In vitro modeling of early mouse development

Previous attempts to recapitulate embryogenesis in a developmentally relevant context started with aggregates composed of a few thousand ESCs, termed embryoid bodies (EB), that upon induction of differentiation reveal a surprising level of autonomous cell fate patterning, in some cases giving rise to spatially arranged ectoderm, mesoderm and endoderm layers. However, cell fate patterning and morphogenesis in EBs is poorly reproducible and cannot be controlled. These limitations necessitate further research on the self-organization potential of ESCs, and in particular the development of more robust in vitro models mimicking post-implantation mouse embryos. This PhD thesis explored three novel approaches towards this goal. Firstly, together with collaborators, I focused on a previously established in vitro model of gastrulation termed gastruloids. Gastruloids emerge from small ESC aggregates that, upon activation of Wnt signaling, break symmetry and undergo axial morphogenesis to create structures that are similar to the developing tail of the mouse embryo. A systematic improvement of the existing culture conditions allowed gastruloids to be cultured for an extended period of time, resulting in an embryo-like patterning along the three major body axes. Furthermore, gene expression analyses, combined with immunohistochemistry and in situ hybridization assays, demonstrated that gastruloids exhibit spatio-temporal activation of Hox genes in a way that is remarkably similar to post-implantation mouse embryos. Secondly, I report a novel gastruloid culture approach, which for the first time promotes the formation of anterior neural tissues. Mouse ESCs were aggregated and treated in high-throughput in a bioengineered microwell array system to generate homogeneous âartificial epiblastsâ that transcriptionally and morphologically resemble post-implantation epiblasts. These epiblast-like (EPI) aggregates break symmetry and undergo axial elongation to establish antero-posterior patterning. I provide evidence for attenuated Wnt signaling and the presence of an epithelium in the starting EPI aggregates as the major determinants for the formation of anterior neural tissues, that are completely absent in conventional gastruloids. Lastly, I systematically explored the role of extraembryonic tissues in the patterning of artificial epiblasts. By combining EPI aggregates with trophoblast stem cell aggregates, hybrid self-organizing structures termed EpiTS embryoids were generated that mimic epiblast and extraembryonic ectoderm, respectively. EpiTS embryoids were found to execute an elaborate self-organization program that is initiated through highly localized gastrulation-like processes at the embryonic-extraembryonic interface. Upon axial elongation, EpiTS embryoids give rise to primitive brain-like and preplacodal ectoderm-like regions. These observations highlight the role of extraembryonic tissues for proper induction of gastrulation and in particular the formation of anterior neural tissues. Altogether, this thesis introduces three innovative and highly versatile stem cell culture technologies to study post-implantation mouse embryonic development. The approaches are amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing experimental tools. This work should contribute to the advancement of the nascent field of synthetic embryology.

In vitro endoderm emergence and self-organisation in the absence of extraembryonic tissues and embryonic architecture

Stefano Davide Vianello

In humans, mice, and other mammals key internal organs such as the gut, the lungs, the pancreas, and the liver all derive from the same embryonic tissue: the endoderm. The development of all of these structures thus depends on a same set of early cells, and on the developmental instructions provided to them by the embryo. One approach to study what these instructions might be is to study the behaviour of endoderm cells when they can not rely on embryonic instructions. That is, when they are generated and made to develop outside of the embryo. The rationale behind this approach, widely embraced by the field of synthetic embryology, is that the unguided development of specific embryonic cell types can provide a window to the observation of intrinsic characteristics of these cells and of fundamental developmental principles which might not be executed or manifested within the regulated environment of the embryo. Accordingly, in the work here described I use gastruloids, synthetic models of development made by aggregation of mouse embryonic stem cells in vitro to characterise the developmental behaviour of endoderm cells, in the absence of embryonic cues and in the absence of the extraembryonic tissues that they would usually rely on in vivo. In the first chapter of this thesis, I provide an introduction, overview, and review of our current understanding of the developmental biology of mouse endoderm as this develops in the embryo. I also introduce gastruloids as stem-cell-based experimental systems to study developmental mechanisms in vitro, decoupled from the constraints of experimental work with animals and animal embryos. Chapter 2 is dedicated to the earliest steps of endoderm development in gastruloids. I show when and where these cells appear, and how they sort as the gastruloid starts to mature. I find that endoderm cells follow similar spatial and temporal developmental patterns as those they would follow if they were in the embryo. I notably find that they always remain in an epithelial state, even as other cell types undergo mesenchymal transition. In Chapter 3 I describe surprising dynamics of endoderm cells over time, and how they self-organise into complex architectures at the core of the gastruloids. The epithelial domain that forms is dynamic and plastic, and partitions the gastruloids into interfacing compartments. Importantly, I reveal a significant endodermal component to this in vitro system, usually considered to be unstructured and neural/mesodermal in character. In Chapter 4 I show how the core endoderm compartment that I see forming in gastruloids is patterned along the longitudinal axis of the aggregate, with different cellular identities at different anterior-posterior position of the domain. By comparison with published embryonic datasets, I identify endodermal identities corresponding to the full spectrum of those that make up the gut tube in the embryo. Specifically, I describe how most endodermal cells adopt an early intestinal fate, and how many acquire anterior foregut identities. The work described in this thesis presents gastruloids as promising in vitro systems for the study of endoderm biology, morphogenesis, and mature fate specification, filling a gap within the field of synthetic embryology. It also notably opens translational possibilities to the use of gastruloids to produce endodermal cell types which may be otherwise difficult to generate using conventional directed differentiation methods.

EPFL2022

Role of canonical Wnt signaling in mammary gland development

Renuga Devi Rajaram

The female reproductive hormones estrogens, progesterone, and prolactin control postnatal breast development and are important to breast carcinogenesis. In particular, exposure to progesterone is tightly linked to breast cancer risk. Using the mouse as a model system to study the mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we demonstrated that progesterone acts by a paracrine mechanism and identified Wnt4 as a target of progesterone important in mediating side branch formation. The mechanisms underlying Wnt4 function remains poorly understood. To understand the pattern of canonical Wnt signaling activation during mammary gland development and to address which cellular compartment of the mammary gland is activated by canonical Wnt signaling, a reporter mouse model was utilized in which the lacZ gene is under the control of endogenous Axin2 promoter, classical target promoter of canonical Wnt signaling. Analysis of reporter mice at different developmental stages of mammary gland development showed the highest reporter activity during early pregnancy which correlated with the time of side branch formation. At this stage, β-galactosidase activity was found exclusively in myoepithelial cells. Overexpression of Wnt1 from MMTV promoter increases reporter activity in the myoepithelium. Gene expression analysis on FACS sorted luminal and myoepithelial cells shows that known canonical Wnt signaling pathway components such as receptors and co receptors as well as targets are specifically expressed in the myoepithelial compartment further supporting the notion that myoepithelial cells are the target cells for Wnt1 and Wnt4 secreted by luminal cells. Consistent with a model whereby progesterone induced Wnt4 secretion results in canonical Wnt signaling activation, reporter activity was induced by progesterone treatment and lacZ expression increased during diestrus when serum progesterone levels are high under physiological conditions. Analysis of contralateral mammary glands engrafted with PR-/-.Axin2 lacZ and PR+/-.Axin2 lacZ epithelium during pregnancy revealed that intact PR signaling is required to activate canonical Wnt signaling. Similar experiments with Wnt4-/-.Axin2 lacZ and Wnt4+/+.Axin2 lacZ mammary epithelium showed that reporter activation relies on Wnt4. Histological analysis on mammary glands in which β-catenin was ablated using MMTV-Cre revealed that β-catenin deleted cells were present only in the luminal epithelial compartment and showed that β-catenin deleted myoepithelial cells cannot participate in side branch formation. Similarly, ectopic expression of dominant negative β-catenin in wt MECs reduced side branching indicating that canonical Wnt signaling mediated by β-catenin is required for side branching. Furthermore, constitutive activation of β-catenin in the mammary epithelium was sufficient to induce side branch formation in virgin mice. In Wnt4 mutant epithelium, proliferation of progesterone receptor positive and negative cells were significantly reduced in response to progesterone treatment. Wnt signaling has been implicated in stem/progenitor activation in several tissues including mammary gland. Consistent with Wnt4 activating stem/progenitors, serial transplantation assays revealed that Wnt4-/- epithelium failed to reconstitute already at 2nd to 3rd generation when the wt has the ability to reconstitute up to seven generations indicating that Wnt4 is required for stem cell function while Wnt4 is dispensable for embryonic and rudimentary mammary gland formation. Histology and gene expression analysis in late pregnancy showed that Wnt4 mutant epithelia were able to differentiate and produce milk proteins, further supporting the thought that Wnt4 function is important for stem/progenitor cell activity and not required for differentiation. Taken together, I report that Wnt4 induces canonical Wnt signaling in myoepithelial cells and activation of canonical Wnt signaling is required and sufficient for side branching. Moreover, Wnt4 function is essential for mammary stem/progenitor cells.

EPFL2010

Role of RIP4 in lung adenocarcinoma differentiation

Jawahar Kopparam

Various studies have attributed NF-kB activation to promote KRAS mediated non-small cell lung cancer progression. In order to identify key genes regulating NF-kB activation and cell survival in lung adenocarcinoma, a synthetic lethal partner screen for KRAS was conducted in cell lines expressing mutant or wildtype KRAS. This screen identified an important role of Receptor-interacting serine/threonine protein kinase 4 (RIP4) in KRAS mediated oncogenesis. But altering RIP4 expression did not affect the viability of KRAS mutant cells, however a novel role of RIP4 in maintaining tumor differentiation was observed. Loss of epithelial differentiation and extracellular matrix remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. Bioinformatics analyses of human lung adenocarcinoma samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. Interleukin 6 (IL6) signaling is activated in lung adenocarcinoma expressing oncogenic KRAS and contributes to cancer invasiveness. Knockdown of RIP4 using shRNA in vitro enhanced activation of IL-6 signaling and also the ability of the cells to invade through collagen. In contrast, overexpression of RIP4 inhibited IL6-mediated Signal Transducer and activator of Transcription 3 (STAT3) activation, which abrogated IL6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. Tumor-specific Rip4 knockdown in an autochthonous mouse model of lung adenocarcinoma initiated by Kras(G12D) expression with loss of p53, progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1 and enrichment of genes regulating extracellular matrix (ECM) remodeling and JAK-STAT signaling. Activated STAT3 can enhance NF-kB activation by interacting with NF-kB and promoting its nuclear retention. Tumors with RIP4 knockdown also exhibited enhanced STAT3- NF-kB interaction suggesting a cross talk between the two pathways. Tail vein injections of cells overexpressing RIP4 showed a reduced potential to invade and form tumors. Analysis of factors regulating RIP4 expression revealed that RIP4 is induced in a p53 dependent manner in response to chemotherapy. Altering the induced RIP4 levels in vitro had a modest effect on chemotherapy induced cell death. Our work has identified that loss of RIP4 enhances IL6 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation.

EPFL2016