Wheat (Triticum aestivum L.) zygotic embryogenesis occurs in a dynamically regulated ovular environment, and in ovulohormones regulate embryogenic processes. Levels of ABA, IAA, and the cytokinins Z, ZR, DHZ, DHZR, iP, and iPA were studied in developing wheat kernels from anthesis to maturity . High cytokinin and low IAA and ABA levels were associated with the early stage of embryo formation and active tissue histodifferentiation. Following histodifferentiation, cytokinin levels declined while IAA accumulated throughout the stage of active grain growth and then declined with grain maturity. ABA levels increased at the soft-dough developmental stage and through to grain maturity. Endogenous +ABA levels in developing wheat grains treated with fluridone, which indirectly blocks ABA synthesis, declined at the soft-dough stage. As a result, mature desiccated fluridone-treated kernels exhibited little dormancy. However, fluridone-treated kernels were not viviparous, suggesting a strong caryopsis-embryo interaction in maintaining embryogenically competent tissues. Induction of embryogenically competent wheat callus cultures was highly variable between genotypes and pre-initiation environments. Genotypic and environmental influences altered endogenous hormone levels and affected embryogenic competence. Establishment of competent embryo explants for somatic embryo induction was favored by a high cytokinin-to-auxin ratio and very low ABA levels throughout histodifferentiation (around 4 to 8 DPA). Similar to embryos forming within the caryopses, competent callus cultures had a high cytokinin (Z)-to-auxin ratio at 7 DPI. Increased frequency of embryogenic cultures was achieved when embryo explants were excised during a narrow window of low hormone levels. Wheat line and pre-initiation environment affected this window. Simulation in vitro of the in ovulo wheat kernel environment improved zygotic embryogenesis in vitro . Embryos exposed to physiologically normal ABA levels and low 0 2 tensions of 2.5 mM (7%) most closely approached morphological and physiological normalcy. The culmination of these studies clearly defines windows of embryo development for explant excision, associated roles of plant hormones in embryogenesis, and in ovulo hormone levels that vastly improve the frequency of successful embryogenesis when simulated in in vitro culture systems.

Research Doctorate - Doctor of Philosophy (PhD) / Somatic embryogenesis (SE) can be induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but not in wild type Jemalong. Wild-type Jemalong will only form callus in the presence of auxin and cytokinin and both Jemalong and 2HA will form roots in response to auxin alone. The F2 analysis of 2HA X Jemalong crosses suggest that a single gene may open the way to SE but additional genes are required to maximise the process. Auxin and cytokinin are required for 2-3 weeks for SE and for de novo root formation auxin is essential for about one week. Abscisic acid (ABA) and ethylene, both stress related hormones, enhance SE induction but inhibit callus and de novo root formation. The WUSCHEL (WUS) gene was investigated in M. truncatula (Mt) and identified by the similarity with Arabidopsis WUS in amino acid sequence, phylogeny, promoter element patterns, and expression patterns in planta. MtWUS is induced by cytokinin after 24-48 h in embryogenic cultures and maximum expression occurs after 1 week which coincides with totipotent stem cell induction. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localises to the embryo and corresponds to the onset of MtCLV3 expression. RNAi studies show that MtWUS expression is essential for callus and somatic embryo production. There is evidence based on the presence of MtWUS promoter binding sites that MtWUS is required for the induction of MtSERF1 which appears to have a key role in the signalling required for SE induced in 2HA. MtWOX5, as for MtWUS, was identified by similarity to Arabidopsis WOX5 based on amino acid sequence, phylogeny, promoter element patterns, and expression patterns in planta. MtWOX5 expresses in the auxin induced root primordium and root meristem and appears to be involved in pluripotent stem cell induction. GA suppresses the MtWOX5 expression in the root apex and suppresses the root primordium induction, consistent with the importance of MtWOX5 in in vitro root formation. The evidence is discussed that the homeotic genes MtWUS and MtWOX5 are “hijacked for stem cell induction which is key to somatic embryo and de novo root induction. In relation to SE, a key role for WUS in the signalling involved in induction is discussed and a model developed.

In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.

Hindsight (HNT) is a zinc finger transcription factor that is required for morphogenesis of the Drosophila embryo, having roles in germ band retraction (GBR) as well as dorsal closure (DC). HNT expression is also found in sensory organ precursors (SOP) of the developing pupal peripheral nervous system, and muscle progenitor cells, but the role of HNT in neurogenesis and myogenesis during embryogenesis has not been investigated in any depth. Microarray analysis of embryos over-expressing HNT during GBR and DC identified 1290 genes with significant changes in expression. This data set included many potential HNT targets, including genes associated with myogensis, and a disruption of muscle development was observed in embryos over-expressing HNT. It is possible that HNT may function to repress muscle identity genes in muscle founder cells. In addition, HNT over expressing embryos were found to resemble the neurogenic class of mutants. Among the potential target genes, D-Pax2 (shaven, sparkling, CG11049) expression, which is known to be expressed in the developing peripheral nervous system, was confirmed to be up-regulated following HNT over-expression. Interestingly, D-Pax2 and HNT expression were found to co-localize at the onset of their expression at stages 10-12 in embryos, but were not co-localized in later stages of embryogenesis. The up-regulation of D-Pax2 by HNT over-expression was further characterized and was found to be associated with strong ectopic HNT expression. The relevance of HNT to the regulation of D-Pax2 during normal development remains to be determined, but it is possible that endogenous expression of HNT is involved in D-Pax2 repression.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Triticum aestivum is of major importance both nutritionally and economically globally. Traditional breeding mechanisms have been unsuccessful at keeping pace with the increasing demand for better yielding and more resilient wheat varieties. The introduction of foreign genes into systems has provided a new tool for crop improvement, but has been difficult to apply to elite wheat varieties mainly as result of their recalcitrance to prerequisite tissue culture. Investigations here demonstrate that modification of the ammonium nitrate content in direct somatic embryogenesis induction medium can increase the number of primary embryos produced by over two fold in the elite hard red wheat cultivar Superb. The number of primary embryos which were capable of transitioning into shoot development also increased by two fold. Biolistic transformation efficiency was also improved when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. / x, 81 leaves : ill. ; 29 cm.

A common event during in vitro morphogenesis (either embryogenesis or shoot organogenesis) is the ability of somatic cells within the explants to de-differentiate and acquire “meristematic identity”. The developmental program of such meristematic cells can then be re-routed to form shoots or embryos depending on the imposed culture environment. The objective of this research is to investigate how the altered expression of Brassica genes regulating meristematic activity in vivo affects in vitro morphogenesis. It is predicted that ectopic expression of positive regulators of the shoot apical meristem, SHOOT MERISTEMLESS (STM) and ZWILLE (ZLL) which increase the pool of meristematic cells within the apical meristem, has a beneficial effect on somatic embryogenesis and shoot organogenesis. Conversely the over-expression of CLAVATA1 (CLV1), a negative regulator which depletes the pool of meristematic cells, should inhibit both processes. Over-expression of the Brassica STM in Arabidopsis enhanced the production of somatic embryos and shoots in vitro possibly by reducing the requirement of the tissue for exogenous auxin, which is the inductive signal for the production of embryogenic and organogenic cells. This was also accompanied by profound alterations in gene expression patterns affecting components of DNA methylation and glutathione metabolism, which are beneficial for embryo formation. The introduction of STM also enhanced Arabidopsis shoot organogenesis through profound transcriptional changes in cytokinin signalling. While the ectopic expression of the Brassica CLV1 inhibited both somatic embryogenesis and shoot organogenesis, the expression of ZLL had no effects on the production of somatic embryos but encouraged the formation of shoots. Taken together these results suggest the existence of similar genetic mechanisms regulating the formation of meristem cells in vivo and embryogenic/organogenic cells in vitro.

Hemoglobins (Hbs) are heme-containing proteins belonging to the globin superfamily that are ubiquitous in most living organisms including prokaryotes and eukaryotes. In addition to the first legHbs found in leguminous plants, there are another three classes of phytoglobins (Pgbs) identified in various plant species including dicots and monocots. The ability of heme groups to bind gaseous ligands such as oxygen, carbon monoxide and nitric oxide (NO) places Pgbs as multifunctional players in various processes during plant growth and development under normal or stress conditions. The objective of this project is to investigate how transcriptional manipulation of ZmPgb1.1 and ZmPgb1.2 influences somatic embryogenesis in maize (Zea mays). Suppression of either of the two genes is sufficient to induce programmed cell death (PCD) through a pathway initiated by accumulation of nitric oxide (NO) and zinc (Zn2+), and mediated by production of reactive oxygen species (ROS). The effect of the death program on the fate of the developing embryos is dependent upon the localization patterns of the two Pgbs. During somatic embryogenesis, ZmPgb1.2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, while ZmPgb1.1 transcripts extend to several embryonic domains. Suppression of ZmPgb1.2 induces PCD in the anchoring cells allowing the embryos to develop further, while suppression of ZmPgb1.1 results in massive PCD leading to embryo abortion. Cells suppressing the Pgb genes are also depleted of endogenous auxin (indole-3-acetic acid, IAA) localization established by polar auxin transport (PAT), thus suggesting a possible involvement of this plant hormone in the observed processes. Collectively, it appears that the cell specific expression of Pgbs has the capability to determine the developmental fate of embryogenic tissue during maize somatic embryogenesis through their effect on PCD. This novel regulation has implications for development and differentiation in other species.

WNT ligands are secreted proteins that act as signals between cells. WNTs activate several interconnected signaling pathways that are required for embryonic development as well as tissue homeostasis in adults. The X-chromosomal Porcn gene encodes a membrane-bound O-acyl transferase that is required for the acylation of all 19 WNT ligands encoded in the mammalian genome. Non-acylated WNTs fail to be secreted from the producing cell and thus do not activate downstream signaling targets. In my thesis research, I have investigated the function of Porcn in mouse embryonic development. In vitro, I have shown that Porcn is required for canonical WNT signaling in ES cells and further, for their differentiation into endodermal and mesodermal derivatives. Taking advantage of a mouse line carrying a conditional (floxed) Porcn allele that I have generated, I have focused my studies on the early embryonic roles of Porcn using Cre recombinase-mediated and X chromosome inactivation-based ablation of Porcn function in vivo. I have found that the earliest requirement for Porcn in mouse development is the induction of gastrulation. In contrast to findings from in vitro studies, I have provided evidence that Porcn is not required for pre-implantation development in vivo. Dissecting embryonic and extra- embryonic roles of Porcn, I have been able to show that Porcn is required in the extra-embryonic chorion in order to mediate chorio-allantoic fusion, whereas ablation in the extra-embryonic visceral endoderm had no apparent effects. The extra-embryonic requirement for Porcn results in a parent-of-origin effect in Porcn heterozygous females due to X chromosome inactivation. In contrast to the placentation defect causing embryonic lethality of maternal allele mutants, deletion of the paternal allele caused variable fetal defects resulting in perinatal lethality with only rare survivors to adulthood. Both fetuses and adults represent a mouse model for Focal Dermal Hypoplasia (FDH), the syndrome caused by mutations in the human PORCN gene. My studies highlight the importance of PORCN-mediated WNT signaling for gastrulation, placentation, and fetal development, but suggest that endogenous WNT secretion does not play an essential role in either implantation or blastocyst lineage specification.