Ingo Burtscher

Foxa2 regulates polarity and epithelialization in the endoderm germ layer of the mouse embryo

In the mouse, one of the earliest events in the determination of cell fate is the segregation of cells into germ layers during gastrulation; however, the cellular and molecular details are not well defined due to intrauterine development. We were able to visualize a clear sequence of events occurring in the process of germ-layer formation, using immunohistochemistry and time-lapse confocal imaging. The T-box transcription factor brachyury (T) and the Forkhead transcription factor Foxa2 specify mesoderm and endoderm in the posterior epiblast. Fate-specified epiblast cells lose their polarity and undergo epithelial-mesenchymal transition to invade into the primitive streak region, where these cell populations quickly separate and differentiate into morphologically and molecularly distinct Foxa2-positive endoderm and T-positive mesoderm populations. The endoderm cells flatten and acquire apical-basal polarity during intercalation into the outside epithelium in order to establish proper intracellular junctions with pre-existing cells. By contrast, the mesodermal cells become spherical during migration and acquire a mesenchymal fate. Interestingly, axial mesodermal cells are descended from Foxa2-positive epiblast cells that upregulate T protein in the anterior primitive streak region. These cells, as well as Foxa2-positive endoderm cells, are highly polarized and epithelialized, suggesting that Foxa2 promotes an epithelial fate and suppresses a mesenchymal fate. This observation is supported by the fact that Foxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium. We propose that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype.

Pitchfork Regulates Primary Cilia Disassembly and Left-Right Asymmetry

A variety of developmental disorders have been associated with ciliary defects, yet the controls that govern cilia disassembly are largely unknown. Here we report a mouse embryonic node gene, which we named Pitchfork (Pifo). Pifo associates with ciliary targeting complexes and accumulates at the basal body during cilia disassembly. Haploinsufficiency causes a unique node cilia duplication phenotype, left-right asymmetry defects, and heart failure. This phenotype is likely relevant in humans, because we identified a heterozygous R80K PIFO mutation in a fetus with situs inversus and cystic liver and kidneys, and in patient with double-outflow right ventricle. We show that PIFO, but not R80K PIFO, is sufficient to activate Aurora A, a protooncogenic kinase that induces cilia retraction, and that Pifo/PIFO mutation causes cilia retraction, basal body liberation, and overreplication defects. Thus, the observation of a disassembly phenotype in vivo provides an entry point to understand and categorize ciliary disease. AUTHOR AUDIO:

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic–erythroid and granulocytic–monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic–erythroid versus granulocytic–monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation

During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior-posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3(-/-) embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.

Sox17-2A-iCre: a knock-in mouse line expressing Cre recombinase in endoderm and vascular endothelial cells.

Sox17 encodes an SRY‐related high‐mobility group (HMG) box transcription factor that is essential for endoderm formation and fetal hematopoietic stem cell maintenance. In the mouse, expression of Sox17 is first observed in the extraembryonic endoderm and is subsequently seen in the definitive endoderm as well as in blood and the endothelial cells of the developing vasculature. To conditionally inactivate genes in these domains, we have targeted the Sox17 locus to generate a bicistronic mRNA linking Sox17 to a codon improved Cre recombinase (iCre) via a viral 2A sequence. Here we report a new Cre knock‐in mouse line, Sox17‐2A‐iCre, with activity in the developing endoderm, the vascular endothelial cells of the cardiovascular system and the hematopoietic system. Our results indicate that the Sox17‐2A‐iCre is active in an early endoderm progenitor and recombination of the Rosa26 reporter was observed in all previously reported expression domains of Sox17. The Sox17‐2A‐iCre line will be an excellent tool to conditionally inactivate genes in the definitive endoderm as well as in the vasculature and hematopoietic system. genesis 47:603–610, 2009.

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Generation of a mouse line expressing Sox17-driven Cre recombinase with specific activity in arteries.

The HMG‐box transcription factor Sox17 has been shown to play important roles in both endoderm formation and cardiovascular development. To conditionally inactivate genes in these domains, we have targeted a codon improved Cre Recombinase (iCre) into exon 1 of the Sox17 gene. Surprisingly, Cre‐mediated recombination in the Rosa26 reporter mouse line revealed largely specific activity within the vasculature rather than in endoderm‐derived tissues. Here we report a new Cre knock‐in mouse line, Sox17iCre with activity in the vascular endothelial cells of arteries in the cardiovascular system but not in veins. Cre‐mediated recombination was also strongly detected in the liver and spleen, the two organs associated with hematopoiesis. Thus, these results indicate that the Sox17iCre would be an appropriate tool for conditional mutagenesis of genes in the vasculature and could be used in studies of blood vessel development and angiogenesis. Additionally, we provide evidence that two different promoters drive Sox17 expression in the endodermal and vascular system. genesis 47:476–483, 2009.

A mouse line expressing Foxa2‐driven Cre recombinase in node, notochord, floorplate, and endoderm

Foxa2 is a forkhead transcription factor expressed in the node, notochord, floorplate, and definitive endoderm and is required in the foregut endoderm for the normal development of the endoderm‐derived organs, such as the liver, lung and pancreas. To conditionally inactivate genes in these tissues and organs, we have targeted a Cre recombinase into Exon 1 of the Foxa2 gene. We show, upon crossing to the ROSA26 reporter mice, that Cre expression from the Foxa2iCre knock‐in allele specifically activates β‐galactosidase expression in the node, notochord, floorplate, and endoderm. In addition, we detect Cre recombination activity in the endoderm‐derived organs including lung, liver, pancreas, and gastrointestinal tract throughout development. These results demonstrate that the Foxa2iCre knock‐in mice are a valuable tool to analyze gene function in endoderm progenitors and endoderm‐derived organs. Moreover, the widespread β‐galactosidase reporter activity in the endoderm suggests that Foxa2 marks a progenitor cell population, which gives rise to the majority of cells in endoderm‐derived organs. genesis 46:515–522, 2008.

The Sox17-mCherry fusion mouse line allows visualization of endoderm and vascular endothelial development

Sox17 is a HMG‐box transcription factor that has been shown to play important roles in both cardio‐vascular development and endoderm formation. To analyze these processes in greater detail, we have generated a Sox17‐mCherry fusion (SCF) protein by gene targeting in ES cells. SCF reporter mice are homozygous viable and faithfully reflect the endogenous Sox17 protein localization. We report that SCF positive cells constitute a subpopulation in the visceral endoderm before gastrulation and time‐lapse imaging reveals that SCF monitors the nascent definitive endoderm during epithelialization. After gastrulation, SCF marks the mid‐ and hindgut endoderm and vascular endothelial network, which can be imaged during establishment in allantois explant cultures. The SCF reporter is downregulated in the endoderm epithelium and upregulated in endothelial cells of the intestine, lung, and pancreas during organogenesis. In summary, the generation of the Sox17‐mCherry reporter mouse line allows direct visualization of endoderm and vascular development in culture and the mouse embryo. genesis 50:496–505, 2012.

Flattop regulates basal body docking and positioning in mono- and multiciliated cells

Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. In this study, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.03842.001