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WIREs Dev Biol

Early development of cephalochordates (amphioxus)

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The Phylum Chordata includes three groups—Vertebrata, Tunicata, and Cephalochordata. In cephalochordates, commonly called amphioxus or lancelets, which are basal in the Chordata, the eggs are small and relatively non‐yolky. As in vertebrates, cleavage is indeterminate with cell fates determined gradually as development proceeds. The oocytes are attached to the ovarian follicle at the animal pole, where the oocyte nucleus is located. The cytoplasm at the opposite side of the egg, the vegetal pole, contains the future germ plasm or pole plasm, which includes determinants of the germline. After fertilization, additional asymmetries are established by movements of the egg and sperm nuclei, resulting in a concentration of mitochondria at one side of the animal hemisphere. This may be related to establishment of the dorsal/ventral axis. Patterning along the embryonic axes is mediated by secreted signaling proteins. Dorsal identity is specified by Nodal/Vg1 signaling, while during the gastrula stage, opposition between Nodal/Vg1 and BMP signaling establishes dorsal/anterior (i.e., head) and ventral/posterior (i.e., trunk/tail) identities, respectively. Wnt/β‐catenin signaling specifies posterior identity while retinoic acid signaling specifies positions along the anterior/posterior axis. These signals are further modulated by a number of secreted antagonists. This fundamental patterning mechanism is conserved, with some modifications, in vertebrates. WIREs Dev Biol 2012, 1:167–183. doi: 10.1002/wdev.11

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Figure 1.

(a) Phylogenetic relations within the Chordata. The three subphyla are Cephalochordata, Tunicata, and Vertebrata. Cephalochordates (amphioxus, lancelets) are basal in the chordates. Although tunicates are the sister group of vertebrates, they are evolving very rapidly and have diverged considerably from other chordates. The three genera of cephalochordates are Asymmetron, which phylogenetic analyses based on mitochondrial DNA sequences place basally in the group,1,3 Branchiostoma and Epigonichthys. (b and c) Photographs of living Branchiostoma floridae (b) and Asymmetron lucayanum (c) taken from the right side. B. floridae has a row of gonads on each side of the body, while A. lucayanum has a single row of gonads on the right side.

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Figure 2.

Migration of male and female pronuclei in the first 14 min after insemination and formation of the germ plasm. (a) The unfertilized ‘egg’ is arrested at second meiotic metaphase with the first polar body closely apposed to the vitelline layer (not shown). At insemination, the second meiotic division resumes with production of the second polar body (2nd pb) at about 10 min after insemination. A female pronucleus does not form. Instead, there are several vesicles containing the maternal chromosomes. The sperm nucleus often enters in the animal hemisphere. Within 45 seconds after insemination at 25°C, the sperm nucleus has been incorporated into the egg and moved beneath the egg cortex. By 5 min after insemination, the sperm nucleus, which is surrounded by mitochondria, is located in the vegetal cytoplasm and has begun to decondense with lighter chromatin surrounding a denser core. By 14 min, the sperm nucleus has fully decondensed and migrated together with a cloud of mitochondria back to the animal hemisphere. The mitochondria tend to cluster along the microtubules, which form the sperm aster (not shown). The male pronucleus and the clusters of female chromosomes move toward each other. The sperm nucleus and clusters of female chromosomes fuse about 15 min after insemination. Then, the resulting nucleus migrates to the center of the zygote. First cleavage typically occurs about 1 h after insemination. At 45 seconds after insemination, the pole plasm (pp), which contains mRNAs for the germ cell markers Vasa and Nanos,11 coalesces in the vegetal cytoplasm. Data from Refs 11 and 16 not depicted are cortical granules, which upon insemination undergo exocytosis, expelling their contents into the perivitelline space. (b) Migration of the pole plasm, also called germ plasm as it is destined to form the germ cells. The Vasa and Nanos expressing pole plasm (asterisks) is segregated into a single blastomere at each cleavage until the mid‐gastrula stage, when it divides into two cells. Subsequently, cells containing the pole plasm migrate posteriorly in the ventral endoderm, dividing into a cluster of at least eight cells as they migrate.11

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Figure 3.

Embryos of amphioxus Branchiostoma floridae, from gastrula to early neurula. Animal pole to the left. The second polar body has detached from these embryos. The blastula is one cell thick, with vegetal cells slightly larger than animal cells. At the onset of gastrulation, the vegetal plate flattens and then invaginates into the blastocoel. By the late gastrula, the blastopore has nearly closed and the neural plate (np) has flattened. In optical cross section, the anterior somites (asterisks) are beginning to form as dorso‐lateral grooves in the archenteron, which will subsequently pinch off starting at the anterior end of the embryo. The arrowhead indicates the future notochord. By the early neurula, the ectoderm adjacent the neural plate has detached from the neural plate and has begun to move over it by means of lamellipodia (arrows).20 Scale bars applicable to all embryos equal 50 µm. Times are for development at 30°C.

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Figure 4.

Fate maps of amphioxus embryos. The animal pole is marked by the second polar body (at left except in the cross section of the late gastrula). Because tissue movements at gastrulation are minimal, the relative positions of the tissue layers do not change except that the presumptive endoderm, somites and notochord invaginate into the blastocoel. The anterior pole is about 20° offset from the animal pole. The animal pole shifts toward the ventral side as the embryo elongates during the gastrula stage. The posterior pole is approximately in line with the equator of the unfertilized egg and blastula.32 Neuroectoderm is in green, non‐neural ectoderm in blue, mesoderm in orange, and endoderm in yellow.

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Figure 5.

Schematic sections of amphioxus embryos showing expression of signaling pathway genes and selected transcription factor genes involved in axial patterning during gastrulation. To facilitate depiction of the patterns, expression domains of Nodal/Vg1, BMPs, and Wnts 4 and 7b are shown inside the outline of the embryos. All other expression domains are shown adjacent to the tissue in which they are expressed. Top row, genes involved in signaling by BMPs, Nodal/Vg1, and FGF pathways. Expression of BMP2/4 and BMP5‐8 is congruent throughout the gastrula stage. FGF8/17/18 is not expressed at the late blastula stage. Expression between late blastula and mid‐gastrula has not been determined. FGF9/16/20 (not shown), is broadly expressed at the mid‐gastrula stage, most strongly in dorsal tissues.40 The three other FGFs (FGFA, FGFC, and FGFE are not expressed during the gastrula stage.40 Middle row, genes involved in canonical Wnt/β‐catenin signaling. β‐catenin is localized to all nuclei at the late blastula stage, but is downregulated in the mesendoderm at the onset of gastrulation. During the late gastrula stage, it is most concentrated in nuclei around the blastopore. Bottom row, genes involved in non‐canonical Wnt signaling [the Wnt/Ca++ and planar cell polarity (PCP) pathways] plus the transcription factors FoxQ2, Hex, Blimp1, goosecoid, Evx, and Sox1/2/3. Data from Refs 12, 13, 21, 22, 28, and 40–45.

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Figure 6.

Diagram of probable gradients of four major signaling pathways (Nodal/Vg1, BMPs, Wnt/β‐catenin, and FGFs) in early amphioxus embryos. Together, signaling by these three pathways establishes and maintains the major embryonic axes. (a) Nodal signaling is high in the animal hemisphere in eggs just after fertilization. BMPs, FGFs, and Wnts signaling through β‐catenin are not expressed in fertilized eggs or cleavage stages. (b) By the mid‐gastrula, Nodal/Vg1 signaling is high dorsally and anteriorly, while BMP signaling is high ventrally and posteriorly. is highest dorsally and posteriorly, while Wnt/β‐catenin signaling is highest posteriorly.

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Figure 7.

Diagram of the Nodal/Vg1 and BMP signaling pathways. Nodal, Vg1, and Activin are closely related members of the TGF‐β superfamily which bind as dimers to the activin type I and type II receptors (ActRI, ActRII), which complex with the Alk4 (also called ActRIB) receptors and with proteins such as Cripto‐I in the EGF‐CFC family of GPI‐linked proteins.54 Activin is not shown in the diagram as it is not expressed in early development in amphioxus.12 Nodal or Vg1 dimers bind to type II and type I receptors, which, as they are serine/threonine kinases, phosphorylate Smad2/3, which in turn binds to Smad4. The complex is translocated to the nucleus and together with transcription factors (TF), including FoxH1,55 regulates transcription of downstream genes such as Mix.56 BMPs (Bone morphogenic proteins) bind as dimers to complexes of type I and type II BMP receptors (BMPRI, BMPRII) together with Alk3/6 receptors. Similarly, these receptors phosphorylate Smad1/5/8, which in turn binds to Smad4 and is translocated to the nucleus where it partners with transcription factors to regulate transcription of target genes. Binding of Nodal/Vg1 to its receptors is inhibited by several secreted proteins including Lefty, Cerberus, and BMP while binding of BMP to its receptors is inhibited by Noggin, Cerberus, Nodal/Vg1, and Chordin. In the cytosol, Smad7 inhibits the Nodal/Vg1 and BMP pathways. Not shown is signaling by TGF‐β, which constitutes a third family of genes in the TGF‐β super family as signaling by TGF‐β appears to be of lesser importance during early development than signaling by Nodal/Vg1 and BMPs. The figure is based on data from vertebrates.54,57–59 Amphioxus has homologs of all of these genes. Although details of TGF‐β signaling pathways have not been determined for amphioxus, they are expected to be similar to those in vertebrates given similar expression and function of Nodal/Veg1 and BMPs in both organisms. Abbreviations: The Vg1 homolog in the frog Xenopus is termed Veg1 for vegetal hemisphere glycoprotein 1. Smad2, mothers against decapentaplegic homolog 2; BMP, bone morphogenetic proteins; ActRII, activins bind specific type II receptor serine kinases; Alk4, activin receptor like 4; Cer, cerberus.

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Figure 8.

Diagram of fibroblast growth factor (FGF) signaling. FGF signals via four major downstream pathways. From left to right, these are (1) SOS‐Ras‐P38MAPK, Rho, or Jun kinase, (2) PLCγ‐IP3‐PKC or Ca2+, (3) Grb2‐PI3K‐PKB/Akt, and (4) SOS‐Ras‐Raf‐MAPK. Two FGF molecules bind to two fibroblast growth factor receptors (FGFR) which dimerize together with heparin sulfate proteoglycan (HSPG). FGFRs are single‐pass transmembrane proteins with an intracellular domain that functions as a tyrosine kinase.60,61 Activated FGFRs transphosphorylate the intracellular tail and tyrosine kinase domains. Several adaptor proteins including Frs2, Grb2, and Gab1 can also be phosphorylated. The main signaling pathway in development, shown at right, leads to a complex of FGFRs with adaptor proteins including Crk, Shc, and SOS , which leads to a signaling cascade involving Ras, Raf, Mek1/2, and a MAPkinase such as Erk1/2. MAPK is translocated to the nucleus and activates transcription factors such as ELK1, which in turn regulate transcription of downstream genes. In a second pathway, depicted at center right, the Grb2, Gab1 complex activates PI3K, which in turn activates PDK1, then PKB/Akt and induces cell survival. A third pathway, center left, is mediated by PLCγ, which activates Ca2+ signaling and PKC, which feeds back into the Ras/MAPK pathway. In this pathway, PIP2 is converted to IP3, which together with DAG activates PKC. The fourth pathway, shown at left, involving a Crk/Shc/SOS adaptor complex is mediated by Ras, Rac, Cdc42, and activates either Jun kinase (pink) or P38MAPK, which promote activation of downstream genes or it activates Rho leading to cytoskeletal rearrangements. There are several inhibitors of FGF‐signaling. These include the transmembrane proteins Sef, which blocks the Frs2‐SOS‐Grb2 complex and interferes with the HSPG/FGFR complex as does FGFRL1. In addition, Sprouty inhibits Ras while Mkp1 and Mkp3 interfere with MAPK function. Abbreviations: Sef: similar to FGFs; FGFRL1: fibroblast growth factor receptor‐like 1; IP3: inositol tris phosphate; MAPK: mitogen‐activated protein Kinase; MKP3: MAPK phosphatase; PI3K: phosphatidylinositol‐3‐kinase; PLCγ: phospholipase Cγ; SOS: son of sevenless; PKC: protein kinase C; Grb2: growth factor receptor‐bound protein 2; Gab1: GRB2‐associated‐binding protein1; Frs2: fibroblast growth factor receptor substrate 2; Crk: CT10 oncogene or p38; Shc: SH2 containing collagen‐related protein; Cdc42: small GTPase in the Rho family; Jun kinase: Jun N‐terminal kinase; Mek1/2: MAPK/Erk kinase; Ras: GTPase; Rho: GTPase; Aklt/PKB: serine/threonine protein kinase; PIP2: phosphatidylinositol 4,5‐biphosphate; Raf: proteo‐oncogene serine/threonine‐protein kinase; DAG: diacylglycerol. The diagram is based on Refs 60–65.

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Figure 9.

Diagram of Wnt signaling through two of the three major signaling pathways important in shaping early embryos: the Wnt/β‐catenin pathway and the Wnt/Jun or Wnt/PCP (planar cell polarity). The first has a role in specification of embryonic axes, while the second is important in changes in tissue shape. The third pathway, the Wnt Ca++ is not shown. In the Wnt/β‐catenin pathway, Wnts, including Wnt1, Wnt3, Wnt6, and Wnt8, bind to Frizzled receptors, which are seven pass transmembrane proteins that partner with LRPs, Dvl (Disheveled), Axin, GSK3β (glycogen synthase kinase 3β), and CK1. In the absence of Wnt ligands, β‐catenin is targeted for degradation. β‐catenin is phosphorylated by CK1 and degraded via β‐TrCP and ubiquitin ligase. In the presence of Wnt ligands, β‐catenin is released from a complex with GSK3β, APC, Axin, Ck1a, and β‐TrCp. Stabilized β‐catenin is translocated to the nucleus and where it functions as a cofactor with the TCF/LEF transcription factor to regulate transcription of target genes. Other cofactors are Brg1, Lgs, and Pygo. In addition, cytosolic β‐catenin associates with cadherin and α‐catenin (white) to form a component of cell junctions. Several inhibitors of Wnt inhibit the pathway. These include WIF, Cer (Cerberus), sFrps, and Dkk1/2/4. In the Wnt/PCP pathway binding of Wnts including Wnt 5, activates the small GTPases RhoA and Rock. RhoA in turn activates JNK and JNK promotes nuclear localization of c‐JUN, which binds to AP‐1 to regulate transcription of downstream genes. In addition, Rock can lead to cytoskeleton rearrangements. Abbreviations: Rock: Rho‐associated kinase; WIF: Wnt inhibitory factor; sFRP: soluble frizzled‐related protein; WTX: Wilm's tumor suppressor; APC: adenomatous polyposis coli; ROCK: Rho‐associated protein kinase. Diagram based on Refs 37, 47, 66–68.

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