Early development in sea urchins
Example of deuterostome (incl. vertebrates) development.
Lytechinus variegatus (green sea urchin) widely studied model organism.
Sea urchins – exceptionally important in studying how genes regulate the formation of the body, first model to provide evidence for:
- Chromosomes needed for development
- DNA and RNA are present in each animal cell
- mRNAs direct protein synthesis
- Stored mRNA provide the proteins for early embryonic development
- Cyclins control cell division
- Enhancers are modular
- Chromatin remodelling concerning histone alterations during development
Also, sea urchin histone protein first cloned eukaryotic gene (1975).
Sea urchin cleavage Exhibit radial holoblastic cleavage; occurs in eggs with sparse yolk, and cleavage furrows extend through the entire egg. First seven cleavages are stereotypic (same pattern in every individual), rest become less regular. (7)
- Meridional (cleavage furrow pass through both animal and vegetable pole)
- Meridional – perpendicular to 1. but still passes through both animal and vegetable
- Equitorial – perpendicular to 1. and 2. => seperates animal and vegetal hemispheres
- The four cells of the animal tier divide meridionally into eight blastomeres each with the same volume = mesomeres. The vegetal tier undergoes an unequal equatorial cleavage to produce four large cells (macromeres) and four smaller micromeres.
- Mesomeres divide equatorially to produce two tiers; an1 and an2. Macromeres divide meridionally, forming a tier of eight cells below an2. Somewhat later, micromeres divide unequally, producing a cluster of four small micromeres at the tip of the vegetal pole, beneath a tier of four large micromeres.
- Small micromeres divide once more, then stop dividing until the larval stage Animal hemisphere cells divide meridionally Vegetal cells divide equatorially, forming two tiers; veg1 and veg2.
- Reversed pattern to 6; Animal divide equatorially, vegetal divide meridionally Results in: Blastula of 120 cells, which form a blastocoel (hollow sphere surrounding a central cavity)
Blastula formation - Sea urchin (9)
- All the cells are the same size and in contact with the proteinaceous fluid of the blastocoel on the inside and with the hyaline layer on the outside.
- Tight junctions unite the once loosely connected blastomeres into a seamless epithelial sheet
- As the cells continue to divide, they remain one cell layer thick, thinning out as they expand (accomplished by their adhesion to the hyaline layer and an influx of water that expands the blastocoel).
- This rapid division last through 9th / 10th division depending on species, cell fate determined by this time.
- Each cell becomes ciliated on the region the cell membrane farthest from the blastocoel; there is an apical (outside) – basal (inside) polarity in each of the embryonic cells (evidence of PAR proteins involvement in distinguishing basal membrane).
- This ciliated blastula begins to rotate within the fertilization envelope.
- Soon after, the cells at the vegetal pole begin to thicken forming a vegetal plate.
- Cells at the animal hemisphere synthesize and secrete a hatching enzyme that digests the fertilization envelope.
- Results in: the embryo is now a free-swimming hatchet blastula.
Fate maps and determination - general
By the 60-cell stage, most of the embryonic cell fates are specified, but the cells are not irreversibly commited.
Cell fates are determined in a two-step process:
- The large micromeres are autonomously specified through inherited maternal determinants.
- These autonomously specified large micromeres can now conditionally specify their neighbours
Gene regulatory network (GRN)
The regulatory logic by which the genes of the sea urchin interact to specify and generate characteristic cell types.
Specification of micromere lineage (and hence the rest of embryo) begins inside the undivided egg.
Initial regulatory inputs are two transcription regulators:
Disheveled and beta-catenin.
After Disheveled and beta-catenin, next regulatory input is Otx TF, enriched in micromere cytoplasm.
Otx functions how?
Otx interacts with beta-catenin/TCF complex at the enhancer of the Pmar1 gene to activate expression shortly after micromere formation.
=> repression of HesC (encodes another repressive TF)
=> repression of genes and TFs involved in micromere specification and differentiation.
Wnt8, autocrine factor, activates the micromeres’ own?
genes for beta-catenin which sets up a positive feedback loop between Blimp1 and Wnt8 establishing a source of beta-catenin for the micromere nuclei.
Double-negative gate ?
when a repressor locks the genes of specification and these can be unlocked by the repressor’s repressor (activation occurs by the repression of a repressor)
Feedforward process
Regulatory gene A product activates both differentiation gene C and regulatory gene B, gene B also activates gene C
Fx genes controlling the differentiation of sea urchin skeleton cells
Subroutine co-option
by a new cell lineage is one of the ways evolution occurs
Fx the recruitment of a pre-existing skeletogenic regulatory system by the micromere lineage gene regulatory system, the skeletogenic subroutine in all other echinoderms is activated late in development.
Specification of the vegetal cells.
The skeletogenic micromeres also produce signals that can induce changes in other tissues: (2)
- Activin: TGF-beta family, paracrine factor, expression controlled by Pmar1-HesC double negative gate, secretion of is critical for endoderm formation.
- Delta: juxtacrine protein, controlled by the double-negative gate, activates Notch on the adjacent veg2 cells (plus later acts on adjacent small micromeres)
- lower veg2 => activates Gcm TF => repression of FoxA TF => become nonskeletogenic mesenchymal cells
- Upper veg2 receives no Delta => becomes endodermal cells.
Sea urchin micromere genes specify their cell fates how?
Sea urchin micromere genes specify their cell fates autonomously, and specify the fates of their neighbours conditionally.
The original inputs come from the maternal cytoplasm and activate genes that unlock repressors of a specific cell fate.
Once the maternal cytoplasmic factors accomplish their functions, the nuclear genome takes over.
Sea urchin AP axis specification
In blastula, the general cell fates (ecto, endo, etc) line up along the animal-vegetal axis (established in the egg prior to fertilization).
Animal-vegetal axis appears to structure the future anterior-posterior axis
(vegetal region sequesters the maternal components necessary for posterior development)
Before sea urchin gastrulation:
- The late sea urchin blastula consists of a single layer of ca 1000 epithelial cells that form a hollow ball, somewhat flattened at the vegetal end.
- The blastomeres are derived from different regions of the zygote and have different sizes and properties.
- It develops from the blastula, through gastrulation, to the pluteus larva stage (characteristic of sea urchins).
- The cells that are destined to become the endoderm (gut) and mesoderm (skeleton) are still on the outside and need to be brought inside the embryo through gastrulation.
Ingression of the skeletogenic mesenchyme
Shortly after the blastula hatches from its fertilization envelope, the descendants of the large macromeres undergo an epithelial-mesenchymal transition.
They move along the blastocoel wall through extension and contraction of long, thin processes (filopodia), at first randomly, but eventually they become localized within the prospective ventrolateral region at two sites.
Here they fuse into syncytial cables, which will form the axis of the calcium carbonate spicules of the larval skeletal rods.
Positional information is provided by the prospective ectodermal cells and their basal lamina.
Filopodia explore and sense the blastocoel wall and appear to sense DV and animal-vegetal patterning cues from ectoderm.
Ingression of the skeletogenic mesenchyme is a result of ?
Ingression is a result of their losing their affinity for their neighbours and the hyaline membrane
Instead they acquire a strong affinity for the basal lamina (proteins that lines the blastocoel, secreted by the cells), through the endocytosis of the original micromere cell membrane and replacement with a new one.
=> migration up into blastocoel
Initially all blastula cells are connected:
- On outer surface to hyaline layer
- On inner surface to basal lamina
- On sides to neighbours
Signals important for migration:
- VEGF paracrine factors: emitted from two small regions of the ectoderm, where the skeletogenic mesenchyme cells congregate.
- FGF (fibroblast growth factor) paracrine factor: made in the equatorial belt between endoderm and ectoderm, becoming defined into the lateral domains where the skeletogenic mesenchyme cells collect.
The skeletogenic mesenchyme cells migrate to these points of VEGF and FGF and arrange themselves in a ring along the animal-vegetable axis.
Skeletogenic mesenchyme cell VEGF R is under control of ?
the micromere GRN network (connecting morphogenesis to cell specification).
Invagination of the archenteron (primitive gut)
First stage:
- As the skeletal mesenchyme cells leave the vegetal region, the cells remaining thicken and flatten to form a vegetal plate.
- Vegetal plate cells remain bound to one another and to hyaline layer of egg.
- Vegetal plate cells move to fill gaps from skeletal mesenchyme cells.
- Vegetal plate involutes (curves) inward by altering its cell shape.
- Actin microfilaments collect in the apical ends of the vegetal cells, causing these ends to constrict, forming bottle-shaped vegetal cells that pucker inward.
- Hyaline layer also buckles inward pga changes in its composition, directed by vegetal plate cells.
- Then it invaginates about ¼-½ of the way into the blastocoel. Invaginated region = archenteron.
- Non-skeletogenic mesenchyme is the first group of cells to invaginate, forms tip of archenteron.
- Opening of archenteron at the vegetal pole is the blastopore.
Invagination of the archenteron (primitive gut)
First stage:
Cell destinies:
- Non-skeletogenic mesenchyme => musculature around gut, pigment cells, contribute to the coelomic pouches
- Endodermal (adjacent to mesenchyme) => foregut (migrates farthest into blastocoel)
- Endodermal (rest) => midgut
- Last row to invaginate => hindgut + anus
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Chapter 147
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Chapter 235
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Chapter 3 - modified for details44
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Part II12
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Chapter 550
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Chapter 747
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Chapter 481
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Chapter 6 - The genetics of axis specification in drosophila61
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Chapter 9 – Early development in vertebrates (birds and mammals)27
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Chapter 10 - Emergence of the ectoderm68
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Chapter 9b54
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Part 326
