David A.F. Loebel

The Transcriptional and Functional Properties of Mouse Epiblast Stem Cells Resemble the Anterior Primitive Streak

Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.

Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo.

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.

Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.

C to U RNA editing mediated by APOBEC1 requires RNA‐binding protein RBM47

Cytidine (C) to Uridine (U) RNA editing is a post‐transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA‐binding protein A1CF. We identify and characterise here a novel RNA‐binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1‐mediated editing in vitro. Editing is further impaired in Rbm47‐deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing.

The mesenchymal architecture of the cranial mesoderm of mouse embryos is disrupted by the loss of Twist1 function

The basic helix-loop-helix transcription factor Twist1 is a key regulator of craniofacial development. Twist1-null mouse embryos exhibit failure of cephalic neural tube closure and abnormal head development and die at E11.0. To dissect the function of Twist1 in the cranial mesoderm beyond mid-gestation, we used Mesp1-Cre to delete Twist1 in the anterior mesoderm, which includes the progenitors of the cranial mesoderm. Deletion of Twist1 in mesoderm cells resulted in loss and malformations of the cranial mesoderm-derived skeleton. Loss of Twist1 in the mesoderm also resulted in a failure to fully segregate the mesoderm and the neural crest cells, and the malformation of some cranial neural crest-derived tissues. The development of extraocular muscles was compromised whereas the differentiation of branchial arch muscles was not affected, indicating a differential requirement for Twist1 in these two types of craniofacial muscle. A striking effect of the loss of Twist1 was the inability of the mesodermal cells to maintain their mesenchymal characteristics, and the acquisition of an epithelial-like morphology. Our findings point to a role of Twist1 in maintaining the mesenchyme architecture and the progenitor state of the mesoderm, as well as mediating mesoderm-neural crest interactions in craniofacial development.

Regionalized Twist1 activity in the forelimb bud drives the morphogenesis of the proximal and preaxial skeleton

Development of the mouse forelimb bud depends on normal Twist1 activity. Global loss of Twist1 function before limb bud formation stops limb development and loss of Twist1 throughout the mesenchyme after limb bud initiation leads to polydactyly, the ulnarization or loss of the radius and malformations and reductions of the shoulder girdle. Here we show that conditional deletion of Twist1 by Mesp1-Cre in the mesoderm that migrates into the anterior-proximal part of the forelimb bud results in the development of supernumerary digits and carpals, the acquisition of ulna-like characteristics by the radius and malformations of the humerus and scapula. The mirror-like duplications and posteriorization of pre-axial tissues are preceded by disruptions to anterior-posterior Shh, Bmp and Fgf signaling gradients and dysregulation of transcription factors that regulate anterior-posterior limb patterning.

Sox17-dependent gene expression and early heart and gut development in Sox17-deficient mouse embryos

Sox17 is a transcription factor that is required for maintenance of the definitive endoderm in mouse embryos. By expression profiling of wild-type and mutant embryos and Sox17-overexpressing hepatoma cells, we identified genes with Sox17-dependent expression. Among the genes that were up-regulated in Sox17-null embryos and down-regulated by Sox17 expressing HepG2 cells is a set of genes that are expressed in the developing liver, suggesting that one function of Sox17 is the repression of liver gene expression, which is compatible with a role for Sox17 in maintaining the definitive endoderm in a progenitor state. Consistent with these findings, Sox17(-/-) cells display a diminished capacity to contribute to the definitive endoderm when transplanted into wild-type hosts. Analysis of gene ontology further revealed that many genes related to heart development were downregulated in Sox17-null embryos. This is associated with the defective development of the heart in the mutant embryos, which is accompanied by localised loss of Myocd-expressing cardiogenic progenitors and the malformation of the anterior intestinal portal.

Context-specific function of the LIM homeobox 1 transcription factor in head formation of the mouse embryo.

ABSTRACT Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity. Summary: Lhx1 mutant mice show head truncations. LHX1 regulates formation of the anterior mesendoderm tissue and limits Wnt pathway activity - acting at least partly via the protocadherin PCDH7.

Rho GTPases in endoderm development and differentiation

The embryonic foregut of the mouse embryo is lined by a layer of endoderm cells whose architecture changes during development. The transition from a squamous to columnar epithelial morphology is accompanied by the upregulation of an atypical Rho GTPase, Rhou. Subsequently, multi-layering of the epithelium at the site of organ bud formation is associated with the downregulation of Rhou. Rho-related small GTPases are known to play multiple roles in establishing and maintaining epithelial polarity, cytoskeletal organization, morphogenesis and differentiation of epithelial tissues, but their role in the early development of the endoderm in mammals is largely unexplored. Our recent study has shown that Rhou is required for maintaining F-actin polarization, epithelial morphogenesis and differentiation of the endoderm. Rhou expression responds to canonical WNT signaling and its activity influences the cytoskeletal organization and differentiation of endodermal cells, possibly via activation of JNK-mediated pathways. In this context, Rhou provides a possible link between β-catenin dependent WNT signaling and cellular processes normally associated with WNT/PCP pathways.

Differential response of epiblast stem cells to Nodal and Activin signalling: a paradigm of early endoderm development in the embryo

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.