2, 5, 10 and 50 ng mL-1) and at different stages of development (1 to 3 and 1 to 9 days post-insemination). Bovine embryos were unresponsive to GM-CSF in terms of IFNτ secretion, pyruvate oxidation, rate of development, blastocyst yield, morphological quality and apoptotic index, irrespective of timing of exposure and/or concentration of GM-CSF. In the second part of the thesis, bovine blastocysts were exposed to a mild heat treatment (42°C for four h) to determine whether heat stress affected IFNτ expression by bovine blastocysts. A novel multiplex reverse-transcription polymerase chain reaction methodology was validated to detect IFNτ and heat shock protein (HSP)70 mRNA in individual bovine embryos relative to an endogenous gene (YWHAZ) and an exogenous mRNA (α-globin) and results were expressed both in absolute terms and in relation to the endogenous control. Heat treatment upregulated IFNτ mRNA expression, suggesting that detrimental environmental factors may influence IFNτ expression. Heat treatment also caused an increase in HSP70 mRNA expression but did not affect blastocyst morphology, suggesting that the level of stress caused by the heat treatment was great enough to activate the cellular stress response, but mild enough not to cause a change in morphology. In addition, the positive correlation between HSP70 and IFNτ transcript levels and the higher IFNτ expression by embryos which showed signs of degeneration and collapse compared to those which progressed in development suggested that IFNτ expression may be indicative of stress. The relationship between IFNτ expression and secretion in vitro with morphology, pyruvate metabolism, apoptotic index and cell number was inconsistent, suggesting that IFNτ production did not correlate with ‘quality’ (defined as an index of viability). Blastocyst yield, day of blastulation and change in morphology index did account for at least part of the variation in IFNτ production, suggesting that some intrinsic factors may regulate IFNτ secretion. These intrinsic factors, however, did not explain all the variation in IFNτ secretion between blastocysts. Therefore, the amount of IFNτ secreted by bovine blastocysts is modulated by both intrinsic and environmental factors. A model was proposed where different levels of stress affect survivability to different extents, and the ability to respond to mild levels of stress may be indicative of improved survivability.

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.

Midline convergence of organ primordia is an important mechanism for shaping the vertebrate body-plan at various stages of development, such as the morphogenesis of the heart and endoderm. Down regulation of wnt or noncanonical wnt signalling components, such as dishelleved (Dvl) or RhoA GTPase (RhoA), impairs midline convergence of the heart primordia and endoderm in zebrafish. This suggests that wnt signaling plays an important role in regulating midline convergence. At the early patterning stage of the zebrafish kidney, the two pronephric-glomerular primodia (PGP), which derive from intermediate mesoderm, converge towards the midline and fuse to form a functional pronephros. In contrast, during development of the mammalian kidney, the pronephros degenerates as the mesonephros develops without midline convergence. The hypothesis is thus that there is/are mechanisms underlying midline convergence of PGP in zebrafish, which is/are in addition to the control of the non-canonical wnt/Dvl/RhoA pathway and specific to kidney morphogenesis. In this study, the aim was to identify genetic factors that are specifically involved in the mechanism of PGP midline convergence by establishing a cell lineage tracing transgenic system with Cre-loxP, followed by the analysis of selected mutant embryos using the cell lineage tracing system, whole-mount in situ hybridization (WISH) and immunostaining. The cell lineage tracing system was generated, and tested. Constructs, in which β-actin promoter drives the transcription of the reporter genes, were microinjected into zebrafish embryos at 1- to 4-cell stages. The mRNAs in microinjected embryos (5 dpf) were analyzed by RT-PCR. The results show that the constructs induced indiscriminate alternative splicing. RNA splicing mechanisms were not affected by transcription termination when the polyA signal was located in introns. To provide an alternative approach, three mutants were selected after screening of available ENU zebrafish mutants. These mutants were chosen not only because of their genetic importance in cell adhesion and motility but also because of their respective developmental defects in tissues surrounding the PGP, such as the notochord (no tail, ntl), somite (spadetail, spt), and endodermal tissues (zygotic oneeyed pinhead, Zoep). Spt and ntl are key targets of fibroblast growth factor (FGF) signalling in the trunk and tail respectively. EGP-CFC gene oep is a Nodal signalling cofactor. Firstly, a rapid genotyping technique was developed, which was applied in identifying the mutant alleles. Since the tools for tracing PGP using transgenes were unavailable, the three mutants were analyzed by WISH and immunostaining. Zygotic mutation of Zoep causes a PGP midline convergence phenotype of variable severity due to maternal Oep effects. In more than 90% of Zoep-/- embryos, PGP midline convergence was impaired. Even though the abnormality could be observed as early as 15 hpf, the differentiation of the PGP was not affected. Heart primordial phenotypes were also observed but they did not correlate with that of the PGP phenotypes. Embryos homozygous for mutations in T-box transcription factors, ntl or spt had normal heart midline convergence phenotypes. PGP midline convergence abnormality was observed in spt-/- but not in ntl-/- prior to 36 hpf. In addition the extracellular matrix (ECM) might play a key role in the mechanisms of PGP midline convergence. Furthermore, PGP midline convergence proceeds from 10 hpf (the specification of intermediate mesoderm) to 48 hpf (fused pronephric glomerulus) in wild type zebrafish embryos. The process was quantified by 2D image analysis of the PGP distance. Prior to 18 hpf, PGP midline convergence is closely correlated with the midline convergence of mesoderm but not at later stages in Zoep-/-. Spt is essential for PGP but not for cardiac primordium midline convergence. Data from this research suggests that there is not one universal mechanism, which controls all the midline convergence of organ primordia. Indeed, specific factors, which depend on tissues and development stages, are also required.

The extraembryonic endoderm of mammals is essential for nutritive support of the foetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimaeras. Here, I further characterise XEN cells and show that these cell lines exhibit high levels of heterogeneity. In an effort for XEN cells to adopt visceral endoderm character different aspects of the in vivo environment were mimicked. I found that BMP4 and laminin promote a mesenchymal-to-epithelial transition of XEN cells with upregulation of epithelial markers and downregulation of mesenchymal markers. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm. Correspondingly, inhibition of Erk and BMP signalling drives XEN cells toward parietal endoderm fate. Finally, I show that BMP4 treatment of freshly isolated parietal endoderm from Reichert’s membrane promotes its visceral endoderm differentiation. This suggests that parietal endoderm is still developmentally plastic and can be transdifferentiated to a visceral endoderm in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.

This thesis investigates the use of micro-injection moulding (μIM), as a high-volume process, for producing three-dimensional, integrated microfluidic devices. It started with literature reviews that covered three topics: μIM of thermoplastic microfluidics, designing for three-dimensional (3-D) microfluidics and functional integration in μIM. Research gaps were identified: Designing 3-D microfluidics within the limitations of μIM, process optimisation and the integration of functional elements. A process chain was presented to fabricate a three-dimensional microfluidic device for medical application by μIM. The thesis also investigated the effect of processing conditions on the quality of the replicated component. The design-of-experiments (DOE) approach is used to highlight the significant processing conditions that affect the part mass taking into consideration the change in part geometry. The approach was also used to evaluate the variability within the process and its effect on the replicability of the process. Part flatness was also evaluated with respect to post-filling process parameters. The thesis investigated the possibility of integrating functional elements within μIM to produce microfluidic devices with hybrid structures. The literature reviews highlighted the importance of quality control in high-volume micromoulding and in-line functional integration in microfluidics. A taxonomy of process integration was also developed based on transformation functions. The experimental results showed that μIM can be used to fabricate microfluidic devices that have true three-dimensional structures by subsequent lamination. The DOE results showed a significant effect of individual process variables on the filling quality of the produced components and their flatness. The geometry of the replicated component was shown to have effect on influential parameters. Other variables, on the other hand, were shown to have a possible effect on process variability. Optimization statistical tools were used to improve multiple quality criteria. Thermoplastic elastomers (TPE) were processed with μIM to produce hybrid structures with functional elements.

Plusieurs composés présents dans les solutions de cryoconservation pour embryons sontsource de préoccupations : soit du point de vue sanitaire à cause de leur origine animale, soitdu fait de leur rôle potentiellement mutagène, notamment pour certains cryoprotecteurspénétrants. Leur retrait ou leur substitution par des composés chimiquement définis pourraitdonc constituer une amélioration sensible des techniques de cryoconservation des embryons.C’est dans ce cadre de réflexion que s’inscrit notre travail.Souvent, la conception et la mise en oeuvre des protocoles de cryoconservation sontempiriques. Il en résulte de nombreuses variations entre les études qui rendent lacomparaison des résultats obtenus d’autant plus difficile. Dans notre démarche, deuxapproches complémentaires ont été associées :•La première, physique, s’appuie sur l’utilisation de la calorimétrie différentielle à balayage(DSC) pour standardiser la comparaison des différentes solutions de congélation lente.Ainsi, les propriétés thermodynamiques de solutions contenant un substituant défini ontété caractérisées et comparées à celles de solutions contenant des produits de référence(sérum de veau foetal ou albumine bovine sérique) ;• La seconde, biologique, a consisté à congeler des embryons de lapins produits in vivo etdes embryons bovins produits in vitro, puis à analyser les taux de survie après culture invitro et/ou transferts. Cette approche a permis d’objectiver les propriétés biologiques dessolutions ainsi définies.Nos résultats confirment qu’il est judicieux d’utiliser une approche thermodynamiquepour sélectionner des molécules chimiquement différentes des composés habituellementutilisés. / Several compounds in embryo cryopreservation solutions are a source of concern:products of animal origin because of the sanitary risks, and the permeating cryoprotectantsbecause of their potential mutagenic effect. Removing or substituting these compounds withchemically defined products might improve embryo cryopreservation technics.Conception and use of cryopreservation protocols are often empirical. This empiricismleads to many variations between the studies which make a comparison between results allthe more difficult. In our study, two complementary approaches were associated:• The first approach (physical) consisted of using the differential scanning calorimetry tostandardize the comparison between different slow-freezing solutions. So, thethermodynamic properties of solutions containing a potential substitute were characterizedand compared to those obtained with solutions containing reference products (fetal calfserum and bovine serum albumin) ;• The second approach (biological) consisted of using freezing of in vivo-produced rabbitembryos or freezing of in vitro-produced bovine embryos in order to evaluate survival