Epiboly in Zebrafish Emrbyos

There are three distinct layers in the zebrafish embryo at the animal pole that are involved in epiboly: the extraembryonic layers: enveloping layer (EVL), the yolk syncytial layer (YSL), and the intermediate deep layer (DL).

Stages of Epiboly in Zebrafish and the Layers Involved http://en.wikipedia.org/wiki/File:EPIBOLY4.jpg

The above figure shows the movements of the three distinctive layers of the Zebrafish embryo during epiboly. The particular movements of these layers are described further below.

Epiboly:

Epiboly begins in the late blastula stage and is defined as the movement of the Yolk Syncytial Layer and blastodisc around the yolk cell.

  • Epiboly starts with the deep layer cells radially intercalating and the yolk cell “doming”. At the end of this initial phase, the embryo has reached 50%-epiboly stage.
  • During the second phase of epiboly, the three layers begin involution around the yolk cell towards the vegetal pole to form the gastrula.
  • Epiboly occurs during the blastula period after the yolk syncytial layer forms (YSL) and the blastula enters midblastula transition (MBT).

Epiboly stages during the Blastula Period: Below are the stages of the embryo during the blastula period.Those with signicant epibolic movements have been highlighted and described in further detail.

  1. 128 cell stage (2.25 h)
  2. 256 cell stage (2.5 h)
  3. 512 cell stage (2.75 h)
  4. 1k-cell stage (3 h)
  5. High stage (3.5 h)
  6. Oblong stage (3.67 h)
  7. Sphere stage (4 h)
  8. Dome stage (4.3 h): epiboly begins indicated by the “doming” of the yolk cell. The E-YSL cells begin thinning and flattening. The nuclei of the YSL being to enlarge
  9. 30% epiboly stage (4.67 h): the blastoderm covers 30% of the entire distance between the animal and vegetal poles. Epiboly has now produced a full blastoderm of even thickness with a thin EVL monolayer and a deep cell multilayer (DEL) 4 cells thick.

Modified from: Kimmel et al., 1955. Developmental Dynamics 203:253-310. Copyright © 1995 Wiley-Liss, Inc. Reprinted only by permission of Wiley-Liss, a subsidiary of John Wiley & Sons, Inc.


Epiboly during the Gastrula Period: Below are the stages of the embryo during the gastrula period. The stages with significant epibolic movement have been highlighted and described in detail.

  1. 50% epiboly stage (5.25 h): the blastoderm covers 50% of the entire distance between the animal and vegetal poles. The blastoderm margin will remain at this position for an hour.
  2. Germ ring stage (5.57 h)
  3. Shield stage (6 h)
  4. 75% epiboly stage (8 h): the blastoderm covers 75% of the entire distance between the animal and vegetal poles.
  5. 90% epiboly stage (9 h): the blastoderm covers 90% of the entire distance between the animal and the vegetal poles. The yolk plug is now visible as the small protrusion of the yolk cell between the blastoderm cells. The blastoderm is thicker on the dorsal side than the ventral side
  6. Bud stage (10 h): Epiboly ends as indicated by the complete coverage of the yolk cell by the blastoderm—100% epiboly. The blastoderm is thicker on the dorsal side then on the ventral side

Modified from: Kimmel et al., 1955. Developmental Dynamics 203:253-310. Copyright © 1995 Wiley-Liss, Inc. Reprinted only by permission of Wiley-Liss, a subsidiary of John Wiley & Sons, Inc.

One of the crucial genes for normal epiboly development is MAPKAPK2 gene identified through the Betty boop mutation.

Betty boop (bbp) is a zebrafish maternal effect mutation, in which homozygous mutant mothers produce embryos with defective epiboly in the embryo. In bbp mutant embryos, at the 50% epiboly stage, the blastoderm rapidly constricts (20 min) at the equator margin and causes the yolk cell to burst, thus halting development.

Compared to wildtype (WT), Bbp mutants also show increased amounts of calcium release at the beginning of the 50% epiboly stage. The increased calcium release is thought to increase the contraction of F-actin at the yolk margin leading to the bursting of the bbp mutant embryos.



Holloway et al. 2009. A Novel Role for MAPKAPK2 in Morphogenesis druing Zebrafish Development. PLoS Genetics 5(3): e1000413.

The above image indicates that compared to the WT embryo, bbp mutant embryos have a higher concentration of calcium at both the early epiboly stage (E) and the 40% epiboly (F).

The mutated gene in bbp mutants is the MAPKAPK2 gene in the maternally supplied RNA. In bbp mutants, mutation in the MAPKAPK2 gene introduces a premature stop codon resulting in a carboxy-terminal truncation of MAPKAPK2. The truncated MAPKAPK2 lacks the sequence containing the nuclear localization signal (NLS), thus abnormally localizing the Bbp protein in the cytoplasm, not allowing the MAPKAPK2 to phosphorylate its nuclear targets resulting in a loss of function.

The kinase activity of the mutated MAPKAPK2 is impaired, in which case the mutated protein fails to phosphorylate a critical target in the yolk cell that regulates epiboly leading to defects in epiboly during embryo development.

In WT embryos p38 MAP kinase phosphorylates MAPKAPK2. The truncated protein lacks the docking site for p38. Because of this lack, the MAPKAPK2 prtotein cannot be activated in mutant bbp embryos. Consequently it is also concluded that p38 is also required for proper functioning of the protein and therefore proper epiboly. Based on these results, it is postulated that MAPKAPK2 modulates actin-based contractility responsible for closing the blastopore during epiboly.

MAPKAPK2 gene of the Zebrafish. Holloway et al. 2009. A Novel Role for MAPKAPK2 in Morphogenesis druing Zebrafish Development. PLoS Genetics 5(3): e1000413.

The image above the shows the MAPKAPK2 protein of the Zebrafish. The nonsense mutation of the bbp mutant occurs at the Q350 site, which results in a turncated protein. This truncation removes the p38 docking site and the NLS region, as shown in the schematic above.

Further research is required to conclude how exactly the MAKKAPK2 and p38 MAP kinase modulates actin and microtubles to influence epiboly during development. Although it can be concluded clearly that MAPKAPK2 and p38 MAP kinase effect microtuble activity, it cannot be concluded whether MAPKAPK2 acts independently of other genes upstream or downstream.

Zebrafish Epiboly Mutants: four mutations tracked

  1. After epiboly, the EVL forms an epithelial monolayer covering the blastoderm and later becomes the embryonic periderm, the embryo’s first skin. The deep cells of the blastoderm rearrange to form the hypoblast and the epiblast, which form the embryonic anlagen. The YSN lead the blastoderm towards the vegetal pole.
  2. Kane and collegues identified four homozygous mutations that arrest epiboly. Three of these four—hab, ava, and law—also cause delayed epiboly and neural tube defects as a zygotic maternal dominant effect phenotype.

i.  Avalanche (ava): Homozygous recessive ava slows down and arrest the epiboly of the deep cells of the blastoderm. Epiboly halts at around 60±10% epiboly stage. Arrest is followed by slow retraction toward the animal pole and the yolk cell eventually lysing near the vegetal pole terminating the embryo. Heterozygote offspring of heterzygote mothers display the mutant phenotype of slow epiboly and detached cells accumulating dorsal to the neural tube. This is indicative of a zygotic-maternal dominant effect phenotype.

ii. Lawine (law): Homozygous recessive law slows down and arrests epiboly around 70±10% epiboly stage. Arrest is followed by slow retraction toward the animal pole and the yolk cell eventually lysing near the vegetal pole terminating the embryo. Heterozygote offspring of heterzygote mothers display the mutant phenotype of slow epiboly and detached cells accumulating dorsal to the neural tube. This is indicative of a zygotic-maternal dominant effect phenotype.

iii. Weg (weg): Homozygous recessive weg slows down and arrests epiboly around 80±10% epiboly stage. Arrest is followed by slow retraction toward the animal pole and the yolk cell eventually lysing near the vegetal pole terminating the embryo. Heterozygote offspring of heterzygote mothers display the mutant phenotype of slow epiboly and detached cells accumulating dorsal to the neural tube. This is indicative of a zygotic-maternal dominant effect phenotype.

iv. Halfbaked (hab): Homozygous recessive hab slows down and arrest epiboly around 80±10% epiboly stage. Arrest is followed by slow retraction toward the animal pole and the yolk cell eventually lysing near the vegetal pole terminating most of the embryos with a few exceptional survivors. Heterozygote offspring of heterzygote mothers display the mutant phenotype of slow epiboly and detached cells accumulating dorsal to the neural tube. This is indicative of a zygotic-maternal dominant effect phenotype. Heterzygote hab offspring have a partial dominant effect phenotype of enlarged hatching gland.

Four Epiboly Mutants in Zebrafish Kane et al. 1996. The Zebrafish Epiboly Mutants. Development 123: 47-55
Relationship between progression of the deep cells of the blastoderm in normal embryos (filled squares) and hab mutants (open circles)

Relationship between progression of the deep cells of the blastoderm in normal embryos (filled squares) and hab mutants (open circles)Kane et al. The Zebrafish Epiboly Mutants. Development 123: 47-55

Development of wild type, heterozygous hab, homozygous recessive hab, and heterozygous ava embryos. Kane et al. 1996. The Zebrafish Epiboly Mutants. Development 123: 47-55

The above images shows that the deep cells of the WT embryo complete epiboly approximately 10 hours postfertilization. However, the deep cells of the hab mutants halt near 70% epiboly stage postfertilization and do not completely epiboly.

The movement of the blastoderm is independent of the movement of the YSL as evidenced by the the recessive mutants. All four of these recessive mutants affect the deep cells of the blastoderm—slowing and stoping their movement toward the vegetal pole. However the movement of the EVL and the YSL was not affected as the YSL continued to “dome” towards the animal pole in homozygous mutants. Also the yolk cell autonomously complete epiboly when the blastoderm is removed and the EVL completes epiboly even when the deep cells do not.

There is on-going research to isolate the genes and proteins in-action behind many epiboly mutant lines of zebrafish. MAPKAPK2 is just one players in a collection of genes responsible for carrying out this crucial process in the developing embryos of all vertebrates. The importance of understanding the process of epiboly is important because of its significance during early development.

References:

Holloway, B.A., S. Gomez de la Torre Canny, Y. Ye, D.C. Slusarski, C.M. Freisinger, R. Dosch, M.M. Chou, D.S. Wagner, and M.C. Mullins. 2009. A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development. PLoS Genetics 5(3): e1000413 http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1000413

Kane, D.A., M. Hammerschmidt, M.C. Mullins, H. Maischein, M. Brand, F.J.M. van Eedeen, M. Furutani-Seiki, M. Granato, P. Haffter, C. Heisenberg, Y. Jiang, R.N. Kelsh, J. Odenthal, R.M. Warga, and C. Nusslein-Volhard. 1996. The Zebrafish Epiboly Mutants. Development 123: 47-55 http://dev.biologists.org/content/123/1/47.full.pdf

Kimmel, C.B., W.W. Ballard, S.R. Kimmel, B. Ullmann, and T.F. Schilling. 1995. Stages of Embryonic Development of the Zebrafish. Developmental Dynamics 203: 253-310

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