Dispersal is a key trait across biology. Within plants, a variety of explosive seed dispersal mechanisms are seen. Whilst ecological and mechanical studies have described this important evolutionary adaptation in many species, a genetic and developmental understanding of explosive seed dispersal is lacking. In this thesis, the morphology and development of the explosive seed pods of Cardamine hirsuta – a member of the Brassicaceae – are characterised in detail, with reference to its close relative, the model organism A. thaliana. Comparison of fruit morphology between these two species and across other Brassicacean species generated hypotheses regarding the function and polarity of morphological features. In order to identify genes that are necessary for C. hirsuta fruit development, a genetic screen was conducted and a range of mutants identified and subsequently characterised. Analysis of the indehiscent valveless (val) mutant revealed a loss of valve tissue and an expansion of valve margin identity in the silique. Mapping and sequencing identified a mutation in the MADS-box gene FRUITFULL (FUL), which results in a truncated protein, as the likely cause of the val phenotype. Consideration of ful mutants in C. hirsuta and A. thaliana allowed comparison of the genetic patterning of the fruit dehiscence zone in these two species. The genetic interactions between fruit mutants characterised in this thesis and mutants in shoot patterning genes revealed common regulatory networks underlying leaf and fruit development in C. hirsuta. Together, comparison of wild-type and mutant C. hirsuta siliques with those of A. thaliana and other Brassicacean species suggests that specialised cell layers within the valve silique region are of key importance to C. hirsuta’s explosive dehiscence mechanism.

smig-1(ev809) is a novel suppressor of multiple distal tip cell (DTC) migration mutants in the nematode Caenorhabditis elegans. In the C. elegans hermaphrodite, the two U-shaped gonad arms develop and form as a result of the migration of two DTCs. smig-1(ev809) suppresses DTC migration defects of mutants encoding components of intracellular glycosylation pathways as well as extracellular basement membrane glycoproteins. The smig-1(ev809) mutation bypasses the requirement for a fully functional chondroitin pathway and MIG-17 metalloprotease in DTC pathfinding. I found that i) suppression of the hypomorphic chondroitin mutant mig-22(k141) is not completely dependent on MIG-17 activity; ii) the smig-1(ev809) mutant is likely to be a loss- of-function suppressor; and iii) SMIG-1 does not visibly affect the localization of poorly glycosylated MIG-17 on the gonad surface. Understanding SMIG-1 function will shed light on the role of glycosylation, the extracellular matrix and basement membranes in cell migration during development.

smig-1(ev809) is a novel suppressor of multiple distal tip cell (DTC) migration mutants in the nematode Caenorhabditis elegans. In the C. elegans hermaphrodite, the two U-shaped gonad arms develop and form as a result of the migration of two DTCs. smig-1(ev809) suppresses DTC migration defects of mutants encoding components of intracellular glycosylation pathways as well as extracellular basement membrane glycoproteins. The smig-1(ev809) mutation bypasses the requirement for a fully functional chondroitin pathway and MIG-17 metalloprotease in DTC pathfinding. I found that i) suppression of the hypomorphic chondroitin mutant mig-22(k141) is not completely dependent on MIG-17 activity; ii) the smig-1(ev809) mutant is likely to be a loss- of-function suppressor; and iii) SMIG-1 does not visibly affect the localization of poorly glycosylated MIG-17 on the gonad surface. Understanding SMIG-1 function will shed light on the role of glycosylation, the extracellular matrix and basement membranes in cell migration during development.

This thesis was inspired by the difficulties of artificial evolutionary systems in finding elegant and well structured, regular solutions. That is that the solutions found are usually highly disorganized, poorly structured and exhibit limited re-use, resulting in bloat and other problems. This is also true of previous developmental evolutionary systems, where structural regularity emerges only by chance. We hypothesise that these problems might be ameliorated by incorporating repeated evaluations on increasingly difficult problems in the course of a developmental process. This thesis introduces a new technique for learning complex problems from a family of structured increasingly difficult problems, Evolutionary Developmental Evaluation (EDE). This approach appears to give more structured, scalable and regular solutions to such families of problems than previous methods. In addition, the thesis proposes some bio-inspired components that are required by developmental evolutionary systems to take full advantage of this approach. The key part of this is the developmental process, in combination with a varying fitness function evaluated at multiple stages of development, generates selective pressure toward generalisation. This also means that parsimony in structure is selected for without any direct parsimony pressure. As a result, the system encourages the emergence of modularity and structural regularity in solutions. In this thesis, a new genetic developmental system called Developmental Tree Adjoining Grammar Guided Genetic Programming (DTAG3P), is implemented, embodying the requirements above. It is tested on a range of benchmark problems. The results indicate that the method generates more regularly-structured solutions than the competing methods. As a result, the system is able to scale, at least on the problem classes tested, to very complex instances the system encourages the emergence of modularity and structural regularity in solutions. In this thesis, a new genetic developmental system called Developmental Tree Adjoining Grammar Guided Genetic Programming (DTAG3P), is implemented, embodying the requirements above. It is tested on a range of benchmark problems. The results indicate that the method generates more regularly-structured solutions than competing methods. As a result, the system is able to scale, at least on the problem classes tested, to very complex problem instances.

A major goal in biology is to understand the genetic basis of morphological variation at different evolutionary scales, for example between and within species. Here I investigate this issue by using plant leaves as an example. Previously comparative studies between the simple leaf model plant Arabidopsis thaliana and its dissected leaf relative Cardamine hirsuta have shown that inter-specific differences in leaf shape mostly result from variation in local tissue growth and patterning (Vlad et al., 2014; Hay et al., 2006; Barkoulas et al., 2008). Here, I aim to elucidate the genetic basis of natural variation in leaf form within species, by using divergent strains of C. hirsuta. I present evidence that variation in six strains collected from geographically diverse locations results from different rates of progression of an age-dependent leaf development programme in a phenomenon known as heteroblasty. By using Quantitative trait loci (QTL) mapping with a recombinant inbred line (RIL) population derived from a cross between the Oxford and Azores strains, I detected six QTL that influence leaflet production on multiple leaves. A QTL located on the 4th linkage group was validated and selected for further analysis. Characterisation of QTL effect indicated that the QTL influences leaf form by altering the rate of heteroblastic development. Subsequently I fine mapped this QTL to a DNA segment of 48 kb containing the gene SQUAMOSA PROMOTER PROTEIN BINDING LIKE 9 (ChSPL9), a previously characterised regulator of age dependent development. The parental alleles of ChSPL9 show variation in their sequence and were transformed into A. thaliana to evaluate whether they contribute to the QTL effect. Resultant phenotypes mirrored the QTL effect suggesting that ChSPL9 does indeed contribute to this QTL effect. These results indicate that age-dependent leaf shape progression underlies variation in leaflet number within species and more broadly suggest that in the case of plant leaves different processes might underlie morphological variation between and within species.

<p>Down syndrome (DS) is a genetic disorder that results from triplication of human chromosome 21 (Hsa21) and occurs in around 1 in 1000 live births. All individuals with DS present with skeletal abnormalities typified by craniofacial features, short stature and low bone mineral density (BMD). Differences between males and females with DS suggest a sexual dimorphism in how trisomy affects skeletal deficits associated with trisomy 21 (Ts21). Previous investigations of skeletal abnormalities in DS have varied methodology, sample sizes and ages making the underlying causes of deficits uncertain. Mouse models of DS were used to characterize skeletal abnormalities, but the genetic and developmental origin remain unidentified. Over-expression <i>Dyrk1a</i>, found on Hsa21 and mouse chromosome 16 (Mmu16) has been linked to cognitive deficits and skeletal deficiencies. Dp1Tyb mice contain three copies of all of the genes on Mmu16 that are homologous to Hsa21, males and females are fertile, and therefore are an excellent model to test the hypothesis that gene dosage influences the sexual dimorphism of bone abnormalities in DS. Dp1Tyb at 6 weeks 16 weeks showed distinctive abnormalities in BMD, trabecular architecture, and reduced bone strength over time that occur generally through an interaction between sex and genotype. Increased gene dosage and sexual dimorphism in Dp1Tyb mice revealed distinct phenotypes in bone formation and resorption. To assess how <i>Dyrk1a</i> influences the activity and function of osteoblasts Ts65Dn female trisomic mice, female mice with a floxed <i>Dyrk1a</i> gene (Ts65Dn, <i>Dyrk1a</i>^fl/+) were be bred to <i>Osx1</i>-GFP::Cre+ mice to generate Ts65Dn animals with a reduced copy of <i>Dyrk1a </i>in mature osteoblast cells. Female Ts65Dn,<i>Dyrk1a^+/+/+</i>and Ts65Dn,<i>Dyrk1a^+/+/-</i>displayed significant defects in both trabecular architecture and cortical geometry. Ultimate force was reduced in trisomic animals, suggesting whole bone and tissue level properties are not adversely affected by trisomy. Reduction of <i>Dyrk1a</i> functional copy number in female mice did not improve skeletal deficits in an otherwise trisomic animal. <i>Dyrk1a </i>may not alter osteoblast cellular activity in an autonomous manner in trisomic female mice. These data establish sex, gene dosage, skeletal site and age as important factors in skeletal development of the skeleton in DS mice, potentially paving the way for identification of the causal dosage-sensitive genes in both male and female animals. </p>

<div>Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. </div><div>To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.</div><div><br></div>

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

<p> Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population.</p> <p> The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. </p> <p> The gene <i>Dual-specificity tyrosine-regulated kinase 1a </i>(<i>Dyrk1a</i>) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic <i>Dyrk1a</i> is related to the emergence of trabecular defects at P30, expression of <i>Dyrk1a</i> in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30.</p> <p> The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background.</p> These data led to the hypothesis that reduction of <i>Dyrk1a</i> gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce <i>Dyrk1a</i> gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic <i>Dyrk1a</i> does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.