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WIREs Dev Biol
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Morphogenesis in sea urchin embryos: linking cellular events to gene regulatory network states

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Gastrulation in the sea urchin begins with ingression of the primary mesenchyme cells (PMCs) at the vegetal pole of the embryo. After entering the blastocoel the PMCs migrate, form a syncitium, and synthesize the skeleton of the embryo. Several hours after the PMCs ingress the vegetal plate buckles to initiate invagination of the archenteron. That morphogenetic process occurs in several steps. The nonskeletogenic cells produce the initial inbending of the vegetal plate. Endoderm cells then rearrange and extend the length of the gut across the blastocoel to a target near the animal pole. Finally, cells that will form part of the midgut and hindgut are added to complete gastrulation. Later, the stomodeum invaginates from the oral ectoderm and fuses with the foregut to complete the archenteron.

In advance of, and during these morphogenetic events, an increasingly complex input of transcription factors controls the specification and the cell biological events that conduct the gastrulation movements. WIREs Dev Biol 2012, 1:231–252. doi: 10.1002/wdev.18

This article is categorized under:

  • Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics
  • Early Embryonic Development > Gastrulation and Neurulation
  • Comparative Development and Evolution > Model Systems

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Figure 1.

Sequence of development of sea urchin embryos. This diagram shows several stages in the development of the embryo up to the pluteus larval stage. Mesomeres, macromeres and micromeres originate at fourth cleavage, which includes an asymmetric cleavage in the vegetal half of the embryo. The micromeres (red) are fated to produce the larval skeleton. The macromeres are fated to produce mesoderm (orange), endoderm (yellow), and a small amount of ectoderm (blue). The animal half of the embryo produces ectoderm and neural tissues. The primary mesenchyme cells (PMCs) ingress at the mesenchyme blastula stage, and shortly thereafter the archenteron begins its invagination. As the skeleton grows the embryo changes shape, first to a prism shape and then to the pluteus larval shape.

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Figure 2.

Endomesoderm gene regulatory network (GRN). Inputs into the enhancer include arrows to indicate an activating input, or repression, indicated by a bar input. The background of each field indicates the subnetwork operating in the micromeres (pink), nonskeletogenic mesoderm (NSM) (purple), and endoderm (yellow and orange). Time roughly is displayed with the first events occurring at the top of the diagram, and later events occurring toward the bottom. Source: Davidson Lab website which is updated periodically as new information becomes available at http://sugp.caltech.edu/endomes/.

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Figure 3.

Micromere gene regulatory network (GRN). This subnetwork represents specification of the skeletogenic lineage. Following initial specification at the top provided by maternal inputs, a series of genes encoding transcription factors are activated sequentially. About 1–1.5 h before ingression the bottom two rows of genes (Tel, Erg, Hex, Tgif, FoxN2/3, Dri, FoxB, FoxO, Twist, Snail) are activated and will control both ingression and events of primary mesenchyme cell (PMC) differentiation. Some of the genes expressing differentiation proteins are shown at the bottom of the figure. Source: Davidson Lab website which is updated periodically as new information becomes available at http://sugp.caltech.edu/endomes/.

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Figure 4.

Ingression of primary mesenchyme cells (PMCs). (a) Diagram based on an ultrastructural analysis of ingression. The yellow cell is the PMC as seen during ingression. (Reprinted with permission from Ref 49. Copyright 1981 John Wiley & Sons, Ltd.) (b) and (c) Ingression as visualized from a time‐lapse sequence. The (b) sequence shows fluorescent PMCs as they assume the teardrop shape and ingress into the blastocoel. The same sequence is shown in (c) with added DIC images to show where the PMCs are located during the ingression process. (d) Two images of ingression in which the plasma membranes of the embryo are labeled with expressed cadherin‐GFP. Note that the ingressed PMCs have lost the cadherin from their surface. (e) An image of PMCs, expressing a PMC marker (green), that have internalized cadherin (red). (f) An enlarged image of an embryo stained as in (e) to show the intracellular vesicles containing cadherin (red). (g) and (h) Two images taken 30 min apart. In (g) an antibody directed against glycosylated MSP130 is first seen in intracellular vesicles as ingression begins (green). (h) Thirty min later MSP130 is expressed on the cell surface of all the PMCs (green) and is joined by neural glia CAM (NgCAM) (red), a protein that is inserted into the PMC membrane after ingression is completed.50 (Reprinted with permission from Ref 51. Copyright 2003 John Wiley & Sons, Ltd.)

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Figure 5.

Genes involved in control of ingression. In each case, either a green‐dyed control micromere or a red‐dyed morpholino‐injected micromere was transferred to the vegetal pole of a control or morpholino‐injected embryo. (a) and (g) A Snail‐morpholino‐injected micromere (red) failed to ingress in a control embryo. (b) and (h) A control green micromere ingressed in an embryo that had been injected with snail morpholino. (c) and (i) Micromeres containing Twist morpholino (red) fail to ingress in a control (green) embryo. (d) and (j) Control micromeres ingress in an embryo containing Twist morpholino. (e) and (k) Control primary mesenchyme cells (PMCs) produce the skeleton in an unlabeled host embryo. (f) and (l) An unlabeled host embryo produces a skeleton but PMCs containing FoxN2/3 morpholino do not participate in the skeleton production due to a loss of a number of PMC functions, beginning with the inability to correctly ingress.44–46

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Figure 6.

Primary mesenchyme cells (PMCs) actively extending thin filopodia. Four frames taken approximately 10 min apart show the dynamics of thin filopodial extension and withdrawal. The four cells to the right in each frame are PMCs. During this time period, a nonskeletogenic cell wanders into the frame. Over time thin filopodia extend and shorten. The filopodia that shorten usually form a kink as they begin to shorten.61

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Figure 7.

Primary mesenchyme cell (PMC) patterning involves filopodial interactions with the ectoderm. (a) An antibody to both PMCs and nonskeletogenic mesoderm (NSM) (at the tip of the archenteron) shows that both cells extend many thin filopodia. (b) The PMCs at the beginning of skeletogenesis stained with an antibody to MSP130. The 64 PMCs form a ring around the archenteron and on the two sides a cluster of PMCs harbors the early triradiate skeleton. Thin filopodia are abundant at the growing tips of the skeleton. (Reprinted with permission from Ref 67. Copyright 1992 John Wiley & Sons, Ltd.) (c) An embryo producing a skeleton. Half the embryo was injected with a dye at the two‐cell stage. (Reprinted with permission from Ref 27. Copyright 1999 John Wiley & Sons, Ltd.) (d) A single thin filopodium extends about 60–70 µm. (e) An embryo fertilized at the same time as the embryo in (b), but grown for 3 h in dilute NiCl2 sufficient to radialize the embryo. (Reprinted with permission from Ref 67. Copyright 1992 John Wiley & Sons, Ltd.) The filopodia produced by the PMCs wrap around the wall of the blastocoel with some up to 150 µm in length. (f) An embryo in which the red half was injected with a truncated cadherin which prevented specification of that half of the embryo. The PMCs migrate but tend to avoid contact with the unspecified, and therefore uninformative half embryo. (Reprinted with permission from Ref 61. Copyright 1999 John Wiley & Sons, Ltd.)

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Figure 8.

Primary mesenchyme cells (PMCs) use ectoderm for patterning information. On the left, half the blue embryo was treated with NiCl2 and after the treatment PMCs were swapped between blue and red embryos. NiCl2‐treated PMCs produce a normal skeleton if placed in a control red embryo (top skeleton). Control red PMCs produce a radialized skeleton if placed in a NiCl2‐treated embryo (bottom, skeleton), demonstrating that the NiCl2 effect is on the ectoderm, and the PMCs receive information for patterning from that ectoderm. On the right, half the red embryos are Lytechinus variegatus and the blue embryos are Tripneustes esculentus. The top half shows the skeletons produced in control embryos of each species. On the bottom PMCs are transferred between species, and again the correct skeleton is produced in each case. The Tripneustes skeleton includes elements not found on L. variegatus skeletons; thus the ectodermal patterning cues from Lytechinus provided the correct positional information and upon reception of those cues, the Tripneustes cells interpreted the information, and produced a skeleton that was correct according to the Tripneustes genotype.67,68

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Figure 9.

VEGF and FGF are two ectodermal signals that contribute to primary mesenchyme cell (PMC) patterning. (a) VEGF is produced by the ectoderm just lateral to the two ventrolateral clusters, and in (e) the VEGF receptor is produced by the PMCs that receive the VEGF signal. (b) NiCl2‐treatment radializes expression of VEGF, and correspondingly in (f) the VEGFR‐expressing PMCs do not form ventrolateral clusters as they do in control embryos in (e). (c) Ectoderm produces FGF just opposite the ventrolateral clusters. Cells in (g) express FGF receptor and form ventrolateral clusters immediately beneath the cells expressing the FGF. (d) A control embryo produces a ring of cells and is initiating skeletogenesis (bright triradiate skeleton at bottom of embryo). If FGF is knocked down, as in (h) the PMCs do not form ventrolateral clusters. (i) Control larva and (j) a larva in which VEGF signaling was eliminated. (k) Control larva, and (l) a larva in which FGF signaling was eliminated. VEGF images are reprinted with permission from Ref 56. Copyright 2007 John Wiley & Sons, Ltd. and FGF and FGFR images are reprinted with permission from Ref 78. Copyright 2008 John Wiley & Sons, Ltd.

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Figure 10.

Sea urchin gastrulation. (a) Primary invagination. Primary mesenchyme cells (PMCs, red) have already ingressed into the blastocoel. At the beginning of primary invagination, cells in the vegetal plate take on a bottle cell shape (orange) helping to buckle the epithelium. (b) Secondary invagination. After the archenteron buckles, the gut lengthens. Secondary mesenchyme cells at the tip of the archenteron (orange) extend filopodia and search the inside of the blastocoel. They make contact to the roof of the blastocoel and stretch the archentron to its full length. (Reprinted with permission from Ref 81. Copyright 2004 John Wiley & Sons, Ltd.)

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Figure 11.

A subnetwork that helps drive restriction of hindgut differentiation state. (Reprinted with permission from Ref 82. Copyright 2009 John Wiley & Sons, Ltd.)

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Figure 12.

Frizzled 5/8 and RhoA are necessary for gastrulation in Lytechinus variegatus. (a) Comparison of control embryos at late gastrula and prism stages (left, top and bottom, respectively) and embryos injected with mRNA coding for a dominant negative form of frizzled 5/8 (FzTM1, right). Inhibiting frizzled 5/8 leads to failure to form the gut. (Reprinted with permission from Ref 91. Copyright 2006 John Wiley & Sons, Ltd.) (b) RhoA is downstream of Frizzled5/8. If embryos are co‐injected with dnfrizzled 5/8 (FzTM1) and mRNA for a constitutively active form of RhoA (RhoAact), normal development is rescued in 28% of the cases. (Reprinted with permission from Ref 91. Copyright 2006 John Wiley & Sons, Ltd.) (c) RhoA promotes gut formation. If a dominant negative form of RhoA (dnRhoA) is injected into embryos, no gut forms (even at later stages, data not shown). If RhoAact is injected a precocious archenteron forms at the hatched blastula stage, at the appropriate location. (Reprinted with permission from Ref 92. Copyright 2006 John Wiley & Sons, Ltd.)

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Figure 13.

Veg1 cells are added to the archenteron late in gastrulation. A lineage tracer labels progeny of a single veg1 cell (the upper daughter of the macromere division at sixth cleavage). Those veg1 cells reach the blastopore in panel (c) when the gut is just beginning to elongate. In (d) the gut has almost completely elongated yet the veg1 cells are still near the blastopore. In (e) after the gut reaches the animal pole, the veg1 cells enter the archenteron and some of the progeny reach as far as the midgut of the archenteron. In (f) the veg1 cells reach as far as the midgut.21

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Figure 14.

Filopodia are not necessary for lengthening the gut up to two‐thirds of its final length. Ablation of filopodia on nonskeletogenic mesoderm (NSM) cells delays archenteron lengthening. Timelapse of an ablation experiment at the onset of archenteron elongation. (a) Gastrula with archenteron at one‐third of its full length, right before the ablation. A prominent secondary mesenchyme cell is marked by an arrow. (b) Immediately following the ablation; remnants of mesenchyme cells can still be seen (arrow); (c) 2 h and 17 min after ablation. Significant elongation occurred. There is a normal aggregation of primary mesenchyme cells into ventrolateral clusters (large arrow). Remnants of severed filopodia ablated after the first series of laser pulses are still visible (small arrows). (Reprinted with permission from Ref 97. Copyright 1988 John Wiley & Sons, Ltd.)

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