Specification: Urchins + Tunicates + Frogs (lec 14)

Question 1

1) What is the major difference between DEUTEROSTOME and PROTOSTOME invertebrates?

2) What kind of invertebrates are urchins (echinoderms) and tunicates?

Answer 1

1) Deuterostome: blastopore —> anus, Protostome: blastopore —> mouth

2) Deuterostome

Question 2

What kind of cleavage do sea urchins undergo?

Answer 2

Radial cleavage (isolecithal –> holoblastic)!

Question 3

(T/F) The animal pole of the sea urchin produces ectoderm, while the vegetal pole produces micromeres.

Answer 3

True!

Question 4

Match the following terms to their definitions:

1) Large micromeres

2) Small micromeres

A) Primordial germ cells

B) Primary mesenchyme cells

Answer 4

Large micromeres: Primary mesenchyme cells

Small micromeres: Primordial germ cells

Question 5

1) What happens when you isolate the animal hemisphere of a sea urchin?

2) What happens when you mix the isolated animal hemisphere with isolated micromeres?

Answer 5

1) An isolated animal hemisphere becomes a ciliated ball of undifferentiated ectodermal cells (what it is normally supposed to be). It develops in an autonomous fashion.

2) When an isolated animal hemisphere is combined with isolated micromeres, a recognizable pluteus larva is formed. It develops in a conditional fashion; micromeres can secrete morphogens to cause a larva to form from cells that are normally autonomous!

Question 6

What happens if micromeres are transplanted from the vegetal pole of an embryo into the animal pole of a host embryo?

Answer 6

The transplanted micromeres invaginate into the blastocoel to create a new set of SKELETOGENIC MESENCHYME CELLS, and they induce the animal pole cells next to them to BECOME vegetal pole endoderm cells.

The transplanted micromeres form SKELETAL RODS while the induced animal cells form a SECONDARY ARCHENTERON. Meanwhile, gastrulation proceeds normally from the original vegetal plate of the host.

*mirror duplication - what happens in the vegetal pole is happening in the animal pole!

Question 7

Fill in the blanks regarding micromere specification of sea urchins.

______, a general transcription factor, and _______ from the maternal cytoplasm are concentrated at the _______ pole of the egg. These transcriptional regulators segregate to the micromeres and activate the ______ gene.

Answer 7

Otx; b-catenin; vegetal; Pmar1

Question 8

What does Pmar1 do?

Answer 8

Pmar1 encodes a repressor of HesC which in turn encodes a repressor of several genes involved in micromere specification; DOUBLE NEGATIVE.

Genes encoding signaling proteins (e.g, Delta) are also under the control of HesC.

Question 9

In which cells is the Pmar1 active and inactive?

Answer 9

In micromeres; Pmar1 is active (micromere specification and signaling genes are active)

In Veg2 cells; Pmar1 is inactive (HesC shuts down the SKELETOGENIC genes)

In cells with Notch; can respond to the Delta signal from the skeletogenic mesenchyme.

Question 10

Differentiate double-negative gate circuits from feedforward circuit.

Answer 10

Double-negative gate circuits: A single gene encodes a repressor of an entire battery of genes. When this repressor gene is repressed, the battery of genes is expressed.

Feedforward circuit: gene product A activated both gene B and gene C, and gene B also activates gene C. Provides an efficient way to AMPLIFY a signal in one direction.

Question 11

(T/F) Skeletogenic mesenchyme cells break through extracellular matrix.

Answer 11

True!

Question 12

What happens to the circumference of the archenteron during early vs late gastrulation in sea urchin embryos?

Answer 12

There is cell rearrangement during EXTENSION (elongation + invagination) of the archenteron in sea urchin embryos.

Early gastrulation: early archenteron with 20-30 cells around its circumference.

Later gastrulation: archenteron has a circumference of 6-8 cells.

Question 13

What kind of cleavage do tunicates undergo?

Answer 13

Bilateral cleavage (isolecithal –> holoblastic)!

Question 14

Briefly describe the four steps of cytoplasmic rearrangement in the fertilized egg of tunicates.

Answer 14

1) Before fertilization, yellow cortical cytoplasm surrounds the gray inner cytoplasm

2) After sperm entry into the vegetal hemisphere of the oocyte, the yellow cortical cytoplasm and the clear cytoplasm derived from the breakdown of the oocyte nucleus contract vegetally TOWARD the sperm.

3) As the sperm PRONUCLEUS migrates ANIMALLY TOWARD the newly formed egg PRONUCLUES, the yellow and clear cytoplasms move with it.

4) The final position of the yellow cytoplasm marks the location where cells will give rise to the tail muscles.

Question 15

Fill in the blanks regarding the gene network leading to notochord development in the early tunicate embryo:

____________ accumulation leads to the expression of the ________ gene, which helps specify the cells to become endoderm and secrete FGFs.

FGFs induce ________ expression in neighboring cells; cells that will become the _________.

The gene expressed ________ regulators of cellular activity such as ________, which regulates cell polarity leading to the _______ _____ of the notochord in the gastrula and neurula stages.

Answer 15

b-catenin; foxd

Brachyury; notochord

Activates; Prickle; convergent extension

*b-catenin —> foxD —> Fgfs —> Brachyury —> Prickle

Question 16

What are the two steps of the two step process for specifying the marginal cells of the tunicate embryo?

Answer 16

The first step involves the cells’ acquisition (or nonacquisition) of the MACHO-1 transcription factor.

The second step involves the reception (or nonreception) of the FGF signal from the endoderm.

Question 17

(T/F) If macho-1 is absent, the marginal cells become the nerve cord/notochord, and when it is present, the marginal cells become the mesenchyme and muscle.

Answer 17

True!

Question 18

Where does gastrulation begin in frog eggs (ie what dictates the location of gastrulation)?

Answer 18

Gastrulation beings in the gray crescent - the region opposite to the point of sperm entry, where the greatest displacement of cytoplasm occurs.

*Point of sperm entry dictates the location of gastrulation.

Question 19

(T/F) 80% into the first cleavage in a frog egg, the cortical cytoplasm rotates 50˚ relative to the internal cytoplasm.

Answer 19

False!

80% into the first cleavage, the cortical cytoplasm rotates 30˚ relative to the internal cytoplasm.

Question 20

Which one of the statements is true regarding cleavage of a frog egg?

1) The second division begins in the animal region of the egg before the first division has divided the vegetal cytoplasm because the vegetal yolk impedes cleavage.

2) The third division is placed toward the vegetal pole.

3) As cleavage progresses, the animal hemisphere contains larger and fewer blastomeres than the vegetal hemisphere.

Answer 20

1!

The third division is placed toward the animal pole.

As cleavage progresses, the vegetal hemisphere contains larger and fewer blastomeres than the animal hemisphere.

Question 21

What are the five key movements during Xenopus (frog) gastrulation?

Answer 21

1) Epiboly (flattening + spreading of epithelial cells to increase the amount of surface they cover)

2) Vegetal rotation

3) Invagination (into bottle cells)

4) Involution and migration (movement of cells inside the embryo as a coherent sheet)

5) Convergent extension (movement of cells toward an axis to extend the axis)

Question 22

What are bottle cells?

Answer 22

Bottle cells are endodermal and contribute to the ARCHENTERON WALL during frog gastrulation (eventually lining the liver).

Question 23

What drives the epiboly of the ectoderm layer toward the vegetal pole to enclose the endoderm in frog eggs?

Answer 23

Radial intercalation (squeezing together of cells leading to expansion)

Question 24

How is radial intercalation occuring from chemotaxis to drive epiboly in frog eggs?

Answer 24

C3a is secreted from the superficial cells as an chemoattractant to C3aR+ deep cells, which guides deep cells to migrate RAPIDLY and OUTWARD, forcing the superficial layer to thin and expand.

This causes the ectoderm layer to enclose the endoderm in late gastrula.

Question 25

Briefly describe the cell movements during frog gastrulation in:

1) Early gastrulation

2) Mid gastrulation

3) End of gastrulation

Answer 25

Early gastrulation: THE BOTTLE CELLS of the margin move INWARD to form the DORSAL lip of the blastopore, and the mesodermal precursors involute under the roof of the BLASTOCOEL.

Mid gastrulation: The ARCHENTERON forms and displaces the blastocoel, and cells migrate from the lateral and ventral lips of the blastopore into the embryo. The cells of the animal hemisphere migrate down toward the vegetal region, moving the blastopore to the region near the vegetal pore.

End of gastrulation: The BLASTOCOEL is OBLITERATED, the embryo becomes surrounded by ectoderm, the endoderm has been internalized and the mesodermal cells are inbetween the two.

Question 26

(T/F) Animal pole changes its position as frog gastrulation continues.

Answer 26

True!

Question 27

Which one of the statements is false regarding early movement of frog gastrulation?

1) At the beginning of gastrulation, the involuting marginal zone (IMZ) forms.

2) Animal rotation pushes the prospective pharyngeal endoderm to the side of the blastocoel.

3) The vegetal endoderm movements push the pharyngeal endoderm forward, driving the mesoderm passively into the embryo and toward the animal pole. The ectoderm begins epiboly.

Answer 27

2!

Vegetal rotation pushes the prospective pharyngeal endoderm to the side of the blastocoel.

Question 28

(T/F) During the formation of blastopore lips in frogs, everything is being pushed inward (involution) like a snow ball effect.

Answer 28

True!

Question 29

What happens in the noninvoluting marginal zone (NIMZ) and the upper portion of the IMZ during frog gastrulation?

Answer 29

Deep (mesodermal) cells are intercalating radially to make a thin band of flattened cells. The thinning of several layers into a few causes CONVERGENT EXTENSION towards the blastopore lip!

After involuting over the lip, intercalation continues, elongating and narrowing the axial mesoderm.

Question 30

What is the role of VegT and Vg1 in frogs?

Answer 30

VegT is maternally deposited in the endoderm and activates nodal to make mesoderm, where zygote produces its own VegT.

Vg1 induces dorsal mesoderm to transcribe genes for several Wnt antagonists.

Question 31

What happens when a dorsal blastopore lip is transplanted into a gastrula in the region that normally becomes ventral epidermis?

Answer 31

The donor tissue INVAGINATES and forms a SECOND ARCHENTERON, and then a SECOND EMBRYONIC AXIS.

Both donor and host tissues are seen in the new neural tube, notochord, and somites.

Eventually, a second embryo forms, joined to the host!

*kind of like micromeres but for frogs

Question 32

What is GSK3? What are Dsh and GBP? What are their roles in the frog egg?

Answer 32

GSK3 suppresses dorsal fate of the frog egg by degrading b-catenin.

Dishevelled (Dsh) and GSK3-Binding Protein (GBP) bind to and block the action of GSK3, preventing the degradation of b-catenin on the dorsal side of the embryo.

Question 33

In the ventral side, b-catenin is ________, while it is _______ in the dorsal side of the frog egg.

Answer 33

Degraded; Stabilized

*Higher b-catenin; site of gastrulation

Question 34

(T/F) If there is an inactive GSK3, there can be a formation of a second dorsal axis.

Answer 34

True!

Question 35

What are the three organizer genes? How does VegT influence these genes?

Answer 35

chordin, noggin and goosecoid

VegT in the endoderm prevents the organizer genes from being expressed outside the organizer area (dorsal).

Question 36

At the late blastula stages, Vg1 and VegT are found in the ________ hemispheres; B-catenin is located in the _______ region.

B-catenin acts synergistically with Vg1 and VegT activate the Xenopus ______-_____ genes, creating a gradient of these proteins (highest in the dorsal region).

Answer 36

Vegetal; dorsal

Nodal related (Xnr)

Question 37

1) Mesodermal regions with little or no Xnr become:

2) Mesodermal regions with intermediate concentrations of Xnr become:

3) Mesodermal regions with high concentrations of Xnr become:

Answer 37

1) Ventral mesoderm

2) Lateral mesoderm

3) Organizer (goosecoid and other dorsal mesodermal genes activated)

Question 38

What is BMP4? What do organizer proteins do to it?

Answer 38

BMP4 is a powerful ventralizing factor.

Organizer proteins in the dorsal region block the action of BMP4, allowing the region to stay dorsal!

Question 39

What happens when you inject NOGGIN mRNA to Xenopus eggs that do not have dorsal structures?

Answer 39

The eggs develop dorsal structures in a dosage-related fashion.

Question 40

What is the difference between regional specificity and temporal specificity of induction?

Answer 40

Regional specificity: Injection of the archenteron roof (where the organizer tissue is) in different regions leads to different tissues being generated.

Temporal specificity: If young gastrula dorsal lip (which form the anterior portion of the organizer) is transplanted, it induces anterior dorsal structures. If older dorsal lips are transplanted, more posterior dorsal structures are induced.