Engrailed genes in mammary development and tumorigenesis

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Engrailed genes in mammary development and tumorigenesis

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryo

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryoSpecification of a unique cell fate during development of a multicellular organism often is a function of its position. The Drosophila central nervous system (CNS) provides an ideal system to dissect signalling events during development that lead to cell specific patterns. Different cell types in the CNS are formed from a relatively few precursor cells, the neuroblasts (NBs), which delaminate from the neurogenic region of the ectoderm. The delamination occurs in five waves, S1-S5, finally leading to a subepidermal layer consisting of about 30 NBs, each with a unique identity, arranged in a stereotyped spatial pattern in each hemisegment. This information depends on several factors such as the concentrations of various morphogens, cell-cell interactions and long range signals present at the position and time of its birth. The early NBs, delaminating during S1 and S2, form an orthogonal array of four rows (2/3,4,5,6/7) and three columns (medial, intermediate, and lateral) . However, the three column and four row-arrangement pattern is only transitory during early stages of neurogenesis which is obscured by late emerging (S3-S5) neuroblasts (Doe and Goodman, 1985; Goodman and Doe, 1993). Therefore the aim of my study has been to identify novel genes which play a role in the formation or specification of late delaminating NBs.In this study the gene anterior open or yan was picked up in a genetic screen to identity novel and yet unidentified genes in the process of late neuroblast formation and specification. I have shown that the gene yan is responsible for maintaining the cells of the neuroectoderm in an undifferentiated state by interfering with the Notch signalling mechanism. Secondly, I have studied the function and interactions of segment polarity genes within a certain neuroectodermal region, namely the engrailed (en) expressing domain, with regard to the fate specification of a set of late neuroblasts, namely NB 6-4 and NB 7-3. I have dissected the regulatory interaction of the segment polarity genes wingless (wg), hedgehog (hh) and engrailed (en) as they maintain each other’s expression to show that En is a prerequisite for neurogenesis and show that the interplay of the segmentation genes naked (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment of NB 7-3 and NB 6-4 cell fate. I have shown that in the absence of either nkd or gsb one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.

Genetische Mechanismen der dorsoventralen Musterbildung im embryonalen Gehirn von Drosophila

In Vertebraten und Insekten ist während der frühen Entwicklung des zentralen Nervensystems (ZNS), welches sich aus dem Gehirn und dem ventralen Nervensystem (VNS) zusammensetzt, die Unterteilung des Neuroektoderms (NE) in diskrete Genexpressions-Domänen entscheidend für die korrekte Spezifizierung neuraler Stammzellen. In Drosophila wird die Identität dieser Stammzellen (Neuroblasten, NB) festgelegt durch die positionellen Informationen, welche von den Produkten früher Musterbildungsgene bereitgestellt werden und das Neuroektoderm in anteroposteriorer (AP) und dorsoventraler (DV) Achse unterteilen. Die molekulargenetischen Mechanismen, welche der DV-Regionalisierung zugrunde liegen, wurden ausführlich im embryonalen VNS untersucht, sind für das Gehirn jedoch weitestgehend unverstanden. rnIm Rahmen dieser Arbeit wurden neue Erkenntnisse bezüglich der genetischen Mechanismen gewonnen, welche die frühembryonale Anlage des Gehirns in DV-Achse unterteilen. So konnte gezeigt werden, dass das cephale Lückengen empty spiracles (ems), das Segmentpolaritätsgen engrailed (en), sowie der „Epidermal growth factor receptor“ (EGFR) und das Gen Nk6 homeobox (Nkx6) für Faktoren codieren, die als zentrale Regulatoren die DV Musterbildung in der Gehirnanlage kontrollieren. Diese Faktoren interagieren zusammen mit den ebenso evolutionär konservierten Homöobox-Genen ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) und muscle segment homeobox (msh) in einem komplexen, regulatorischen DV-Netzwerk. Die im Trito (TC)- und Deutocerebrum (DC) entschlüsselten genetischen Interaktionen basieren überwiegend auf wechselseitiger Repression. Dementsprechend sorgen 1) Vnd und Ems durch gegenseitige Repression für eine frühe DV-Unterteilung des NE, und 2) wechselseitige Repression zwischen Nkx6 und Msh, als auch zwischen Ind und Msh für die Aufrechterhaltung der Grenze zwischen intermediärem und dorsalem NE. 3) Sowohl Ind als auch Msh sind in der Lage, die Expression von vnd zu inhibieren. Ferner konnte gezeigt werden, dass Vnd durch Repression von Msh als positiver Regulator von Nkx6 fungiert. Überdies beeinflusst Vnd die Expression von ind in segment-spezifischer Art und Weise: Vnd reprimiert ind-Expression im TC, sorgt jedoch für eine positive Regulation von ind im DC durch Repression von Msh. Auch der EGFR-Signalweg ist an der frühen DV-Regionalisierung des Gehirns beteiligt, indem er durch positive Regulation der msh-Repressoren Vnd, Ind und Nkx6 dazu beiträgt, dass die Expression von msh auf dorsales NE beschränkt bleibt. Ferner stellte sich heraus, dass das AP-Musterbildungsgen ems die Expression der DV-Gene kontrolliert und umgekehrt: Ems ist für die Aktivierung von Nkx6, ind und msh in TC und DC erforderlich ist, während Nkx6 und Ind zu einem späteren Zeitpunkt benötigt werden, um ems im intermediären DC gemeinsam zu reprimieren. Überdies konnte gezeigt werden, dass das Segmentpolaritätsgen en Aspekte der Expression von vnd, ind und msh in segment-spezifischer Art und Weise reguliert. En reprimiert ind und msh, hält jedoch vnd-Expression im DC aufrecht; im TC wird En benötigt, um die Expression von Msh herunter zu regulieren und somit die Aktivierung von ind dort zu ermöglichen.rnrnZusammengenommen zeigen diese Ergebnisse, dass AP Musterbildungsfaktoren in umfangreichen Maß die Expression der DV Gene im Gehirn (und VNS) kontrollieren. Ferner deuten diese Daten darauf hin, dass sich das „Konzept der ventralen Dominanz“, welches für die DV-Musterbildung im VNS postuliert wurde, nicht auf das genregulatorische Netzwerk im Gehirn übertragen lässt, da Interaktionen zwischen den beteiligten Faktoren hauptsächlich auf wechselseitiger (und nicht einseitiger) Repression basieren. Zudem scheint das Konzept der ventralen Dominanz auch für das VNS nicht uneingeschränkt zu gelten, da in dieser Arbeit u.a. gezeigt werden konnte, dass dorsal exprimiertes Msh in der Lage ist, intermediäres ind zu reprimieren. Interessanterweise ist gegenseitige Repression von Homöodomänen-Proteinen im sich entwickelnden Neuralrohr von Vertebraten weit verbreitet und darüberhinaus essenziell für den Aufbau diskreter DV-Vorläuferdomänen, und weist insofern eine große Ähnlichkeit zu den in dieser Arbeit beschriebenen DV-Musterbildungsvorgängen im frühembryonalen Fliegengehirn auf.rn

Analysis of Wnt/beta-catenin signaling in kidney tubulogenesis

The β-catenin/Lef/Tcf-mediated Wnt pathway is central to the developmental of all animals, stem cell renewal, and cancer progression. Prior studies in frogs and mice have indicated that the ligand Wnt-4 is essential for the mesenchyme to epithelial transition that generates tubules in the context of kidney organogenesis. More recently, Wnt-9b in mice, was likewise found to be required. Yet despite the importance of Wnt signals in renal development, the corresponding Frizzled receptor(s) and downstream signaling mechanim(s) are unclear. My work addresses these knowledge gaps using in vitro (Madin-Darby Canine Kidney cells) and in vivo (Xenopus laevis and zebrafish pronephros) tubulogenic kidney model systems. Employing established reporter constructs of Wnt/β-catenin pathway activity, I have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions. I have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/β-catenin pathway using β-Engrailed and dnTCF-4, constructs that suppress this pathway. I have further found that MDCK cells express the Frizzled-6 receptor, and that Wnt-4 forms a biochemical complex with Frizzled-6, yet does not appear to transduce Wnt-4's canonical signal. Additionally, I demonstrate that standard Hepatocyte Growth Factor (HGF)-mediated (non-physiologic) induction of MDCK tubulogenesis in collagen matrices is not altered by activation or suppression of β-catenin signaling activity; however, β-catenin signaling maintains cell survival in this in vitro system. Using a Wnt/β-catenin signaling reporter in Xenopus laevis, I detect β-catenin signaling activity in the early pronephric epithelial kidney tubules. By inhibiting the Wnt/β-catenin signaling pathway in both zebrafish and Xenopus , a significant loss of kidney tubulogenesis is observed with little or no effect on adjoining axis or somite development. This inhibition also leads to the appearance of severe edema that phenocopies embryos depleted for Wnt-4. Tubulogenic loss does not appear to be caused by increased cell death in the Xenopus pronephric field, but rather by lessened expression of tubule epithelium genes associated with cellular differentiation. Together, my results show that Wnt/β-catenin signaling is required for renal tubule development and that Wnt-4 is a strong candidate for activating this pathway. ^

Oncogenic transformation induced by the Qin protein is correlated with transcriptional repression

The retroviral oncogene qin codes for a protein that belongs to the family of the winged helix transcription factors. The viral Qin protein, v-Qin, differs from its cellular counterpart, c-Qin, by functioning as a stronger transcriptional repressor and a more efficient inducer of tumors. This observation suggests that repression may be important in tumorigenesis. To test this possibility, chimeric proteins were constructed in which the Qin DNA-binding domain was fused to either a strong repressor domain (derived from the Drosophila Engrailed protein) or a strong activator domain (from the herpes simplex virus VP16 protein). The chimeric transcriptional repressor, Qin–Engrailed, transformed chicken embryo fibroblasts in culture and induced sarcomas in young chickens. The chimeric activator, Qin–VP16, failed to transform cells in vitro or in vivo and caused cellular resistance to oncogenic transformation by Qin. These data support the conclusion that the Qin protein induces oncogenic transformation by repressing the transcription of genes which function as negative growth regulators or tumor suppressors.

Siamois is required for formation of Spemann’s organizer

Spemann’s organizer develops in response to dorsal determinants that act via maternal components of the wnt pathway. The function of siamois, a wnt-inducible homeobox gene, in Spemann’s organizer development was examined by fusion of defined transcriptional regulatory domains to the siamois homeodomain. Similar to native siamois, a VP16 activator fusion induced axis formation, indicating that siamois functions as a transcriptional activator in axis induction. Fusion of the engrailed repressor generated a dominant inhibitor that blocked axis induction by Xwnt8, β-catenin, and siamois, and repressed wnt activation of the goosecoid promoter. Dorsal injection of the engrailed-siamois fusion resulted in complete inhibition of dorsal development and organizer gene expression, an effect rescued by siamois, but not by Xwnt8 or β-catenin. Thus, as a zygotic mediator of maternal dorsal signals, siamois function is required for development of Spemann’s organizer.

Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp), Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost. We make further inferences concerning the evolution of heads and Hox genes in arthropods.

Role of the iroquois3 homeobox gene in organizer formation

In zebrafish, the organizer is thought to consist of two regions, the yolk syncytial layer (YSL) and the shield. The dorsal YSL appears to send signals that affect formation of the shield in the overlying mesendoderm. We show here that a domain of dorsal deep cells located between the YSL and the shield is marked by expression of the iro3 gene. As gastrulation proceeds, the iro3 positive domain involutes and migrates to the animal pole. Iro3 expression is regulated by Nodal and bone morphogenic protein antagonists. Overexpression of iro3 induced ectopic expression of shield-specific genes. This effect was mimicked by an Iro3-Engrailed transcriptional repressor domain fusion, whereas an Iro3-VP16 activator domain fusion behaved as a dominant negative or antimorphic form. These results suggest that Iro3 acts as a transcriptional repressor and further implicate the iro3 gene in regulating organizer formation. We propose that the iro3-expressing dorsal deep cells represent a distinct organizer domain that receives signals from the YSL and in turn sends signals to the forming shield, thereby influencing its expansion and differentiation.