Katsuyoshi Takaoka

Cell fate decisions and axis determination in the early mouse embryo

The mouse embryo generates multiple cell lineages, as well as its future body axes in the early phase of its development. The early cell fate decisions lead to the generation of three lineages in the pre-implantation embryo: the epiblast, the primitive endoderm and the trophectoderm. Shortly after implantation, the anterior-posterior axis is firmly established. Recent studies have provided a better understanding of how the earliest cell fate decisions are regulated in the pre-implantation embryo, and how and when the body axes are established in the pregastrulation embryo. In this review, we address the timing of the first cell fate decisions and of the establishment of embryonic polarity, and we ask how far back one can trace their origins.

Origin and role of distal visceral endoderm, a group of cells that determines anterior–posterior polarity of the mouse embryo

Anterior–posterior polarity of the mouse embryo has been thought to be established when distal visceral endoderm (DVE) at embryonic day (E) 5.5 migrates toward the future anterior side to form anterior visceral endoderm (AVE). Lefty1, a marker of DVE and AVE, is asymmetrically expressed in implanting mouse embryos. We now show that Lefty1 is expressed first in a subset of epiblast progenitor cells and then in a subset of primitive endoderm progenitors. Genetic fate mapping indicated that the latter cells are destined to become DVE. In contrast to the accepted notion, however, AVE is not derived from DVE but is newly formed after E5.5 from Lefty1− visceral endoderm cells that move to the distal tip. Concomitant with DVE migration, all visceral endoderm cells in the embryonic region undergo global movement. In embryos subjected to genetic ablation of Lefty1-expressing DVE cells, AVE was formed de novo but the visceral endoderm including the newly formed AVE failed to migrate, indicating that DVE guides the migration of AVE by initiating the global movement of visceral endoderm cells. Future anterior–posterior polarity is thus already determined by Lefty1+ blastomeres in the implanting blastocyst.

Antagonism between Smad1 and Smad2 signaling determines the site of distal visceral endoderm formation in the mouse embryo

The anterior–posterior axis of the mouse embryo is established by formation of distal visceral endoderm (DVE) and its subsequent migration. The precise mechanism of DVE formation has remained unknown, however. Here we show that bone morphogenetic protein (BMP) signaling plays dual roles in DVE formation. BMP signaling is required at an early stage for differentiation of the primitive endoderm into the embryonic visceral endoderm (VE), whereas it inhibits DVE formation, restricting it to the distal region, at a later stage. A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal. DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal. An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.

Removal of maternal retinoic acid by embryonic CYP26 is required for correct Nodal expression during early embryonic patterning

The abundance of retinoic acid (RA) is determined by the balance between its synthesis by retinaldehyde dehydrogenase (RALDH) and its degradation by CYP26. In particular, the dynamic expression of three CYP26 genes controls the regional level of RA within the body. Pregastrulation mouse embryos express CYP26 but not RALDH. We now show that mice lacking all three CYP26 genes manifest duplication of the body axis as a result of expansion of the Nodal expression domain throughout the epiblast. Mouse Nodal was found to contain an RA-responsive element in intron 1 that is highly conserved among mammals. In the absence of CYP26, maternally derived RA activates Nodal expression in the entire epiblast of pregastrulation embryos via this element. These observations suggest that maternal RA must be removed by embryonic CYP26 for correct Nodal expression during embryonic patterning.

TTC25 deficiency results in defects of the outer dynein arm docking machinery and primary ciliary dyskinesia with left-right body asymmetry randomization

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

A Wnt5 activity asymmetry and intercellular signaling via PCP proteins polarize node cells for left-right symmetry breaking.

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.

RBM14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity

Formation of a new centriole adjacent to a pre‐existing centriole occurs only once per cell cycle. Despite being crucial for genome integrity, the mechanisms controlling centriole biogenesis remain elusive. Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes. Depletion of RBM14 in human cells induces ectopic formation of centriolar protein complexes through function of the STIL/CPAP complex. Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity. Moreover, we find that, upon RBM14 depletion, a part of the ectopic centriolar protein complexes in turn assemble into structures more akin to centrioles, presumably by incorporating HsSAS‐6, a cartwheel component, and cause multipolar spindle formation. We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre‐existing centrioles. This study sheds light on the possibility that ectopic formation of aberrant structures related to centrioles may contribute to genome instability and tumorigenesis.

Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6‐/‐ mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2–IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6−/− mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6‐dependent mechanism.

Origin of cellular asymmetries in the pre-implantation mouse embryo: a hypothesis

The first cell fate decision during mouse development concerns whether a blastomere will contribute to the inner cell mass (ICM; which gives rise to the embryo proper) or to trophectoderm (TE; which gives rise to the placenta). The position of a cell within an 8- to 16-cell-stage embryo correlates with its future fate, with outer cells contributing to TE and inner cells to the ICM. It remains unknown, however, whether an earlier pre-pattern exists. Here, we propose a hypothesis that could account for generation of such a pre-pattern and which is based on epigenetic asymmetry (such as in histone or DNA methylation) between maternal and paternal genomes in the zygote.

A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO

Lee et al. show that PGAP6 is a glycosylphosphatidylinositol (GPI)-specific phospholipase A2 expressed on the cell surface. PGAP6 selectively acts on a GPI anchor of CRIPTO, but not its close homologue CRYPTIC, and modulates Nodal signaling during embryonic development.