Appendage Development: Flies and Mammals + Development & Disease (lec 18/19)

Question 1

What is an imaginal disc?

Answer 1

Population of cells that grow/pattern together and then undergo metamorphosis to give rise to the adult appendages.

Question 2

Fill in the blanks regarding the sequence of leg imaginal disc development in Drosophila:

1) Specification of the disc type occurs within the _________.

2) Proliferation of the disc cells and specification as to the type of leg cell that each will produce are accomplished in the _______ _______.

3) Elongation of the disc takes place in the ____ ________ stage.

4) Differentiation of the leg tissues occurs while the insect is a _______.

Answer 2

1) Embryo

2) Larval stages

3) Early pupal (prepupa)

4) Pupa

Question 3

Briefly answer the following questions regarding compartmentalization + anterior-posterior patterning in wing imaginal disc:

1) Where is the anterior-posterior axis formed? How is it recognized?

2) What does the engrailed transcription factor do?

3) What is the role of hedgehog?

Answer 3

1) In the first instar larva, the anterior-posterior axis has been formed and can be recognized by the expression of the engrailed gene in the posterior compartment.

2) The transcription factor activates the hedgehog gene.

3) Hedgehog acts as a SHORT-range paracrine factor to activate decapentaplegic (dpp) in the ANTERIOR cells adjacent to the posterior compartment, where Dpp, acts over a longer range.

Question 4

Fill in the blanks regarding compartmentalization + anterior-posterior patterning in wing imaginal disc:

1) Dpp proteins create a concentration gradient of ______-like signalling, measured by the __________ of ____.

2) High concentration of Dpp near the source activates both the _____ and _____ genes.

3) Lower concentrations activate ______ but not _____.

4) When Dpp levels drop below a certain threshold, ______ (the antagonist of Dpp) is no longer repressed.

Answer 4

1) BMP; phosphorylation of Mad (pMad)

2) spalt (sal); optomotor blind (omb)

3) omb; sal

4) brinker (brk)

Question 5

(T/F) L2-L5 mark the longitudinal wing veins, with L5 being the most anterior.

Answer 5

False!

L2-L5 mark the longitudinal wing veins, with L2 being the most anterior.

Question 6

What are AER and ZPA?

Answer 6

AER: apical ectodermal ridge (impt for proximal-distal axis)

ZPA: zone of polarizing activity (impt for posterior-anterior axis)

Question 7

In the hand, the dorsal is the _______ while the ventral is the _________.

The anterior is the ______, while the posterior is the _______.

Answer 7

upper-side; underside

thumb; pinky

Question 8

Match the following:

1) Stylopod
2) Zeugopod
3) Autopod

A) carpals + digits
B) humerus
C) ulna + radius

Answer 8

Stylopod: humerus

Zeugopod: ulna + radius

Autopod: carpals + digits

Question 9

Briefly answer the following questions regarding the forelimb field of the salamander:

1) What kind of cells does the central area contain?

2) What do the cells surrounding the free limb give rise to?

3) What would happen to the cells outside these regions if the central tissues were destroyed?

Answer 9

1) The central area contains cells destined to form the LIMB (the free limb).

2) The cells surrounding the free limb give rise the PERIBRACHIAL FLANK tissue and the SHOULDER GIRDLE.

3) There is a ring of cells outside these regions (^) that is not included in the limb, but can form a limb if the more central tissues were destroyed.

Question 10

What is the role of hox genes in limb development?

Answer 10

Hox genes play a very important role in providing identities in which bones along the proximal-distal axis along our limbs will become depending on which combinations of hox genes are being expressed.

Question 11

Fill in the blanks regarding Hox paralogues in the forelimb:

1) _____ and _____ paralogues specify the humerus (stylopod).

2) _____ paralogues to a lesser extent in the radius and ulna (zeugopod).

3) _____ paralogues are chiefly responsible for patterning the zeugopod.

4) _______ and _______ paralogues function in the autopod, with _____ paralogues functioning primarily in the wrist and to a lesser extent in the digits.

Answer 11

1) Hox9; Hox10

2) Hox10

3) Hox11

4) Hox12; Hox13; Hox12

*similar pattern seen in hindlimb except hox9 not present.

Question 12

What happens when there is a forelimb mutation that results in the lack of functional Hoxa11 and Hoxd11 genes?

Answer 12

The ulna and radius are severely reduced or absent in the mutant!

Hox11 is highly expressed in zeugopod (and some in autopod).

Question 13

Human polysyndactyly (“many fingers joined together”) results from a:

Answer 13

Homozygous mutation at the HOXD13 loci.

*Hox13 critical for wrist and digits development

Question 14

Tiktaalik roseae was a fish with WRISTS and FINGERS.

1) What indicated that it could breathe air?

3) What did the fossilized bones reveal?

Answer 14

1) The external nostrils on its snout.

2) They revealed the beginnings of digits, wrists, elbows, and shoulders and suggest that this amphibian-like fish could propel itself on stream bottoms and perhaps live on land for short durations.

Question 15

The forelimb bud initiation is almost identical for chickens, mice, and fish.

However, the hindlimb bud initiation is slightly different for chickens and mice. Differentiate them.

Answer 15

Many of the essential factors patterning hindlimb development between chick and mouse are the same and also generate positive feedback loops of signalling.

However, some of the initiation factors for hindlimb induction do differ. Islet1 is required for the mouse but not the chick hindlimb.

Question 16

Instead of Islet1 used in mice hindlimb bud initiation, _____ is used for chick hindlimb bud initiation.

For all, ____ is used for forelimb bud initiation.

_______ is a key molecule that dictates the outgrowth of the limb.

Answer 16

Tbx4

Tbx5

Fgf8

Question 17

1) Where is Fgf10 expressed?

2) What do Tbx5-containing limb buds produce compared to Tbx4-containing limb buds?

3) If a new limb bud is induced with an FGF-secreting bead, the type of limb formed depends on?

4) What happens if the FGF-secreting bead is placed between the regions of Tbx4 and Tbx5 expression?

Answer 17

1) Fgf10 becomes expressed in the lateral plate mesoderm in precisely the positions where limbs normally form.

2) Tbx5-containing limb buds produce wings, whereas Tbx4-containing limb buds generate legs.

3) If a new limb bud is induced with an FGF-secreting bead, the type of limb formed depends on which Tbx gene is expressed in the limb bud.

4) If placed between the regions of Tbx4 and Tbx5 expression, the bead will induce a limb bud expressing Tbx5 anteriorly and Tbx4 posteriorly and will generate a chimeric limb!

Question 18

In the normal 3-day chick embryo, Fgf8 is expressed in the ______ of both the forelimb and hindlimb buds.

Expression of Fgf8 RNA in this location is the source of ______ ______ to the underlying _______.

Answer 18

AER

Mitotic signals; mesoderm

Question 19

What happens to limb development when:

1) AER removed

2) Extra AER

3) Nonlimb mesenchyme replacing leg mesenchyme

4) AER replaced by FGF bead

Answer 19

1) AER removed: limb development ceases

2) Extra AER: wing is duplicated

3) Nonlimb mesenchyme replacing leg mesenchyme: AER regresses; limb development ceases

4) AER replaced by FGF bead: normal wing! NEED THE FGF NOT THE AER CELLS.

Question 20

What happens when you transplant limb bud tips to the head region of another chick embryo when the 1st chick is in:

1) Stage 19-20 (youngest)

2) Stage 21-22 (older)

3) Stage 24 (oldest)

Answer 20

Stage 19-20 (youngest): full structure of the hand

Stage 21-22 (older): proximal parts missing

Stage 24 (oldest): tiny little digit (distal)

*as the cells are developing, the cells at the top give rise to the more distal parts.

Question 21

Retinoic Acid (RA) _________ the bones forming from the transplanted mesenchyme.

Answer 21

proximalizes

Question 22

FGFs/Wnts are found in the _______ parts of the hand, while Retinoic Acid is found in the _______ parts.

These both antagonize each other!

Answer 22

Distal (fingers); Proximal (shoulders)

Question 23

What happens when a ZPA of one embryo is transplanted into another region (anterior) of a second embryo already with a ZPA?

Answer 23

Duplicated digits emerge as a mirror image to the normal digits!

Question 24

1) What morphogen is expressed in the ZPA?

2) What happens when this morphogen is implanted into the anterior portion of limb bud with a ZPA already in its normal position?

Answer 24

1) Sonic hedgehog

2) Duplication of digits as a mirror image to the normal digits! No need for the entire ZPA; hedgehog is sufficient to cause duplication!

*ZPA is not found in the anterior portion of limb bud.

Question 25

Lmx1b is found in the _______ regions of the forelimb.

What happens when there is a loss of lmx1b?

Answer 25

Dorsal

Loss of lmx1b ventralizes the forelimb; there is footpads present on both sides of the paw in the mutant phenotype.

*footpad present in the ventral side

Question 26

________ induces dorsal cell fates of the limb bud through _________, whereas ______ signalling functions through ___________ to regulate ventral limb patterning.

Answer 26

Wnt7a; Lmx1b

BMP; Engrailed-1 (En1)

Question 27

Fetal mouse brains were infected with Zika virus:

1) What does Zika infection upregulate?

2) What does Zika infection downregulate?

3) Are neurons affected in the Zika virus-infected brains?

Answer 27

1) Zika infection upregulates the miR-9 microRNA, which is associated with cell death and decreased neuron formation.

2) Subsequent downregulation of GDNF, a neural growth factor that is a target of miR-9.

3) There is a decrease in CORTICAL NEURONS in the Zika virus-infected brains.

Question 28

What happened when a mice embryo was exposed to alcohol?

Answer 28

The anterior NEURAL TUBE FAILED to close, resulting in EXENCEPHALY (brain tissue is exposed to the exterior).

Later in development, the exposed brain tissue erodes away, resulting in ANENCEPHALY.

Prenatal alcohol exposure (fetal alcohol syndrome) can also affect FACIAL DEVELOPMENT, resulting in a small nose and an abnormal upper lip.

Question 29

How are zebrafish embryos exposed to crude oil components different than normal zebrafish?

Answer 29

Zebrafish embryos exposed to oil spill components produced larvae with severe developmental anomalies, including REDUCTION IN THE SIZE OF head, gill, and thoracic cartilages.

Question 30

What happens to a Xenopus tadpole that is raised in environmentally relevant concentrations of GLYPHOSATE?

Answer 30

The herbicide causes the PHARYNGEAL ARCHES and MIDLINE FACIAL CARTILAGE to fail to develop properly.

Question 31

What happens to frog testis with:

1) 0.5 parts per billion ATRAZINE (a herbicide)

2) 0.8 parts per billion ATRAZINE

Answer 31

1) Testis contained three lobules that are developing both SPERM and an OOCYTE.

2) Severe TESTICULAR DYSGENESIS (defective development) in 28% of population –> incapable of reproducing

Question 32

(T/F) Atrazine has no effect on testosterone levels in frogs.

Answer 32

False!

Atrazine-treated males have plasma testosterone levels at or below those of control females!

Question 33

What is diethylstilbestrol (DES)?

Answer 33

Des was prescribed to pregnant women between 1940 and 1971 to prevent miscarriage, premature labour, and related complications of pregnancy.

However, it led to the altered morphology of the female genitalia.

Question 34

How does DES lead to the altered morphology of the female genitalia?

Answer 34

During normal morphogenesis, Hoxa10 and Hoxa11 genes in the mesenchyme are activated and maintained by Wnt7a from the epithelium. Wnt7a also induces Wnt5a in the mesenchyme, and Wnt5a protein both maintains Wnt7a expression and mesenchymal cell proliferation. Together, these factors specify and order the morphogenesis of the uterus.

DES, acting through the ESTROGEN RECEPTOR, BLOCKS Wnt7a expression. Proper activation of the Hox genes and Wnt5a in the mesenchyme DOES NOT OCCUR, leading to a radically altered morphology of the female genitalia.

Question 35

1) What is Phocomelia?

2) How was it caused?

Answer 35

1) Phocomelia is the lack of proper limb development.

2) It was caused when the mothers took the drug THALIDOMIDE during pregnancy in the early 1960s. Thalidomide disrupts different structures at different times of human development.

Question 36

_______ _____ prevents and decreases folate and neural tube defects.

Answer 36

Folic acid

Question 37

What activates the Hedgehog pathway when Shh is a mitogen (induces mitosis)?

Answer 37

When Shh is a mitogen, loss of function mutations in the Hh ligand PATCHED (PTCH1) or gain of function mutations in the PATCHED INHIBITOR, SMOOTHENED (SMOH) activates the hedgehog pathway even in the absence of Shh or another Hedgehog protein.

Question 38

In the autocrine model, tumour cells both _______ and ______ to the Hh ligand.

In the paracrine model, tumour cells _____ and _____ the Hh ligand, and the surrounding stromal cells receive protein. The stromal cells respond by producing _______ ______ such as _______ or ______ that support tumour growth and survival.

Answer 38

Produce; Respond

Produce; Secrete; Growth Factors; VEGF; IGF

Question 39

What is GLI?

Answer 39

GLI is the transcription factor activated by the Hh pathway.

Question 40

What are the two types of cells that cancer stem cells produce?

Answer 40

A TUMOUR CELL and a SUPPORT CELL that is not a tumour cell, but forms part of the tumour niche!

The support cell can synthesize and secrete a function Wnt protein. When the Wnt paracrine factor binds to the Wnt receptor on a tumour cell, it activates the cell’s Wnt pathway, which drives tumour cell proliferation.

Question 41

(T/F) While traditional therapy includes things like surgery, chemo- or radiotherapy, differentiation therapy includes paracrine or hormonal factors.

Answer 41

True!

Question 42

What concept is differentiation therapy based on?

Answer 42

Differentiation therapy is based on the concept that a tumor is a developmental disorder and can be induced to become a normal “adult” cell.

Question 43

________ _____ can induce malignant nasopharyngeal carcinoma cells to become normal adult cells.

Nasopharyngeal carcinoma cells are like undifferentiated _____ cells, and the enzyme _________ is epigenetically suppressed by a complex containing enhancer of ______ __________ ___.

This treatment prevents the formation of the _________ ______ and restores _______ expression.

Answer 43

Retinoic acid

Neck; IKKα; Zeste homologue 2 (EXH2)

Inhibitory complex; IKKα