Embryonic Induction

Abstract: INTRODUCTION Embryonic development and tissue homeostasis depend on cooperation between specialized cell types. Resident macrophages are professional phagocytes that survey their surroundings; eliminate unfit cells, microorganisms, and metabolic waste; and produce a large range of bioactive molecules and growth factors. Resident macrophages also serve tissue-specific purposes: For example, microglia in the central nervous system support neuronal circuit development, Kupffer cells scavenge blood particles and dying red blood cells in the liver, and alveolar macrophages uptake surfactant and remove airborne pollutants and microbes from the airways. Resident macrophage diversity in adult mice is reflected in tissue-specific gene expression profiles, which may be due to responses to specific cues from their microenvironment, different developmental processes, and the contribution of distinct progenitors cell types. Altogether, the mechanisms responsible for the generation of tissue-resident macrophage diversity remain unclear. RATIONALE Tissue-resident macrophages originate, at least in part, from mesodermal erythro-myeloid progenitors (EMPs) from the yolk sac, which invade the embryo proper at the onset of organogenesis. These tissue-resident macrophages are also self-maintained in postnatal tissues, independently of definitive hematopoietic stem cells (HSCs) in a steady state. We therefore hypothesized that resident macrophages represent a founding cell type within most organ anlagen. In this model, the generation of macrophage diversity, as observed in the tissues of postnatal mice, may be integral to organogenesis. RESULTS To test this hypothesis and explore the molecular basis of macrophage diversity in mammals, we performed a spatiotemporal analysis of macrophage development in mice, from embryonic day 9 (E9) to 3 weeks after birth. Unbiased single-cell RNA sequencing (RNA-seq) analysis of CD45 + cells, combined with RNA-seq analyses of sorted cell populations, genetic fate mapping, and in situ analyses, revealed that EMPs give rise to a population of premacrophages (pMacs) that colonize the whole embryo from E9.5, as they acquire a core macrophage differentiation program that includes pattern recognition, scavengers, and cytokine receptors. The chemokine receptor Cx3cr1 is up-regulated in pMacs and is important for embryo colonization, which is delayed in Cx3cr1 -deficient embryos. Fate mapping of pMacs using a Tnfrsf11a –Cre reporter labels homogeneously fetal and adult tissue-resident macrophages but not HSCs and their progeny. Transcriptional regulators that identify postnatal tissue-resident macrophages in the brain, liver, kidney, skin, and lung were specifically up-regulated immediately after colonization. These dynamic changes mark the onset of diversification into adult macrophages. We identified Id3 as a Kupffer cell–specific transcriptional regulator. Deletion of Id3 in pMacs resulted in Kupffer cell deficiency but did not affect development of microglia and kidney macrophages. CONCLUSION Our study shows that EMP-derived precursors colonize embryonic tissues and simultaneously acquire a full core macrophage program. This is followed by their diversification into tissue-specific macrophages during organogenesis, likely via the expression of distinct sets of transcriptional regulators. These results indicate that differentiation of tissue-resident macrophages is an integral part of organogenesis and identify a spatiotemporal molecular road map for the generation of macrophage diversity in vivo. Our findings provide a conceptual framework to analyze and understand the consequence(s) of genetic variation for macrophage contribution to development, homeostasis, and disease pathogenesis in different tissues and will support efforts to differentiate specialized macrophages in vitro.

Abstract: The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP(-)Flk1(-), GFP(+)Flk1(-) and GFP(+)Flk1(+) cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.

Abstract: Vertebrate eye development proceeds by a series of reciprocal tissue interactions between derivatives of the head surface ectoderm and the forebrain neuroectoderm. In particular, the process of lens induction has been studied as a model system to explore general mechanisms underlying embryonic induction (for review, see Jacobson and Sater 1988). Soon after the turn of this century, a few classical experiments using amphibian embryos led to the idea that the optic vesicle has an instructive role in inducing lens formation in the overlying surface ectoderm (Spemann 1901; Lewis 1904, 1907a,b). According to this original model, the optic vesicle is potentially able to induce a lens from ectoderm anywhere on the embryo. Subsequently, however, other studies using different species and experimental conditions raised doubts about this hypothesis, and rather supported the idea that the optic vesicle is not essential for lens formation (for review, see Saha et al. 1989). Furthermore, a key experiment using a host–donor cell-marking technique revealed that the optic vesicle is insufficient to induce lens in ectopic ectoderm taken from outside the lens field; the induced lens in the recombinants was derived from the optic rudiment, rather than from the grafted ectopic ectoderm (Grainger et al. 1988). The current model, based mainly on experiments using Xenopus embryos, proposes that lens induction proceeds through multiple intermediate states, starting early in development from the gastrula stage (for review, see Grainger 1992). Evidence from many in vivo and in vitro studies indicates that the lens can be formed in the absence of the optic vesicle in several vertebrate species (for review, see Jacobson and Sater 1988; Saha et al. 1989; Henry and Grainger 1990). The role of the optic vesicle during lens induction has, therefore, been considered minor—merely to establish the precise location of the lens within the head ectoderm (Grainger 1992). Several lines of evidence, however, suggest that, in vivo, there is apparent species specificity in the extent to which lens formation is dependent on the optic vesicle. Ablation of prospective retinal neuroectoderm in chick embryos abolishes lens formation (Li et al. 1994; Kamachi et al. 1998). Furthermore, lens formation is completely absent in mouse embryos lacking a functional Lhx2 gene, which encodes a LIM-homeodomain protein and is expressed in the forebrain, including the forming optic vesicle neuroectoderm. In these embryos, impairment of optic vesicle development results in failure of the optic vesicle to contact the surface ectoderm (Porter et al. 1997). Therefore, it appears that lens formation in higher vertebrates requires the presence of the optic vesicle in vivo. Like many other embryonic induction processes, secreted signaling molecules are likely to have critical roles during lens induction. In spite of the extensive experimental studies in the amphibian system described above, however, no actual signaling molecules have yet been identified. Likewise, although a number of genes have been implicated in mammalian eye development by molecular and genetic approaches (for review, see Graw 1996; Oliver and Gruss 1997), characterization of their precise in vivo function in many cases awaits further studies. One exception is the Small eye mutant in mouse (Pax6Sey) and rat (rSey), which have been studied extensively in relation to lens induction, as homozygous mutant embryos completely lack lens formation (Hill et al. 1991; Matsuo et al. 1993). Mutations in the Pax6 gene, which encodes a paired-type homeodomain protein, have also been associated with congenital eye defects in humans (Jordan et al. 1992; Glaser et al. 1994; Hanson et al. 1994). Tissue recombination experiments using rSey mutant embryos have revealed that homozygous mutant ectoderm does not form a lens when recombined with a wild-type optic vesicle, whereas the reciprocal recombination allows lens formation in wild-type ectoderm (Fujiwara et al. 1994). This clearly implies a requirement for PAX6 in the head ectoderm for competence to respond to the optic vesicle signal, and indicates that the optic vesicle retains lens inducing activity in the absence of PAX6 function. The underlying mechanisms of PAX6 function in the ectoderm and inductive signaling factors involved in lens induction are still unknown. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily of secretory signaling molecules, have been implicated in many aspects of embryonic tissue interactions (for review, see Hogan 1996). Several BMP family members have been reported to be expressed during mouse eye development (Dudley and Robertson 1997). Furthermore, Bmp7 is required for normal embryonic eye development in the mouse (Dudley et al. 1995; Luo et al. 1995). Here, we report that another member of the Bmp gene family, Bmp4, has critical roles during the lens induction process. We show that the optic vesicle is the major source of BMP4 in the early developing eye, and that it is required for lens induction. A series of explant culture experiments suggest that BMP4 is an essential factor to manifest lens inducing activity of the optic vesicle. We also show that BMP4 regulates specific gene expression in the optic vesicle neuroectoderm. We discuss a model in which BMP4 has multiple roles during mammalian lens induction.

Abstract: The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells1. From mouse blastocysts, it is possible to derive both trophoblast2 and embryonic stem-cell lines3, which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal–KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation. Trophoblast and embryonic stem cells interact in vitro to form structures that resemble early blastocysts, and the embryo provides signals that drive early trophectoderm development and implantation.