FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.     

X chromosome inactivation revealed by the X-linked lacZ transgene activity in periimplantation mouse embryos

Using H253 mouse stock harboring X-linked HMG-lacZ transgene, we examined X chromosome inactivation patterns in sectioned early female embryos. X-gal staining patterns were generally consistent with the paternal X inactivation in the trophectoderm and the primitive endoderm cell lineages and random inactivation in the epiblast lineages. The occurrence of embryonic visceral endoderm cells apparently at variance with the paternal X chromosome inactivation in 7.5 dpc embryos was explained by the replacement of visceral endoderm cells with cells of epiblast origin. The frequency of cells negative for X-gal staining in 4.5-5.5 dpc XmXp* embryos fluctuated considerably especially in the extraembryonic ectoderm and the primitive endoderm, whereas it was less variable in the embryonic ectoderm. We could not, however, determine whether it is a normal phenomenon revealed for the first time by the use of HMG-lacZ transgene or an abnormality caused by the multicopy transgene.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.     

The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130(-/-) embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130(-/-) delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.     

Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge

In the gastrula stage embryo, the epiblast migrates toward the primitive streak and ingresses through the primitive groove. Subsequently, the ingressing epiblast cells undergo epithelial-mesenchymal transition (EMT) and differentiate into the definitive endoderm and mesoderm during gastrulation. However, the developmental mechanisms at the end of gastrulation have not yet been elucidated. Histological and genetic analyses of the ventral ectodermal ridge (VER), a derivative of the primitive streak, were performed using chick and mouse embryos. The analyses showed a continued cell movement resembling gastrulation associated with EMT during the early tailbud stage of both embryos. Such gastrulation-like cell movement was gradually attenuated by the absence of EMT during tail development. The kinetics of the expression pattern of noggin (Nog) and basal membrane degradation adjacent to the chick and the mouse VER indicated a correlation between the temporal and/or spatial expression of Nog and the presence of EMT in the VER. Furthermore, Nog overexpression suppressed EMT and arrested ingressive cell movement in the chick VER. Mice mutant in noggin displayed dysregulation of EMT with continued ingressive cell movement. These indicate that the inhibition of Bmp signaling by temporal and/or spatial Nog expression suppresses EMT and leads to the cessation of the ingressive cell movement from the VER at the end of gastrulation.     

Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.     

The Rab5 effector EEA1 interacts directly with syntaxin-6.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.     

KIF16B delivers for transcytosis

EMBO J 32 15, 2125–2139 doi:10.1038/emboj.2013.130; published online June072013Protein sorting pathways control correct delivery of membrane proteins to specific compartments of the plasma membrane and are required to maintain the physiological functions in all epithelia. Most clathrin-dependent cargoes require the adaptor protein complexes AP-1A and AP-1B for proper sorting to the basolateral plasma membrane. In this issue of The EMBO Journal, Perez Bay et al (2013) shed light on the mechanism of basal-to-apical protein transport, or transcytosis, of the transferrin receptor in natively AP-1B-deficient epithelia. In AP-1B-deficient epithelia, the transferrin receptor transcytoses through the apical recycling endosome, and requires Rab11. Furthermore, they characterize a novel and specific role for the endosomal microtubule motor Kinesin KIF16B in transferrin receptor apical transport. These findings constitute the first characterization of a specific microtubule motor involved in basal-to-apical transcytosis in epithelia.Epithelial cells present a compartmentalized plasma membrane, where the composition of each compartment is tightly controlled by a precise protein and lipid sorting machinery (Folsch, 2008). The two most conspicuous compartments are the apical and basolateral domains, which generate and segregate from each other through the formation of apically localized junctional complexes. Protein sorting mechanisms ensure delivery of newly synthesized or recycled, protein components to their proper localization in either the apical or basolateral plasma membrane domains. Vectorial transport of proteins requires sorting determinants that are present in the cytoplasmic, transmembrane or extracellular domains. Most of the information that we have about these sorting determinants comes from the basolateral traffic, which depends on clathrin adaptor proteins (APs) AP-1A/B, AP-3 and AP-4 (Gonzalez and Rodriguez-Boulan, 2009). Specific APs bind to cytoplasmic sorting motifs in transmembrane proteins and recruit clathrin-coat components, which sequentially induce membrane curvature, clathrin oligomerization, vesicle budding and fission (Ohno, 2006; Hirst et al, 2011). Mammalian cells present five different AP complexes (AP1–5), each constituted by a heterotetramer of one α-, γ-, δ-, ɛ- or ζ-subunit, one β(1–5) subunit, one σ(1–5) subunit and one μ(1–5) subunit. How these clathrin-coated vesicles deliver membranes to precise compartments in the cell to regulate protein sorting is still poorly understood. The AP1 complex is a key regulator of basolateral polarity (Folsch et al, 1999; Gan et al, 2002; Gravotta et al, 2012). The AP1 complex μ-subunit presents two isoforms μ1A and μ1B, which define the formation of two different complexes, AP-1A and AP-1B, both required for basolateral polarity. AP-1A is ubiquitously expressed in different tissues and localizes mainly to the trans-Golgi network. In contrast, AP-1B is primarily localized to common recycling endosomes (CRE) and is specifically expressed in the majority of epithelial tissues, with the remarkable exception of retinal pigment epithelium and the proximal convoluted tubule in the nephron, which sort most of the basolateral cargo to the apical surface.A wide array of model membrane proteins requires AP-1B to properly localize to the basolateral membrane, including the low-density lipoprotein receptor (LDLR), the VSV-G protein and the transferrin receptor (TfR). Furthermore, the expression of μ1B in μ1B-deficient epithelial cell line LLC-PK1 is sufficient to prevent apical sorting of TfR, indicating that AP-1B is a main player in this clathrin-mediated basolateral sorting pathway. Interestingly, the results of the present study suggest that transcytosis (a membrane trafficking pathway that transports apical or basolateral proteins to the opposite domain in the plasma membrane) is the main mechanism for apical transport of clathrin-dependent cargoes in AP-1B-deficient cells. Basal-to-apical transcytosis of the polymeric IgA receptor (pIgAR) is the best-known transcytotic pathway, and requires several steps in which the receptor complex traverses multiple compartments before reaching a Rab11-positive apical recycling compartment, from where it is sorted to the apical plasma membrane (Golachowska et al, 2010). Polymeric IgA receptor transcytosis requires the function of cytoskeletal proteins for its correct delivery to the apical membrane, including microtubules and actin binding motors. However, no specific microtubule motor has ever been described associated with transcytosis.In the present study, Perez Bay et al (2013) analyse how the TfR is transported to the apical membrane in μ1B-deficient epithelia using as model system the retinal pigment epithelium cell line, which lacks AP-1B, and MDCK cells. They show that basolateral administration of labelled Tf results in its endocytosis and transcytosis towards the apical membrane in AP-1B-depleted MDCK cells, following a pathway that involves Rab11-positive apical recycling endosomes (AREs), and requires Rab11 for its correct delivery. Additionally, they find that TfR transport into AREs depends on microtubules and the kinesin KIF16B, a specific microtubule motor present in the CRE (Figure 1). KIF16B is a plus-end microtubule motor that binds to PtdIns(3)P and GTP-bound Rab14 and regulates the distribution of early endosomes (Hoepfner et al, 2005; Ueno et al, 2011). Surprisingly though, apical transport of pIgAR is not affected by the expression of a KIF16B-dominant negative mutant, which suggests that assembly of KIF16B/TfR carriers occurs downstream of cargo separation during transcytosis. It is also tempting to speculate that more than one transcytosis pathways are at play, and while TfR uses the KIF16B-dependent pathway, pIgAR is transported through a KIF16B independent mechanism. This article is the first study of KIF16B in epithelial cells, and the first showing involvement of a microtubule motor in transcytosis, more than 20 years after the pioneering studies that characterized the role of microtubules in this process (Hunziker et al, 1990).Open in a separate windowFigure 1KIF16B controls basal-to-apical transcytosis of transferrin receptor in AP-1B-deficient epithelia. In AP-1B-expressing epithelia (such as MDCK cells), transferrin receptor (TfR) is endocytosed and sorted to common recycling endosomes, where AP-1B-clathrin-vesicles assemble and transport the protein to the basolateral plasma membrane. In AP-1B-deficient epithelia (such as RPE cells), internalized TfR is instead sorted by the plus-end directed microtubule motor KIF16B towards the ARE, and then transcytosed to the apical plasma membrane through a Rab11-regulated pathway. Polymeric IgA receptor is internalized into the same basolateral endosomes, but it uses a KIF16B-independent pathway to reach the apical membrane.As a whole, this paper represents a significant advance in our understanding of the protein sorting machinery in epithelial cells, and importantly, opens new questions that will be addressed in future studies. First, is the KIF16B-dependent recycling/sorting pathway required for other cargoes, especially in AP-1B-positive epithelia? Second, why TfR, but not pIgAR, requires KIF16B for correct sorting? Although KIF16B is not required for pIgAR transcytosis, its transport route still requires microtubules, thus opening the possibility for discovery of additional microtubule motors involved in transcytosis. And finally, what is the mechanism of KIF16B binding to TfR-positive recycling endosomes? It is possible that the mechanism depends on the activation of Rab14, which has been characterized as a regulator of lipid-raft transport from the Golgi apparatus to recycling endosomes (Ueno et al, 2011).     

Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3

The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell-cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.     

The Primitive Endoderm Segregates from the Epiblast in β1 Integrin-Deficient Early Mouse Embryos

We analyzed the mechanism of developmental failure in implanted β1 integrin-null blastocysts and found that primitive endoderm cells are present but segregate away from, instead of forming an epithelial layer covering, the inner cell mass. This cell segregation phenotype was also reproduced in β1 integrin-null embryoid bodies, in which primitive endoderm cells segregated and appeared as miniature aggregates detached from the core spheroids, and a primitive endoderm layer failed to form on the surface. Restricted β1 integrin gene deletion in embryos using Ttr-Cre or Sox2-Cre indicated that the loss of integrin function in the cells of the inner core rather than the outer layer is responsible for the failure to form a primitive endoderm layer. We conclude that β1 integrin is essential for the attachment of the primitive endoderm layer to the epiblast during the formation of a basement membrane, a process concurrent with the transition from cadherin- to integrin-mediated cell adhesion.     

Distinct GATA6- and laminin-dependent mechanisms regulate endodermal and ectodermal embryonic stem cell fates

This study investigates the establishment of alternative cell fates during embryoid body differentiation when ES cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. We report that endoderm differentiation and endoderm-specific gene expression, such as expression of laminin 1 subunits, is controlled by GATA6 induced by FGF. Subsequently, differentiation of the non-polar primitive ectoderm into columnar epithelium of the epiblast is induced by laminin 1. Using GATA6 transformed Lamc1-null endoderm-like cells, we demonstrate that laminin 1 exhibited by the basement membrane induces epiblast differentiation and cavitation by cell-to-matrix/matrix-to-cell interactions that are similar to the in vivo crosstalk in the early embryo. Pharmacological and dominant-negative inhibitors reveal that the cell shape change of epiblast differentiation requires ROCK, the Rho kinase. We also show that pluripotent ES cells display laminin receptors; hence, these stem cells may serve as target for columnar ectoderm differentiation. Laminin is not bound by endoderm derivatives; therefore, the sub-endodermal basement membrane is anchored selectively to the ectoderm, conveying polarity to its assembly and to the differentiation induced by it. Unique to these interactions is their flow through two cell layers connected by laminin 1 and their involvement in the differentiation of two epithelia from the same stem cell pool: one into endoderm controlled by FGF and GATA6; and the other into epiblast regulated by laminin 1 and Rho kinase.     

Dual roles for the Dab2 adaptor protein in embryonic development and kidney transport

The Disabled-2 (Dab2) gene has been proposed to act as a tumor suppressor. Cell culture studies have implicated Dab2 in signal transduction by mitogens, TGFbeta and endocytosis of lipoprotein receptors. To identify in vivo functions of Dab2, targeted mutations were made in the mouse. In the absence of Dab2, embryos arrest prior to gastrulation with a phenotype reminiscent of those caused by deletion of some TGFbeta signal transduction molecules involved in Nodal signaling. Dab2 is expressed in the extra-embryonic visceral endoderm but not in the epiblast. Dab2 could be conditionally deleted from the embryo without affecting normal development, showing that Dab2 is required in the visceral endoderm but dispensable in the embryo proper. Conditionally mutant Dab2(-/-) mice are overtly normal, but have reduced clathrin-coated pits in kidney proximal tubule cells and excrete specific plasma proteins in the urine, consistent with reduced transport by a lipoprotein receptor, megalin/gp330, in the proximal tubule. This evidence indicates that Dab2 is pleiotropic and regulates both visceral endoderm function and lipoprotein receptor trafficking in vivo.     

Kinesin-1 plays a role in transport of SNAP-25 to the plasma membrane

The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874-894. Overexpression of a fragment of KIF5B (residues 594-910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events.