Integration of Shh and Fgf signaling in controlling Hox gene expression in cultured limb cells

During embryonic development, fields of progenitor cells form complex structures through dynamic interactions with external signaling molecules. How complex signaling inputs are integrated to yield appropriate gene expression responses is poorly understood. In the early limb bud, for instance, Sonic hedgehog (Shh) is expressed in the distal posterior mesenchyme, where it acts as a mediator of anterior to posterior (AP) patterning, whereas fibroblast growth factor 8 (Fgf8) is produced by the apical ectodermal ridge (AER) at the distal tip of the limb bud to direct outgrowth along the proximal to distal (PD) axis. Here we use cultured limb mesenchyme cells to assess the response of the target Hoxd genes to these two factors. We find that they act synergistically and that both factors are required to activate Hoxd13 in limb mesenchymal cells. However, the analysis of the enhancer landscapes flanking the HoxD cluster reveals that the bimodal regulatory switch observed in vivo is only partially achieved under these in vitro conditions, suggesting an additional requirement for other factors.

Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd

Denis Duboule

The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question utilises the murine Hox gene clusters encoding homeobox- containing proteins. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and might be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, here we analyse the nuclear behaviour of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. We show that this transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation and to break Hoxd colinearity in the primitive streak region of the early embryo. Significantly, in rhombomere 4, the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that reorganisation of chromosome territories and chromatin decondensation can be uncoupled from transcription itself and suggests that they can therefore operate upstream of gene expression.

2008

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

In vitro modeling of early mouse development

Mehmet Ugur Girgin

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.