Para mejorar la eficiencia de regeneración en dos variedades africanas de camote (Tanzania y Wagabolige) se probó un protocolo vía organogénesis de dos fases en Tanzania y de dos, tres y cuatro fases en Wagabolige. La primera fase, en ambas variedades, consistió en el mantenimiento de los explantes en un medio con auxinas y las siguientes, con citoquininas. Se probó tres tipos de explantes: hojas con peciolo, peciolos y raíces. Todos los regenerantes fueron obtenidos a partir de hojas con peciolo. Los brotes emergentes fueron de apariencia normal y anormal. Se logró resultados favorables al trabajar con bajas concentraciones de 2,4-D y en Tanzania, al suplementar AG3 a la primera etapa, se incrementó la frecuencia de regeneración de brotes normales; sin embargo, ambas variedades presentaron un bajo índice de regeneración: 3.33% en Tanzania y 1.46% en Wagabolige.To improve the regeneration efficiency of two sweetpotato African varieties (Tanzania and Wagabolige), a two-stage organogenesis regeneration protocol was tested in Tanzania; while a two, three, four-stages was tested in Wagabolige. The first stage, in both varieties, maintained the explants in a medium supplemented with auxines, and the next stages, with cytokinines. Three different explants were tested: leaves with petiole, petioles and roots. All shoots came from leaves with petioles. Shoots presented normal or anomalous morphology. The best results were obtained working with low concentrantions of 2,4-D. For Tanzania, the addition of GA3 in the first stage was found to increase normal shoot frequency, nevertheless both varieties showed low regeneration efficiencies: 3.33% in Tanzania, and 1.46% in Wagabolige.

Dentre os fatores relacionados à capacidade superior de regeneração in vitro de Solanum peruvianum, está a presença do alelo dominante Rg1. O gene RG1, envolvido na formação de gemas caulinares a partir de raízes e outros explantes, foi mapeado no cromossomo 3 entre os genes BETA-CAROTENE HYDROXYLASE (CrtR-b) e PHYTOENE SYNTHASE (PSY1). Variações alélicas também são conhecidas para genes CrtR-b e PSY1. white flower (wf) é o alelo de CrtR-b que produz pétalas brancas e yellow flesh (r) é o alelo de PSY1 que produz frutos amarelos. Os alelos wf e Rg1 com r foram introgredidos na cultivar Micro-Tom (Solanum lycopersicum L). Através da transferência sequencial de explantes de SIM para MB e de RIM para SIM, verificou-se que MT-Rg1 reduz o tempo necessário para a indução de gemas em um dia (6 dias em SIM para MT), devido a redução no tempo necessário para a aquisição de competência em um dia (2 dias para MT). Foram obtidos 30 possiveis recombinantes advindos do cruzamento Rg1/rg1 Wf/wf x rg1/rg1 wf/wf, baseados nas características de pétala branca (efeito do alelo wf) e maior ramificação (efeito do alelo Rg1). Todos os possíveis recombinantes foram testados in vitro quanto à capacidade de formar gemas caulinares em SIM e raízes em RIM. Com isso, 7 linhagens recombinantes foram confirmadas e utilizadas para o mapeamento fino da região na qual o gene RG1 está localizado no cromossomo 3. Para o mapeamento foram testados marcadores do tipo CAPS e SCAR, sendo utilizado o DNA genômico extraído de S. pennellii, S. peruvianum, Micro- MsK, MT, MT-Rg1, MT-wf e das 7 linhagens recombinantes, constatando-se que alguns marcadores moleculares desenhados para S. pennellii podem ser utilizados para S. peruvianum. Estas análises também evidenciaram que o segmento de introgressão de MT-Rg1 está entre o marcador P5 e o gene CrtR-b, totalizando 136 genes, entre os quais GRAS 10 destacou-se como principal candidato para a função gênica de Rg1. Complementarmente, foi realizada a análise de RNA-Seq (plataforma SOLiD) para a identificação de genes diferencialmente expressos entre MT e MT-Rg1. Com isso, observou-se que existem mais genes regulados negativamente do que positivamente durante a aquisição de competência. Devido ao grande número de genes diferencialmente expressos, alguns parâmetros foram utilizados para classificar os genes que poderiam ser quantificados por qRT-PCR em um experimento de regeneração in vitro. Entre os 361 genes classificados, 5 foram selecionados para a quantificação de sua expressão. Destes 5 genes, GRAS 10 e Serine/threonine protein phosphatase 7 foram os que apresentaram-se mais positivamente expressos e aparentemente são os que estão mais intimamente ligados a fase de aquisição de competência. Como existem muitos genes GRAS, foi feita uma árvore filogenética para posicioná-lo e identificar genes homólogos a ele. Com base na análise filogenética foi possível identificar o gene GRAS 10 como homológo ao gene SCL8 já identificado em Arabidopsis. Porém, pouco se sabe a respeito de SCL8. Desse modo, ainda é necessário confirmar a identidade do gene RG1, o que possibilitará a compreensão do processo de aquisição de competência. / The dominat allele Rg1 is related to the greater in vitro regeneration capacity of Solanum peruvianum L. The Rg1 gene is required for shoot formation from roots and others explants and was mapped on chromosome 3 between BETA-CAROTENE HYDROXYLASE (CrtR-b) and PHYTOENE SYNTHASE (PSY1) genes. Allelic variants are also known for the genes CrtR-b and PSY1. white flower (wf) is the allele of CrtR-b that produces white petals and yellow flesh (r) is the allele of PSY1 that produces yellow fruits. The alleles wf and Rg1 with r were introgressed into the Micro-Tom cultivar (Solanum lycopersicum L). Through the sequential transfer of explants from SIM to BM and RIM to SIM, we found that MT-Rg1 reduces the time required for the induction of shoots in one day (6 days on SIM for MT), due the reduction of the time required for competence acquisition in one day (2 days to MT). It was obtained 30 putative recombinants from crossing Rg1/rg1 Wf/wf x rg1/rg1 wf/wf. The recombinant lines were scored based on the presence of white petals (wf allele\'s effect) and increased shoot branching (Rg1 allele\'s effect). All putative recombinant lines were tested in vitro for their capability to form shoots in SIM and roots in RIM. Thus, 7 recombinant lines were selected and used to fine mapping the region where RG1 gene is located. CAPS and SCAR markers designed to S. pennellii were tested to fine-mapping, using as template genomic DNA samples from S. pennellii, S. peruvianum, Micro-MsK, MT, MT-Rg1, MT-wf and the 7 recombinant lines. These results confirmed that the molecular markers designed for S. pennellii can be successfully used for S. peruvianum. These analyzes also suggest that the introgressed segment of MT-Rg1 is located between the P5 marker and the CrtR-b gene. Within this region, there are 136 genes, including the GRAS 10 gene whichis the main candidate to RG1 gene function. In addition, we performed RNA-Seq analysis (SOLiD platform) to identify genes differentially expressed in MT and MT-Rg1. It was observed more down-regulated than up-regulated genes in the competence acquisition stage. Due to the large number of differentially expressed genes, some parameters were used for selection of genes that would have their expression validated by qRT-PCR in an in vitro regeneration test. Five genes among 361 genes differentialy expressed were selected to test their expression. Of these 5 genes, GRAS 10 and Serine / threonine protein phosphatase 7 were the most up-regulate genes and showed to be closely related to the stage of competence acquisition. Since there are many GRAS genes, we performed a phylogenetic analysis to identify homologous genes. Based on the phylogenetic analysis, GRAS 10 was identified as homologous to SCL8, a gene already identified in Arabidopsis. However, little is known about SCL8. Thus, it is still necessary to confirm the RG1 gene identity. This approach will provide a better understanding of competence acquisition process.

Diversos estudos têm demonstrado o envolvimento benéfico da utilização do aminoácido glutamina em meios de cultura, favorecendo a organogênese dos tecidos vegetais cultivados. Sabe-se que as fontes de nitrogênio podem influenciar na produção endógena de fitormônios, entretanto o papel exato da glutamina ainda não está bem estabelecido. Em Ananas comosus (L.) Merr., a adição de glutamina ao meio de cultura exerceu efeito promotor sobre a taxa de organogênese e o vigor do crescimento das gemas caulinares a partir de bases foliares. Além da glutamina, discute-se se o estresse resultante da explantação também poderia estar envolvido com a indução do processo organogenético, acarretando na produção de espécies reativas de oxigênio e na alteração do estado redox endógeno. Esse estresse para ser benéfico, entretanto, deveria estar restrito a certo limite. O presente trabalho visou compreender o efeito favorável da glutamina na organogênese adventícia em bases foliares de abacaxizeiro cultivadas in vitro. O envolvimento da glutamina com uma possível diminuição do estresse oxidativo durante o período de indução da organogênese também foi abordado. Para tanto, buscou-se correlacionar a influência do suprimento de glutamina no meio de cultura com os teores endógenos de peróxido de hidrogênio, glutationa e ascorbato. O estado redox da glutationa e do ascorbato durante o período de indução da organogênese adventícia também foi analisado. Além disso, foram analisadas as atividades de duas enzimas antioxidantes nesses explantes foliares, a superóxido dismutase e a catalase. Tentativamente, a glutationa foi adicionada ao meio de cultura, contendo ou não glutamina, visando conhecer o efeito desse antioxidante no processo organogenético. Os resultados mostraram que a glutationa substituiu, mas não intensificou, o efeito benéfico da glutamina sobre a taxa de organogênese das bases foliares de abacaxizeiro. Esse antioxidante não substituiu o efeito positivo do aminoácido no ganho de massas fresca e seca dos eixos caulinares formados, no entanto atuou favoravelmente na formação de um maior número de gemas adventícias por explante inoculado. Ao que parece, o cultivo in vitro das bases foliares gerou um estresse oxidativo nesses tecidos logo no início do período de cultivo, a julgar pela alta concentração de H2O2 detectada nas primeiras 24 horas. Entretanto, essa possível condição estressante foi controlada ao longo do período de cultivo, retornando a uma homeostase do tecido e conferindo condição para que as células se reprogramassem para seguir a uma rota de organogênese caulinar. A glutamina pareceu favorecer a manutenção de um estado redox reduzido tanto de ASC quanto de GSH durante o período em que houve o possível estresse oxidativo. Os resultados das atividades das enzimas antioxidantes sugeriram que a CAT pode ter sido responsável pela regulação do conteúdo endógeno de H2O2, já que a SOD não apresentou alterações expressivas ao longo do período de indução da organogênese tanto em SIM quanto em SIMGln. Em conjunto, os resultados sugerem que o estresse oxidativo causado pelo cultivo in vitro pode ter gerado uma sinalização importante para que a organogênese se inicie, sendo que a glutamina exerceria um papel de manter o estado redox dos tecidos foliares reduzido no momento da maior concentração de H2O2 endógeno. / A positive influence of glutamine on organogenesis of in vitro cultured plant tissues has been demonstrated by several studies. It is well known that the endogenous synthesis of phytohormones can be influenced by nitrogen sources, although it is not completely established in which way glutamine acts in this process. The addition of this amino acid to the culture medium has enhanced the organogenesis rate and resulted in a better vigor of the shoots that were originated from the leaf bases of Ananas comosus (L.) Merr. cultured in in vitro conditions. It is also suggested that the tissue excision may result in a stressful condition by increasing the production of reactive oxygen species and changing the endogenous redox state, which might be involved in the induction of organogenic process. However, this stress should be beneficial only if restricted. The aim of this work was to comprehend the positive influence of glutamine on the in vitro adventitious organogenesis of pineapple leaf bases. It was also attempted to determine whether the glutamine would be involved on a possible oxidative stress decrease during the organogenesis induction. In order to answer these questions, we tried to correlate the presence of glutamine in the culture medium and the endogenous hydrogen peroxide, glutathione, ascorbate levels. The redox state of these antioxidants is also analyzed during the induction of adventitious shoot organogenesis. Moreover, two antioxidants enzymes activities are quantified in the leaf explants: catalase and superoxide dismutase. The glutathione influence on the process was also investigated, considering the glutamine presence or not. It was done in order to establish the effect of this antioxidant in the organogenic process. The results showed that glutathione could replace, but not enhance, the positive effect of glutamine on the organogenesis rate of pineapple leaf bases. This antioxidant did not substitute the positive effect presented by the glutamine on the acquisition of fresh and dry masses by the new shoots. On the other hand, glutathione enhanced the number of adventitious buds per explant. Apparently, the excision of the leaf bases and its subsequent cultivation in the induction culture medium resulted in the tissue oxidative stress early in the first 24 hours of incubation. This could be inferred by the high H2O2 concentrations detected during this period. However, this possible stressful condition was controlled during the culture period, leading to the return of the homeostasis of the tissue and allowing the cells to become determined to shoot organogenesis. During the probable period of oxidative stress, glutamine seemed to maintain the reduced redox state on both ASC and GSH. The results of the antioxidant enzymes activities suggested that CAT may have been responsible for the regulation of the endogenous H2O2 levels, while SOD did not showed significant changes during the induction of organogenesis of leaf bases cultivated either in SIM or SIMGln. Taken together, the results obtained in this work suggest that the oxidative stress caused by the excision of the leaf tissues and its in vitro cultivation may be an important signal to the induction of the leaf organogenesis. Furthermore, the glutamine may have a role in the maintenance of the reduced redox state when higher levels of endogenous H2O2 are present in the tissues.

Dentre os fatores relacionados à capacidade superior de regeneração in vitro de Solanum peruvianum, está a presença do alelo dominante Rg1. O gene RG1, envolvido na formação de gemas caulinares a partir de raízes e outros explantes, foi mapeado no cromossomo 3 entre os genes BETA-CAROTENE HYDROXYLASE (CrtR-b) e PHYTOENE SYNTHASE (PSY1). Variações alélicas também são conhecidas para genes CrtR-b e PSY1. white flower (wf) é o alelo de CrtR-b que produz pétalas brancas e yellow flesh (r) é o alelo de PSY1 que produz frutos amarelos. Os alelos wf e Rg1 com r foram introgredidos na cultivar Micro-Tom (Solanum lycopersicum L). Através da transferência sequencial de explantes de SIM para MB e de RIM para SIM, verificou-se que MT-Rg1 reduz o tempo necessário para a indução de gemas em um dia (6 dias em SIM para MT), devido a redução no tempo necessário para a aquisição de competência em um dia (2 dias para MT). Foram obtidos 30 possiveis recombinantes advindos do cruzamento Rg1/rg1 Wf/wf x rg1/rg1 wf/wf, baseados nas características de pétala branca (efeito do alelo wf) e maior ramificação (efeito do alelo Rg1). Todos os possíveis recombinantes foram testados in vitro quanto à capacidade de formar gemas caulinares em SIM e raízes em RIM. Com isso, 7 linhagens recombinantes foram confirmadas e utilizadas para o mapeamento fino da região na qual o gene RG1 está localizado no cromossomo 3. Para o mapeamento foram testados marcadores do tipo CAPS e SCAR, sendo utilizado o DNA genômico extraído de S. pennellii, S. peruvianum, Micro- MsK, MT, MT-Rg1, MT-wf e das 7 linhagens recombinantes, constatando-se que alguns marcadores moleculares desenhados para S. pennellii podem ser utilizados para S. peruvianum. Estas análises também evidenciaram que o segmento de introgressão de MT-Rg1 está entre o marcador P5 e o gene CrtR-b, totalizando 136 genes, entre os quais GRAS 10 destacou-se como principal candidato para a função gênica de Rg1. Complementarmente, foi realizada a análise de RNA-Seq (plataforma SOLiD) para a identificação de genes diferencialmente expressos entre MT e MT-Rg1. Com isso, observou-se que existem mais genes regulados negativamente do que positivamente durante a aquisição de competência. Devido ao grande número de genes diferencialmente expressos, alguns parâmetros foram utilizados para classificar os genes que poderiam ser quantificados por qRT-PCR em um experimento de regeneração in vitro. Entre os 361 genes classificados, 5 foram selecionados para a quantificação de sua expressão. Destes 5 genes, GRAS 10 e Serine/threonine protein phosphatase 7 foram os que apresentaram-se mais positivamente expressos e aparentemente são os que estão mais intimamente ligados a fase de aquisição de competência. Como existem muitos genes GRAS, foi feita uma árvore filogenética para posicioná-lo e identificar genes homólogos a ele. Com base na análise filogenética foi possível identificar o gene GRAS 10 como homológo ao gene SCL8 já identificado em Arabidopsis. Porém, pouco se sabe a respeito de SCL8. Desse modo, ainda é necessário confirmar a identidade do gene RG1, o que possibilitará a compreensão do processo de aquisição de competência. / The dominat allele Rg1 is related to the greater in vitro regeneration capacity of Solanum peruvianum L. The Rg1 gene is required for shoot formation from roots and others explants and was mapped on chromosome 3 between BETA-CAROTENE HYDROXYLASE (CrtR-b) and PHYTOENE SYNTHASE (PSY1) genes. Allelic variants are also known for the genes CrtR-b and PSY1. white flower (wf) is the allele of CrtR-b that produces white petals and yellow flesh (r) is the allele of PSY1 that produces yellow fruits. The alleles wf and Rg1 with r were introgressed into the Micro-Tom cultivar (Solanum lycopersicum L). Through the sequential transfer of explants from SIM to BM and RIM to SIM, we found that MT-Rg1 reduces the time required for the induction of shoots in one day (6 days on SIM for MT), due the reduction of the time required for competence acquisition in one day (2 days to MT). It was obtained 30 putative recombinants from crossing Rg1/rg1 Wf/wf x rg1/rg1 wf/wf. The recombinant lines were scored based on the presence of white petals (wf allele\'s effect) and increased shoot branching (Rg1 allele\'s effect). All putative recombinant lines were tested in vitro for their capability to form shoots in SIM and roots in RIM. Thus, 7 recombinant lines were selected and used to fine mapping the region where RG1 gene is located. CAPS and SCAR markers designed to S. pennellii were tested to fine-mapping, using as template genomic DNA samples from S. pennellii, S. peruvianum, Micro-MsK, MT, MT-Rg1, MT-wf and the 7 recombinant lines. These results confirmed that the molecular markers designed for S. pennellii can be successfully used for S. peruvianum. These analyzes also suggest that the introgressed segment of MT-Rg1 is located between the P5 marker and the CrtR-b gene. Within this region, there are 136 genes, including the GRAS 10 gene whichis the main candidate to RG1 gene function. In addition, we performed RNA-Seq analysis (SOLiD platform) to identify genes differentially expressed in MT and MT-Rg1. It was observed more down-regulated than up-regulated genes in the competence acquisition stage. Due to the large number of differentially expressed genes, some parameters were used for selection of genes that would have their expression validated by qRT-PCR in an in vitro regeneration test. Five genes among 361 genes differentialy expressed were selected to test their expression. Of these 5 genes, GRAS 10 and Serine / threonine protein phosphatase 7 were the most up-regulate genes and showed to be closely related to the stage of competence acquisition. Since there are many GRAS genes, we performed a phylogenetic analysis to identify homologous genes. Based on the phylogenetic analysis, GRAS 10 was identified as homologous to SCL8, a gene already identified in Arabidopsis. However, little is known about SCL8. Thus, it is still necessary to confirm the RG1 gene identity. This approach will provide a better understanding of competence acquisition process.

Diversos estudos elucidaram mecanismos envolvidos com a organogênese in vitro, porém pouco é conhecido a respeito da fase de aquisição de competência, fundamental para que a regeneração ocorra. Alguns genes já foram identificados por interferirem na fase de aquisição de competência em tomateiro (Solanum lycopersicum), mas ainda existem diversas lacunas a serem esclarecidas. Para investigar a expressão de genes e o controle hormonal na fase de aquisição de competência, foram utilizados os mutantes de tomateiro, sob o background genético da cultivar Micro-Tom (MT), MT-Rg1 e MT-pro (procera), os quais afetam positiva ou negativamente a organogênese in vitro, respectivamente. Embora a resposta constitutiva a giberelina no mutante MT-pro seja conhecida, a identidade molecular do gene RG1 permanece indefinida. O mutante MT-Rg1 apresenta aumento tanto na formação de gemas caulinares quanto de raízes e reduz o tempo necessário para a indução desses órgãos, devido à diminuição do período para a aquisição de competência. A partir do estabelecimento das fases de aquisição de competência e indução da organogênese in vitro para MT e MT-Rg1, foram identificados genes diferencialmente expressos entre estes genótipos. Entre estes genes, CELL DIVISION CYCLE ASSOCIATED 7 e LACCASE 1A estão regulados positivamente em MT-Rg1 e todos estão fortemente relacionados à fase de aquisição de competência, e a alterações na proliferação de células do protoxilema durante o início da organogênese. Por outro lado, a resposta constitutiva à giberelina no mutante MT-pro reduz a formação de gemas caulinares e raízes e aumenta a formação de calos in vitro, sem afetar o tempo requerido para a indução de gemas caulinares e raízes. De forma oposta a MT-Rg1, o gene CDCA7 apresenta expressão reduzida durante a fase de aquisição de competência em MT-pro, diminuindo o número de células do protoxilema em divisão. Outro fator importante para a divisão celular no mutante MT-pro é o aumento da expressão do gene WUS, causando um aumento da proliferação das stem cells, que são células indiferenciadas relacionadas à formação de novos órgãos. Esta proliferação celular inadequada, somada a uma alteração desfavorável na homeostase das citocininas, justifica o efeito negativo do alelo pro na formação de gemas caulinares, o que permitiu a criação de um novo modelo para organogênese in vitro / Several studies have enabled the discovery of mechanisms to achieve in vitro organogenesis; however, little is known about the phase of acquisition of competence, essential for regeneration. A few genes have been identified to interfere in the acquisition of the competence phase in tomato (Solanum lycopersicum), but there are still many gaps to be filled. We have used the mutants, under the genetic background of the Micro-Tom cultivar, MT-Rg1 and MT-pro (procera), which positively or negatively affect in vitro organogenesis, respectively, to investigate gene expression and the hormonal control in the phase of acquisition of competence. Despite the fact that the constitutive gibberellin response in the procera mutant is well-established, the molecular identity of RG1 gene remains unknown. The MT-Rg1 mutant presents an increase in the formation of both shoot and roots and a reduced period for the induction of these organs, because of the reduced time required for acquisition of competence.We searched for the identity of differentially expressed genes between MT and MT-Rg1 after the establishment of the competence acquisition phase and organogenesis induction stages. Among those genes, CDCA7 and LAC1A are upregulated in MT-Rg1 and these genes appear to be strongly related with the acquisition of competence phase and changes in proliferation of protoxylem cells during early organogenesis. The constitutive response to gibberellin in the MT-pro mutant decreases the formation of shoot and roots and increase in vitro calli formation, without reducing the induction phase of shoots and roots. Unlike MT-Rg1, MT-pro reduces the CDCA7L expression during the acquisition of competence phase, causing a reduction of the protoxylem dividing cells. Another important factor for cell division in MT-pro mutant is the increased expression of the WUS gene, leading to an abnormal proliferation of stem cells. Thereby, this abnormal cell proliferation, in addition to an unfavorable change in the cytokinin homeostasis, justify the negative effect of the pro allele in the shoot formation, which enabled the proposal of a new model for in vitro organogenesis

<p>Sonic hedgehog (Shh) is a critical secreted signaling molecule that regulates many aspects of organogenesis. In the absence of Shh, many organs, including the foregut, larynx, palate, cerebellum and heart do not form properly. However, the cellular details of the roles of Shh, including the relevant domains of Shh expression and reception, have not been elucidated for many of these processes. </p><p>The single embryonic foregut tube must divide into the trachea and esophagus, which does not occur in the Shh-null mutant. In Chapter 5, I use Cre-Lox technology to determine that the ventral foregut endoderm is the relevant source of Shh for this process and the mesoderm must directly receive that Shh signal. Surprisingly, this signaling event appears to occur two days before the foregut begins to divide, indicating an early essential role for Shh in foregut division. </p><p>Shh is also expressed at later stages in the maturing trachea and esophagus. In Chapter 6, I demonstrate that these domains serve to establish differentiated mesoderm. In the trachea, Shh from the endoderm signals directly to the mesoderm to form the tracheal cartilage rings. In the esophagus, the roles of Shh are more complex. Shh regulates the size of the esophagus and controls patterning of the concentric rings of esophageal mesoderm, however this process seems to be indirect, requiring autocrine Shh signaling within the esophageal endoderm. </p><p>The laryngeal apparatus is entirely absent in the Shh-null mouse. I n Chapter 3, I dissect the domains of Shh expression and reception required for laryngeal development and demonstrate that loss of endodermal Shh expression causes laryngotracheoesophageal clefts and malformed laryngeal cartilages. As much of laryngeal morphogenesis poorly understood, I also utilize dual mesodermal and neural crest fate maps to determine the embryonic origins of various laryngeal tissues. Finally, as Shh signaling often occurs in concert with Bone Morphogenic Protein (BMP) signaling, I investigate the roles of BMP signaling in laryngeal development. </p><p>Much of Shh signaling occurs at the primary cilium, to which Smoothened, a critical pathway member, must translocate upon Shh signal transduction. This process requires a Smo-Kif3a-&#946;arretin complex in mammalian cell culture. However, the roles of &#946;arrestins in mouse development, and their relationship to Shh signaling have not been investigated in vivo. To do so, in Chapter 4, I analyze the phenotypes of the &#946;arr1/&#946;arr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling. </p><p>Altogether, my work dissects the cellular details of Shh signaling during multiple organ systems in the mouse embryo. I further analyze the consequences of absent or misregulated Shh signaling across multiple developmental contexts and determine that Shh plays critical and diverse roles in organogenesis.</p> / Dissertation

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.

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.

In this study, optimization of a transformation and regeneration system via indirect organogenesis in cotyledonary petiole tissue of lentil (Lens culinaris Medik.) was investigated. Eight different medium types differing in their plant growth regulator compositions were employed to examine the callus induction potency of cotyledonary petiole. Except two, all other tested medium yielded more than 80% callus induction. Nine different medium types were studied to test the potencies of callus structures for shoot induction. Only the callus induced in medium H (1 mg/L Zeatin riboside + 1 mg/L Naphthalane acetic acid) yielded shoots at 8 to 40 % frequency. The most responsive medium was MS basal medium with no growth regulators. Also five and three different medium types were employed to examine callus induction potency of epicotyl tissues respectively. Each medium type yielded 90% callus induction. Only the callus induced in medium H yielded shoots At 6 to 26% frequency. Preliminary studies were carried out for somatic embryogenesis in cotyledonary petiole. Effects of salicylic acid on somatic embryogenesis were also investigated. Salicylic acid at 200&micro / M was found to enhance the percentage of somatic embryos by 25 % and reduce the necrosis 24 %. However none of the globular and heart shape embryos were able to regenerate. Transient GUS expression efficiencies of roots, shoot tips, and cotyledonary petioles were tested after Agrobacterium-mediated transformation. Transformation frequencies were 26, 74, and 38 % for cotyledonary petiole, shoot tips, and roots respectively.

Abstract Secreted signalling molecules are important for the regulation of developmental cell responses. In the developing kidney, signalling occurs between epithelial ureteric bud and metanephric mesenchyme and in between their derivatives. Wnt6 gene activity was localized to the ureteric bud and newly formed branches of the ureteric tree during early stages of kidney development. In a classic organ culture system, Wnt6 signalling induced the activation of marker genes for early nephrogenesis. The metanephric mesenchymes isolated from the Wnt4 deficient embryos were also induced, and the Wnt4 gene became activated in the presence of a Wnt6 signalling source. We propose that Wnt-6 is involved as a metanephric inducer and controls nephrogenesis. Wnt4 is essential for nephrogenesis in mouse and we indicate an additional role for Wnt4 in the control of periureteric stromal differentiation. A failure in vascular development was also found. Bmp4 expression in the medullar stroma of the Wnt4-deficient kidneys was absent concomitantly with a loss of expression of the smooth muscle marker, α-SMA. In vitro Wnt4 signalling induced Bmp4 expression and local α-SMA production. Hence, we conclude that lack of Wnt4 signalling leads to a loss of the periureteric smooth muscle cells, and Wnt4 may locally regulate this cell population in normal kidneys via regulation of Bmp4 signalling. The pluripotent neural crest cells are proposed to play regulatory roles in the early metanephros. Here, the use of transgenic animals allowed visualisation of the lumbo-sacral neural crest (NC) cells in close proximity to the early metanephros. The NC cells, however, disappeared in most part of the kidney by E12.5. The Splotch embryos lack the NCs from the early urogenital region. A developmental defect in the kidneys of Splotch embryos was not observed in vivo or in vitro. The results suggest that the neural crest is not essential for early embryonic kidney development. In sum, the work presented indicates an important role for Wnt6 in the induction of kidney tubules in vitro, for Wnt4 in the specification of kidney smooth muscle cells and for endothelial development in kidney. The neural crest cells apparently have no active morphogenetic role in early kidney development.