Vertebrate Axial Patterning: From Egg to Asymmetry

Abstract

The emergence of the bilateral embryonic body axis from a symmetrical egg has been a long-standing question in developmental biology. Historical and modern experiments point to an initial symmetry-breaking event leading to localized Wnt and Nodal growth factor signaling and subsequent induction and formation of a self-regulating dorsal "organizer." This organizer forms at the site of notochord cell internalization and expresses primarily Bone Morphogenetic Protein (BMP) growth factor antagonists that establish a spatiotemporal gradient of BMP signaling across the embryo, directing initial cell differentiation and morphogenesis. Although the basics of this model have been known for some time, many of the molecular and cellular details have only recently been elucidated and the extent that these events remain conserved throughout vertebrate evolution remains unclear. This chapter summarizes historical perspectives as well as recent molecular and genetic advances regarding: (1) the mechanisms that regulate symmetry-breaking in the vertebrate egg and early embryo, (2) the pathways that are activated by these events, in particular the Wnt pathway, and the role of these pathways in the formation and function of the organizer, and (3) how these pathways also mediate anteroposterior patterning and axial morphogenesis. Emphasis is placed on comparative aspects of the egg-to-embryo transition across vertebrates and their evolution. The future prospects for work regarding self-organization and gene regulatory networks in the context of early axis formation are also discussed.

Keywords: Anterior visceral endoderm; Anteroposterior patterning; Axis formation; Cortical rotation; Dorsoventral patterning; Embryonic induction; Gastrulation; Nieuwkoop center; Spemann organizer; Vertebrate embryology.

Fig. 1

Vertebrate axial organization. (a) Diagram of a sagittal section through a Xenopus gastrula, showing the involution of the dorsal mesoderm (d.m., dark red) at the dorsal lip. The neural plate (n.p., blue) overlies the dorsal mesoderm. bl blastocoel, v.l.m. ventrolateral mesoderm (orange), e endoderm (yellow). (b) Sagittal (left panel) and coronal (right panel) diagrams of a tailbud-stage Xenopus embryo showing the elongated anterior-to-posterior axis and organization of tissues within. The neural tube is located dorsally and will form the entire central nervous system (c.n.s.). The dorsal mesoderm gives rise to the notochord and somites, ventrolateral mesoderm (v.l.m.) will form the kidneys, body wall muscles and vascular system. The endoderm forms the gut and its derivative organs. The cement gland (c.g.), a larval amphibian anchoring structure, is shown at the anterior end. After Hausen and Riebesell (1991)

Fig. 2

Gray crescent formation in amphibians. Top panel, diagram of an amphibian egg (e.g., Rana) before (left) and after fertilization (right). The heavily pigmented animal pole (an) and the paler vegetal pole (veg) are indicated. After fertilization, corticocytoplasmic movements opposite to the sperm entry point (s.e.p.) result in the appearance of the gray crescent (g.c.) on the prospective dorsal side. Bottom panel, images of a Rana egg at fertilization (a), and at 20 min post-fertilization, showing the gray crescent (b; dorsal view, arrow). Bottom panel reproduced from Rugh (1951)

Fig. 3

Events of cortical rotation in Xenopus. Microtubules are disassembled during oocyte maturation, and are absent from the egg cortex (left panels). Certain RNAs are localized to the vegetal cortex during oogenesis (blue) and encode proteins critical for cortical rotation and dorsalization (e.g., trim36, wnt11b). After fertilization, the incoming sperm pronucleus and associated centrosome initiate astral microtubule assembly. Cortical microtubule assembly also begins, forming a network by 40 min post-fertilization. A shear zone forms and microtubules associate with the yolky cytoplasmic core (not shown) and cortical rotation begins, under the action of kinesin-like proteins (kinesins). Relative cortical movement occurs dorsally, possibly the result of nudging by ventrally positioned astral microtubules, and rapidly orients microtubule plus ends dorsally (Olson et al. 2015) (middle panel). Microtubule assembly and organization becomes robust by 60 min post-fertilization and full cortical rotation commences, continuing until first cleavage. Rapid transport of dorsalizing activity occurs along parallel microtubule arrays using kinesin-like motors (right panel). The corresponding bottom panels show live images of microtubules labeled with Enconsin microtubule-binding domain tagged GFP (EMTB-GFP), showing progressive assembly and alignment during cortical rotation (Olson et al. 2015)

Fig. 4

Dorsal determinant transport in zebrafish. (a) Sequence of events in wildtype embryos. RNAs and other dorsal determinants are localized vegetally during oogenesis (blue). After fertilization, cytoplasm streams to the animal pole, forming the future blastoderm. Microtubule assembly initiates ~20 min post-fertilization at the vegetal pole of the yolk cell; localized RNAs and Syntabulin protein (Sybu) are shifted toward the future dorsal side. Microtubule networks in the lateral cortex facilitate global transport animal-ward, which on the dorsal side would contain axis determinants. (b) In hecate (hec) mutants lacking Grip2a, maternal vegetal localization occurs, but cortical rotation and microtubule assembly are deficient post-fertilization. This image is reproduced and modified from, Ge X, Grotjahn D, Welch E, Lyman-Gingerich J, Holguin C, Dimitrova E, et al. (2014) Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction. PLoS Genet 10(6): e1004422. doi:10.1371/journal.pgen.1004422, under the terms of the Creative Commons Attribution License (CC BY 4.0)

Fig. 5

Model for establishment of asymmetry in bird eggs. Left, sectional view of a uterine chicken egg viewed from the sharp end. The direction of rotation is indicated; because of this rotation, the lighter blastoderm cytoplasm is maintained off angle as it continually floats to the highest point. The blastoderm is exposed to the subgerminal cytoplasm, which is hypothesized to contain axis determinants (blue). At this stage, the blastoderm is several thousand cells and has not formed the area pellucida epiblast. Right, top view of 2–3 day embryo showing anterior-to-posterior axial polarity. This embryo would conform to von Baer’s rule, with head oriented away with the blunt end positioned left. ant. anterior, post. posterior

Fig. 6

Early bias of mouse blastomeres towards lineage fate but not axial polarity. Two-cell blastomeres undergo rotational cleavage (dotted white lines indicate cleavage planes), generating a fraction of embryos with a tetrahedral cell arrangement. In this formation, vegetal blastomeres are biased towards contributing to the trophectoderm (dark gray) in the blastocyst. The corresponding animal blastomeres are biased towards contributing to the inner cell mass (blue). After Zernicka-Goetz et al. (2009)

Fig. 7

Generalized Wnt signaling networks. In the absence of activating Wnt ligands (top panel, -Wnt), beta-catenin protein (Ctnnb1) is phosphorylated by destruction complex components and tagged for proteasomal degradation. In the nucleus, Tcf7l1/Tcf3 represses Wnt target promoter activity through recruitment of Groucho. Upon stimulation with Wnt ligand, a variety of pathways are activated (see text for details). Predominantly positive-acting components with respect to beta-catenin regulation are shown in green, negative components in red, beta-catenin-independent components are light blue. Beta-catenin is shown in yellow. Circles indicate component nodes, lines indicate edges, or interacting components. This arrangement is not meant to convey specific exact binding relationships or stoichiometry. Wnt1 is shown as a beta-catenin-activating ligand, whereas Wnt5 is shown as a Wnt/PCP and Wnt/Calcium-stimulating ligand. Plot was generated with iGraph in R (Csardi and Nepusz 2014). txn transcription

Fig. 8

Models for Wnt/beta-catenin activation in Xenopus and zebrafish. (a) During cortical rotation in Xenopus (top) and zebrafish (bottom), beta-catenin stabilizing dorsalizing activity is transported into the equatorial region of the embryo by microtubule-mediated rotation of the cortex and through transport along microtubule arrays. Candidates for this activity include wnt11b and Lrp6/Dvl particles in Xenopus and wnt8a in zebrafish. (b) By the cleavage stages (16–128-cell stage), beta-catenin be-comes activated and enriched in dorsal vegetal and marginal nuclei until MBT. In Xenopus, priming of Wnt target genes occurs through dimethylation of Histone3 at arginine 8 (H3R8). In zebrafish, beta-catenin accumulates in dorsal marginal and dYSL nuclei, and is antagonized by multiple antagonists and calcium signaling mediators. (c) During the peri-MBT stages, beta-catenin activates direct Wnt targets and cooperates with maternal T-domain proteins (Vegt, Eomes) to activate nodal initially on the dorsal side. The combination of nodal and BMP antagonism induced by beta-catenin induce the formation of the organizer (gsc, chrd)

Fig. 9

Models for axis induction signaling in chick and mouse. (a) In the chick blastoderm (left panel, top/dorsal view, ~stage X, Eyal-Giladi and Kochav 1976) the outer marginal zone of the epiblast expresses Wnt8a in a posterior-to-anterior gradient (purple shading). In the PMZ, Pitx2 (yellow) activates Gdf1 expression (green). Subsequently (middle panel), the newly formed hypoblast (below the plane of the page) begins anterior migration and Gdf1 + Wnt8a cooperate to induce Lef1 in the PMZ and Nodal in the adjacent epiblast. Gata2 is expressed in the anterior marginal zone and antagonizes Gdf1 long-range. Nodal (magenta) is antagonized by Cerberus (Cer), which is expressed in the hypoblast. By the initial primitive streak stage (right panel, stage 2+, Hamburger and Hamilton 1951), the anterior migration of the hypoblast and migration of the endoblast beneath the posterior epiblast removes the inhibition of Nodal and allows feed-forward signaling leading to primitive streak formation. The same signaling molecules are ex-pressed in the primitive streak and induce organizer genes in Hensen’s node (dotted circle, Gsc, Chrd) at the anterior tip of the streak. (b) In the mouse, the earliest asymmetries are the expression of Lefty1 and Cerl (red-brown) at the tip of the postimplantation AVE (left panel, ~E5.0). These genes are regulated by Nodal (green) and Tdgf1, and Tdgf1 is regulated by beta-catenin in the absence of secreted Wnt ligand activity (stop symbol). Lefty1 and Cerl antagonize Nodal and feedback regulation drives AVE migration towards the proximal egg cylinder on one side (right panel, ~E5.5). Nodal activity is restricted to the posterior epiblast and is responsible for Wnt3 expression (blue), which in turn maintains Nodal. These signals cooperate to induce the primitive streak, which induces Hensen’s node toward the distal tip later in gastrulation. a anterior, p posterior, ExE extraembryonic ectoderm, VE visceral endoderm, ParE parietal endoderm

Fig. 10

The organizer experiment of Spemann and Mangold. (a) Diagrammatic model of Spemann and Mangold’s dorsal lip transplantation from a lightly pigmented species (light gray) to the ventral region of a darker species (dark gray). (b) Image of a Xenopus tadpole following the successful grafting of an early gastrula dorsal lip, showing the endogenous axis (1° axis) and the induced partial axis (2° axis). The dark pigment in the head of the 2° axis is related to abnormal head development in the induced axis. (c) Diagram of a cross section through an embryo resulting from a dorsal lip transplant as in (a). The typical lineage contribution of the donor lip is lightly shaded reflecting the species origin, indicating contributions to the notochord, floor plate and medial somite (c is after Spemann and Mangold 1924). v ventral, d dorsal

Fig. 11

Dorsoventral patterning of the gastrula. (a) Image of an early gastrula Xenopus embryo (left image) showing gsc mRNA expression in the organizer region (purple). This area expresses BMP antagonists Chrd, Fst, and Nog in a dorsoventral gradient (blue shading), and is complementary to a gradient of BMP signaling (red shading), resulting in a graded pattern of phospho-Smad1/5 (p-Smad1/5). On the right is a late gastrula showing continued ventroposterior pattering by BMP signaling, specifying in progressively later fashion (differing line thicknesses) the anterior neural crest (a.n.c), posterior neural crest (p.n.c.) and epidermis (ep.), in addition to the underlying germ layers (not shown). (b) Simplified network model of secreted protein interactions acting in dorsoventral patterning. Chrd secreted by the organizer antagonizes Bmp activity, mediated by Bmp4/7 ventrally and Admp within the organizer. Bmp activity inhibits chrd expression. Tld/Bmp1 acts as a Chrd inhibitor via proteolysis; Szl is a Tld inhibitor, indirectly promoting Chrd activity in the extreme ventral region (dotted arrow). The critical reciprocal control interactions responsible for self-organization are indicated with blue lines; Bmp4/7 positively controls its own expression but inhibits admp expression. Bmps also upregulate cv2/bmper, a ventral Bmp antagonist, and inhibit chrd dorsally. Model after De Robertis (2009)

Fig. 12

Models for anteroposterior axis patterning in vertebrates. (a) Depiction of Nieuwkoop’s ectodermal fold implantation experiments (Nieuwkoop 1952; Nieuwkoop and Nigtevecht 1954). Dorsal posterior view of a neurula stage amphibian embryo; neural fate is represented as a gradient from light-to-dark with darker color indicating more posterior fates; the epidermis is yellow. The implanted folds are shown as boxes, divided to show the approximately position of induced neural fates. Each fold is characterized by a distal epidermal portion (ep.) bounded by general neural/neural plate border (activated tissue); this is followed proximally by graded neural fates, reflecting the hypothesized influence of a transforming gradient (as opposed to a distinct inducer at each AP level). (b) Molecular interpretation of Nieuwkoop’s model. In the gastrula, neural induction is accomplished by BMP antagonism, which induces neural tissue with forebrain character. (c) During later gastrulation, the expression of Wnts directly induces posterior fates in anterior neural-fated tissue in a dose-dependent fashion. FGF signaling is required in a permissive role. Wnt antagonists expressed in the anterior mesendoderm limit the extent of Wnt signaling and the anterior remains forebrain

Fig. 13

Model for origin and role of the AVE in anteroposterior pattering in the mouse. (a) In the peri-implantation blastocyst (E5.0), a subpopulation of primitive endoderm (PE) expressing Lefty1 and Cerl arise stochastically positioned asymmetrically in the distal egg cylinder. This population requires Nodal and Tdgf1 in the epiblast and is inhibited by Bmp4 in the extraembryonic ectoderm. (b) As the conceptus grows after implantation (E5.25), the AVE begins to also express Wnt antagonists and is repelled by Nodal and Wnt signals; the action of BMP is limited to the proximal epiblast and PE, allowing migration of AVE in the distal region. (c) As AVE migration proceeds, a second set of Lefty1; Cerl-expressing cells is induced in the distal VE (2°AVE) by Tdgf1-independent Nodal signaling. These cells and the 1°AVE migrate in a coherent stream toward the presumptive anterior, inhibiting Nodal and Wnt signaling in the epiblast and specifying the anterior neuroectoderm. Progressive loss of Nodal from the anterior limits activity to the posterior, where Nodal and Wnt maintain and amplify each other’s expression through BMP4, inducing the primitive steak (PS) in the prospective posterior. ParE parietal endoderm, PE/VE primitive/visceral endoderm, ExE extra-embryonic ectoderm. Images were modified and adapted from: Bedzhov I, Graham SJL, Leung CY, Zernicka-Goetz M (2014) Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo. Philosophical Transactions of the Royal Society B: Biological Sciences 369:20130538. doi:10.1098/rstb.2013.0538 under the terms of the Creative Commons Attribution License CC BY 3.0

Fig. 14

Model for the evolution of axis formation in vertebrates. Highly generalized schematic diagrams of different vertebrate embryos during early (upper panels) and middle (lower panels) gastrulation. In the basal vertebrates (left panels; vegetal views), gastrulation initiates at the organizer with induction by the Nieuwkoop center (high/early Nodal signaling). The initial internalization movements are through involution. By mid-gastrulation, ingression of mesendoderm progresses around the vegetal cells (yolk plug) and forms the nascent blastopore. Involuted dorsal mesendoderm undergoes convergent extension via mediolateral cell intercalation under the control of Wnt/PCP signaling. During the evolution of amniotes (middle panels; top/dorsal views), eggs increased in size and yolk content and began to undergo meroblastic cleavage. Organizer induction by Nieuwkoop center molecules is retained in early gastrulation. Ingression proceeds through the horizontal slit of the blastoporal plate/blastopore and does not circumferentially envelop the non-cleaving yolk (not shown). In the evolution of modern birds and mammals, gastrulation initiates with ingression at the primitive streak, which would be homologous to the later ventrolateral blastopore in ancestral forms. The organizer (Hensen’s node) is induced later by Nodal signaling from the middle primitive streak. The heterochrony in the pattern of gastrulation morphogenesis and organizer formation could result from several main events; the hypoblast/anterior endoderm segregating from the epiblast as opposed to forming from cleaving vegetal cells, the loss of early organizer induction, the apparent emergent behavior of polonaise-like movements leading to primitive streak formation and the relatively later induction of Hensen’s node by the primitive steak