Assignments

Question 1

In the space below define and describe what it means for a cell to be specified vs. determined
towards a certain cell fate. Give an example of an experiment using the early frog embryo that
would distinguish between the two types of cellular potential. You can use a diagram to help
illustrate your answer. [4 marks]

Answer 1

-Both terms describe the developmental state of a cell as a measure of the level of commitment a
cell has towards a certain fate [0.5].
-Specified describes the developmental status/state of a group of cells such that when the cells are
isolated from an embryo [0.5], or another developmental context, and placed in a neutral
environment such as a culture dish [0.5], these cells will develop according to their normal
developmental fate.
-The cells said to be capable of differentiating autonomously [0.5], that is, they do not require
further cues from the environment in order to differentiate [0.5].
-Specification is the first stage [0.5] and is a labile phase such that the commitment to a cell fate
is still capable of being reversed [0.5].
-The second stage of commitment is determination [0.5].
-A cell or tissue is said to be determined when it is capable of differentiating autonomously even
when placed into another region of the embryo [0.5].
-A transplant experiment [0.5] is an
example of this.
-If it is able to differentiate according to its original fate even under these circumstances, it is
assumed that the commitment is irreversible [0.5].
-This suggests that a cells fate can no longer
be changed according to environmental cues.

Question 2

From the experiments performed by Wilhem Roux and Hans Dreisch two forms of development
were modeled termed mosaic and regulative, respectively. Define each term and explain how
each experiment can be used to explain each form of development. As a part of your answer,
describe how each scientist explained their different results. [6 marks]

Answer 2

-Mosaic development: the embryo appears to be constructed of independent self-differentiating
parts. [0.5]
-The embryo develops through the distributions of cytoplasmic (morphogenetic)
determinants [0.5].
-Determinants are often proteins or messenger RNA [0.5] that are placed in
different regions of the egg cytoplasm [0.5] and are apportioned/localized to the different cells as
the embryo divides [0.5].
-These cytoplasmic determinants specify the cell type.
-When one cell of the two-cell embryo is destroyed one half of the embryo develops.
-This implies
that both cells have enough information to form one respective half of the embryo [0.5].
-It also
implies that they develop independently [0.5] and that if one cell is removed, the other will not
change their fate to compensate for its loss [0.5]. [3 marks]

-Regulative development: explains the ability of an embryo to develop normally even when cells
are removed or rearranged [0.5].
-This describes the ability of the embryonic cells to change their
fates [0.5] to compensate for the missing parts [0.5].
-This form of development implies that cells
in the embryo have the competence to differentiate into cell-types other than their normal fate
[0.5].
-In the sea urchin embryo, the remaining cell is able to form the cells that would normally develop
from the missing cell.
-This suggests 1) the cells can change their fate in response to a change in the
early embryo, this is an indicator of commitment, [0.5] -2) cells somehow communicate such that
the missing parts are detected and a compensation in fate of all of the cells can be made [0.5] and
-3) the potential of each of the cells in the embryo is greater than their normal fate, this is an
indicator of potency [0.5]. [3 marks]

Question 3

As our understanding of embryo development has progressed the terms of mosaic vs. regulative
development have been modified. Autonomous specification vs. conditional specification seems
to be a more accurate means to explain the scientist’s results. Describe autonomous specification
and conditional specification. Define the term autonomous by contrasting it to non-autonomous
development and use cells of the early frog embryo to explain your answer. Explain what
conditional specification might suggest about a cell’s potential to form a specific cell fate in the early
sea urchin embryo. [6 marks]

Answer 3

-Autonomous specification, if a cell or groups of cells is removed and placed in isolation the cells
will produce the same cells that it would have made if it were still part of the embryo. -This implies
that the cell has all the information or factors it requires to differentiate into its normal fate and
that no environmental signals or factors are required [1].
-The embryo from which that cell is
taken will lack those cells (and only those cells) that would have been produced by the missing
cells.
-This indicates that the other cells are unable to compensate for the cell loss. -This is likely a
combination of absent cellular communication and limited competence [1].
-Autonomous means that a cell is self-regulating.
-It does not require cues from other cells or the
environment in order to be specified towards a particular cell fate [1].
-In the frog, the one half of the embryo can develop independently of the other half. [1]
-Conditional specification, involves interactions with the environment or neighbouring cells.
-This is
also a description of potential such that each cell originally has the ability to become many
different cell types.
-The interactions of the cell with other cells restricts the fate of one or both of
the participants.
-The fate of a cell depends upon the conditions in which the cell finds itself. [1]
-If a blastomere is removed from the sea urchin embryo, the remaining embryonic cells alter their
fates so that the roles of the missing cells can be taken over.
-Cells recognize a missing cell type and
the remaining cells change their fates to compensate for the missing parts. [1]

Question 4

During the first division of the C. elegans embryo, several events occur that allow
asymmetric distribution of cytoplasmic determinates. Using what is known about the
cytoskeleton of the egg cortex and the function of the PAR proteins, describe the steps that
regulate this asymmetry.

Initiation of asymmetry in the egg. [2 marks]

Answer 4

-Following fertilization, the sperm DNA and centrosome move to the pole closest to where
the sperm entered the elliptical egg. [1]
-The sperm centrosome [0.5] abuts the cortex and
specifies this end of the zygote as the posterior [0.5].

Question 5

During the first division of the C. elegans embryo, several events occur that allow
asymmetric distribution of cytoplasmic determinates. Using what is known about the
cytoskeleton of the egg cortex and the function of the PAR proteins, describe the steps that
regulate this asymmetry.

Establishing Polarity: the function and localization of the PAR proteins. [10 marks]

Answer 5

-The PAR genes and PKC-3 encode a variety of cortically enriched scaffolding [0.5] and
signaling proteins [0.5].
-PAR proteins segregate to distinct anterior and posterior [0.5]
cortical domains [0.5]
-As polarization occurs, contractile cortical ruffles that initially appear throughout the
cortex become limited to the anterior, leaving the posterior cortex smooth. [1]
-The multi-PDZ domain protein PAR-3, the PDZ and CRIB domain protein PAR-6 and the
atypical protein kinase C (aPKC) PKC-3 localize to the contractile anterior cortex. [3.5]
-The serine-threonine kinase PAR-1 and the RING protein PAR-2 occupy a complementary
domain in the smooth posterior cortex . [2.5]
-The kinase PAR-4 and the 14-3-3 protein PAR-5 are cortically enriched but remain
symmetrically distributed. [1]
-PAR-6 clears away from the posterior cortex and moves anteriorly, and at the same time
PAR-2 fills in the posterior cortical domain devoid of PAR-6 [1].
-The boundary between the anterior PAR6 and posterior PAR-2 domains stabilizes once it
reaches the middle of the embryo. [1]
-‘Anterior’ and ‘posterior’ PAR proteins polarize the zygote by signaling to effectors that
alter the distribution of developmental determinants [1] and regulate spindle
positioning [1].
-These effectors include MEX-5 and MEX-6 (muscle excess) [1], which are cytoplasmic zincfinger proteins required for the posterior enrichment of several proteins that are
important for germline [1]
-P-granules composed of mRNA and proteins required for
development of the germline [1]
-Cortically enriched GPR-1 and GPR-2 (G protein regulator) proteins, which control the
asymmetric positioning of the mitotic spindle. [1]

Question 6

During the first division of the C. elegans embryo, several events occur that allow
asymmetric distribution of cytoplasmic determinates. Using what is known about the
cytoskeleton of the egg cortex and the function of the PAR proteins, describe the steps that
regulate this asymmetry.

The mechanism behind the movement of cell components. [3 marks]

Answer 6

-Networks of actin filaments and motor protein myosin (actomyosin) form the contractile
cortex just under the egg’s cell membrane. [1]
-PAR domains form in the C. elegans zygote through an asymmetric contraction of an
actomyosin network at the cell cortex [1]
-Before polarization, nonmuscle myosin II heavy chain (NMY-2, hereafter ‘myosin’) and F- actin are found throughout the cortex as large foci and interconnecting filaments.
-Following fertilization, actomyosin foci form, contract and turn over with no net
directionality, causing cortical ruffling [1].
-As polarization initiates, actomyosin
contractility is inhibited at the posterior cortex, causing the remaining tensile network to
contract anteriorly. [1]
-Actomyosin contraction creates an anteriorly directed cortical flow
and induces the anterior translocation of PAR-3, PAR-6 and PKC-3 [1]
-anterior flow of the cortex causes posterior-ward flow of the cytoplasmic components (P- granules) [1]

Question 7

During the first division of the C. elegans embryo, several events occur that allow
asymmetric distribution of cytoplasmic determinates. Using what is known about the
cytoskeleton of the egg cortex and the function of the PAR proteins, describe the steps that
regulate this asymmetry.

The function of the PAR proteins in polarity maintenance. [3 marks]

Answer 7

-PAR domain maintenance is mediated by Rho GTPase signaling [0.5] and through
reciprocal inhibitory interactions between anterior and posterior PAR proteins.
-a negative feedback loop [0.5] involving two kinases, aPKC, at the anterior [1] and PAR-1 at
the posterior [1] maintain two distinct domains
-each kinase inhibits specific proteins from
associating locally [0.5]
-The Rho GTPase CDC-42 regulates polarity maintenance.
-CDC-42 becomes enriched at the
anterior cortex as polarity is established.
-CDC-42 regulates polarity maintenance largely through PAR-6
-PKC-3 keeps PAR-2 off of the cortex by phosphorylating a domain within PAR-2 that has
been shown to mediate its cortical localization
-LGL-1 [a homolog of Lethal giant larvae (Lgl)] functions redundantly with PAR-2 to keep anterior
PAR proteins from returning to the posterior cortex during the maintenance phase

Question 8

During the first division of the C. elegans embryo, several events occur that allow
asymmetric distribution of cytoplasmic determinates. Using what is known about the
cytoskeleton of the egg cortex and the function of the PAR proteins, describe the steps that
regulate this asymmetry.

Give one example of how similar proteins are regulating Drosophila development. Briefly
explain how these proteins function. Give at least one similarity and one difference
between the two systems. [4 marks]

Answer 8

• In C. elegans, PAR-3 protein regulates the cell’s polarity
• For Drospohila, a similar protein to PAR-3 is Bazooka (Baz)
• Baz received its name due to its disruptions in the epithelial making holes in the cuticle
• one similarity between PAR-3 and Baz is in their sequence, being homologous to each other
• Drosophila and C. elegans have similar systems in the sense that they share homologous proteins involved in polarization of the cells (PAR-1, aPKC=PKC-3, PAR-6, Lkbl (PAR-4), Bazooka (PAR-3))
• a difference between Drosophila and C. elegans’ system is the specification of the axis in the cell
• for Drosophila, the oocyte’s axis specification happens before fertilization
• on the other hand, C. elegan’s axis specification occurs after fertilization
• Bazooka’s function is to establish apical-basal polarity in epithelial cells, as well as helping with making single cells asymmetrical

Question 9

Why is asymmetric cell division an important process during development?

Answer 9

-cells use components of the cytoskeleton to create polarity within the cell.
-This polarity creates asymmetric distribution of membrane-bound and cytoplasmic molecules,
which at cytokinesis, can be unequally inherited by each of the daughter cells.
-important in early fate specification and
patterning of the early C. elegans and Drosophila embryo

Question 10

PAR proteins are important in which processes?

Answer 10

• during first cell division in C. elegans
• stem cell divisions
• cell migration
• neural development

Question 11

What experiment did Wilhem Roux perform and what were his results?

Answer 11

• used a hot needle to kill one of the cells of the two-cell stage of the frog embryo
• The remaining cell was allowed to continue to develop and a half frog embryo resulted
• concluded that the early frog embryo must use a form of development called mosaic development

Question 12

What experiment did Hans Driesch perform and what were his results?

Answer 12

-separated the sea urchin two-cell embryo which resulted in
death of one of the cells
-The remaining cell continued to develop and resulted in a complete embryo although it approximately half of the size of normal embryo.

Question 13

Cell fate definition

Answer 13

describes what a cell will normally develop into

Question 14

Planar cell polarity

Answer 14

used to explain how cells within a sheet, such as an epithelial
layer, can sense direction within the plane of a tissue.

Question 15

Epithelium

Answer 15

• a continuous sheet of cells that adhere together to form a barrier
• Cells within this sheet form strong cell-cell adhesions to maintain the structure of the layer.
• Within the sheet these cells have two major types of polarity: apico-basal polarity (apical to basal) and planar polarity

Question 16

Apico-basal polarity (apical to basal)

Answer 16

the basal surface is in contact with an extracellular

matrix called the basal lamina

Question 17

Planar polarity

Answer 17

This is polarity within the plane of the epithelium.

Question 18

Imaginal discs

Answer 18

-The Drosophila wing develops from a structure in the larvae called an imaginal disc.
-These are sac-like groups of cells that will grow and change shape during metamorphosis to
create the organs and tissues of the adult

Question 19

Trichomes

Answer 19

-The bristles, also called trichomes, of the wing are small hair like structures that follow a proximo-distal polarity or orientation

Question 20

List the five core proteins that exhibit asymmetric subcellular localization and the one
core protein that is symmetrically localized. For each, briefly describe the function and
localization pattern of each protein within the cells on the dorsal surface of the wing

Answer 20

-seven-pass transmembrane protein Frizzled (Fz) is confined to distal cell junctions along
with the cytosolic proteins Dishevelled (Dsh) and Diego (Dgo) [1.5]
-four-pass transmembrane protein Strabismus (Stbm, also known as Van Gogh; Vang) and the cytosolic protein Prickle (Pk) are localised proximally [1]
-the seven-pass transmembrane cadherin Flamingo (Fmi, also known as Starry Night; Stan)
is present both distally and proximally [1]
-Fz proximal membrane of each cell, next to Stbm in distal membrane of adjacent cell
-these are transmembrane proteins [1]
-Dsh, Dgo and Pk in the cytoplasm interacting with Fz and Stbm. [0.5]

Question 21

Planar cell polarity

Answer 21

a form of
development where cell-cell communication causes two or more cells to adopt a
coordinated polarity

Question 22

In general terms, explain how cells are communicating their polarity with adjacent cells.
[2 marks]

Answer 22

-The core protein asymmetric localisations are thought to result from intracellular
feedback interactions (also refered to as self-organization) between proximally and
distally localising components [1]
-The cell-cell coordination of this asymmetry involves the formation of asymmetric
intercellular contacts [1]

Question 23

In Box 2H and Figure 2 and 7 of the review there are several examples, using the fly wing
or frog epidermis, where cells that are defective in one of the core proteins cause a non- autonomous defect in planar cell polarity. In some cases a core protein is missing (these are
sometimes called loss of function mutant) while in others they are being over-expressed
(the protein is present in new locations or at levels higher than normal). Choose one of
these examples and explain how polarity of the affected cell and adjacent cells is influenced.
How might this explain how cells are coordinating their polarity. [2 marks]

Answer 23

-Complete loss of activity of any of the core proteins leads to a loss of planar polarity, with trichomes initiating in the cell centre. [1]
-Groups of cells that lack Fz induce neighbouring cells to point their hairs towards the
mutant cells, whereas loss of Stbm causes neighbouring cells to point their hairs away [1]
-mutant cells and their normal neighbours can only assemble asymmetric junctional
complexes of a particular polarity, and this polarity is then propagated to neighbouring
cells [1]

Question 24

Give one other example of a tissue or organ that displays multicellular planar polarity.
You can use other tissues in the fly or vertebrates as examples. [1 mark]

Answer 24

ommatidia in the fly eye or hair follicles in mammalian skin

Question 25

Who are Hans Spemann and Hilde Mangold, and what research did they carry out?

Answer 25

-In 1935 Hans Spemann received the Nobel Prize for Physiology or Medicine for his foundational work on amphibian embryo development. -This was the first award given for embryological research. -With the help of his student Hilde Mangold, in 1924 Hans Spemann published a paper describing the properties of the organizer. -It was later determined that Hilde Mangold was in fact the individual who performed many of the experiments. -Unfortunately, Hilde Mangold perished in a gas stove explosion and was unable to receive full credit for her achievement. -In this paper, the Spemann-Mangold organizer was grafted to a different gastrula staged embryo.

Question 26

Explain what is meant by inductive development. [1 mark]

Answer 26

-Induction is a type of regulatory development. -This is a process where one tissue directs the development of another tissue.

Question 27

In the space below outline how the experiment was performed. Include the stage and type of tissue that was grafted. How were the scientists able to distinguish between the donor and the host tissues? Describe the results of the experiment and provide at least three important findings that resulted from the careful analysis of the resulting embryos. [5 marks]

Answer 27

- used newt embryos of two different species with a differing pigment colour [1] - grafted of the blastopore lip of one newt onto another [1] - the blastopore is the opening formed in early gastrulation through which cell migrate inside - the graphed blastopore lip (i.e. Spemann-Mangold organizer) can induce the formation of an ectopic axis (a complete embryonic body) [1] - the graphed donor tissue is able to induce host tissue to form parts of new embryo - this induction changes the normal developmental fate of the host tissue

Question 28

You perform an experiment on the 32-cell staged Xenopus embryo as depicted in the top panel of the figure to the left. mRNA for a specific protein is injected into the future ventral axis of the embryo (top panel) and it is noted that a twinned embryo results (bottom panel) with a duplicated dorsal- anterior axis. Of the following five factors, β-catenin, GSK-3β, Vg1, Noggin, XWnt-11, which would give the same experimental result if the mRNA for this gene was injected into the same area of the embryo. Justify your answer by explaining how each of the five the factor functions during mesoderm induction and dorsal-ventral axis specification of the Xenopus embryo. [10 marks]

Answer 28

-Injection of ß –catenin and XWnt-11 would result in a twinned embryo [2.0]. -Maternal ß –catenin is sufficient to induce a second axis when injected into the ventral side of the embryo [0.5]. -ß-catenin is maternally contributed and stabilized on the dorsal side of the embryo upon cortical rotation. [0.5] -XWnt-11 would activate the canonical Wnt pathway through the Xfz7 receptor [0.5]. -Xfz7 is necessary for ß-catenin activation in the dorsal region of the embryo [0.5]. -Together they would stabilize maternal ß-catenin in the ventral side of the embryo. [0.5] -GSK-3ß is part of a complex responsible for degradation of ßcatenin. [1] -It is normally present in the ventral region and would have no effect on the development of the embryo. [1] -Vg1 is a Transforming Growth Factor ß molecule and is required for mesoderm induction [1]. -Vg-1 is already maternally deposited in this region and would likely not have an affect on the embryo. [1] -Sufficiently high levels may activate enough nodal signalling to cause more dorsallike structures to form, but without ß-catenin will not produce sufficient levels to induce a second organizer [1]. -Noggin is normally expressed in Spemann organizer [0.5]. -It binds to and inhibits the BMP-4 protein that signals to ventralize the embryo [1]. -Noggin protein can rescue UV-irradiated ventralized embryos, therefore, injection into the ventral side would cause dorsalization of the ventral tissue [1] and a second axis would form. -B-catenin and VegT activate siamois to form Nieuwkoop center. [1.0]

Question 29

What worked was done by Martin Evans?

Answer 29

-In 2007 Martin Evans was awarded the Nobel Prize in Physiology or Medicine for his foundational work on embryonic stem cells. -Cell lines can be created through the isolation of cells from the inner cell mass of the mammalian embryo. -Conditions were established where these cells could be grown indefinitely in the laboratory and hence were called embryonic stem cells. -Embryonic stem (ES) cells can be manipulated in the laboratory and re-introduced into the inner cell mass of a host embryo to create chimeric embryos. -Chimeric embryos have a mix of donor and host tissue. -If the donor cells contribute to a healthy adult germ line, the manipulated cells can generate many generations of animals called clones. -This technique was used to generate transgenic mice and provided a means for the mouse genome to be manipulated. -Much of what we know about mammalian development has been established using this cloning technique. -In many parts of the world, using similar techniques to create human embryonic stem cells and chimeric humans is not supported by federal funding or is illegal.

Question 30

What is stem cell research? What is the main goal?

Answer 30

-Using our understanding of early vertebrate embryo development, embryonic stem cells can be convinced to differentiate into certain cell fates such as neurons, heart or pancreas. -This has become an area of developmental biology research called stem cell research -scientists use developmental pathways that guide the embryonic stem cells towards terminal differentiation -As a long-term goal it is hoped that such techniques can be used to cure genetic diseases, repair damaged tissues and create “made-to-order” organs within the laboratory.

Question 31

Who is Shinya Yamanaka and what is his research about?

Answer 31

-In 2012 Shinya Yamanaka was awarded the Nobel Prize for his foundational work on induced pluripotent stem (iPS) cells. -Yamanaka’s research group carefully defined the conditions required to transform terminally differentiated human cells, such as skin fibroblasts, into cells that behaved like human embryonic stem cells. -Terminally differentiated cells can be re- programmed to erase their developmental history and regain the pluripotent competency of the inner cell mass of the human embryo.

Question 32

What were the four transcription factors that are sufficient to reprogram mammalian somatic cells into stem cells that are pluripotent? [2 marks]

Answer 32

Oct4, Sox2, Klf4 and c-myc

Question 33

If you were a stem cell researcher, give at least two ways you could test if reprogrammed iPS cells were in fact pluripotent? Define pluripotent and provide justifications to your answer. [3 marks]

Answer 33

-Pluripotent cells can give rise to all cell types of the embryo proper (all cells except the extra-embryonic tissue). [1] -ES cells injected into an adult will give rise to teratomas. -These are cancer-like masses of cells that divide and differentiate spontaneously into cell types from all three germ layers. -The ability to take on fates of any of the three germ layers suggests that these cells are pluripotent in their competence [1]. -iPS cells can be injected into a blastocyst staged embryo to create a chimeric animal. -If the cells contribute to the inner cell mass and contribute to all cell types of the adult, they are pluripotent [1].

Question 34

Primordial germ cells (PGCs) are the cells that are set aside in the early mammalian embryo that will give rise to the germline and are considered to be totipotent (Chapter 10). Dnmt3a2 is a methyl transferase that methylates DNA. Methylation of DNA tends to occur in regions of heterochromatin and is a stable epigenetic mark. Why did the authors compare the methylation status of the genomic DNA of the iPSs cells and PGCs. Explain why the structure of chromatin within a cell might affect the potency of iPS, ES or PG cells. [2 marks]

Answer 34

-Pluripotent cells can contribute to all cells of the embryo proper. -In chimeric mice pluripotent stem cells can give rise to the germline. -Cells of the germline need to be able to maintain their totipotency through embryo development. [1] -iPS cells selected for Fbx15 [0.5] expression do not produce adult chimeric mice [0.5] and do not contribute to the germline of chimeric animals [0.5]. -iPS Fbx15 cells do not express the same genes as pluripotent ES cells [0.5] and do not display the same DNA methylation patterns [0.5]. -Heterochromatin has a chromatin conformation that is largely closed [0.5] to gene transcription. -DNA methylation can influence which genes are competent to be expressed [0.5]. -An open conformation would suggest a higher level of competence [0.5] -PGCs can contribute to the germline. -Imprinting occurs in mammals where genes in the diploid embryo are differentially methylated and expressed depending upon the sex of origin. -During gamete formation, imprinted genes, particularly genes on the chromosomes from the opposite sex parent, need to be erased and re- established (i.e. reprogrammed) based on the sex of the progeny. -The authors compared the methylation status of imprinted genes, H19 and Igf2r, in the iPS nanog cells to determine if imprinting was a factor in the ability of these cells to contribute to the germline of chimeric embryos.

Question 35

The authors determine the influence of two transcription factors nanog and c-myc expression during the formation of iPS cells. nanog is normally expressed in the inner cell mass of the blastocyst staged embryo. c-myc is potent activator of cell growth and division and linked to the progression of many human cancers. Briefly summarize the major conclusions of their findings. [2 marks]

Answer 35

-Nanog expression is required for more efficient reprogramming of iPS cells into cells that more closely resemble embryonic stem cells. -These cells can contribute to adult chimeric mice and the germ line of chimeric animals. -Nanog is expressed in the pluripotent epiblast. -Expression is associated with their self-renewing and undifferentiated state. [1] -In 20% of progeny that were derived from iPS nanog cells, c-myc was reactivated and contributed to tumour formation. -Retroviral infection of fibroblasts resulted in reactivation of c-myc in subsequent offspring. Normally, the transgenes donated through retroviral transfection are silenced through DNA methylation. [1]

Question 36

Why do you think these studies are important? How close is the scientific community in being able to use iPS cells for medical treatment. Explain. [2 marks]

Answer 36

-Importance: immune rejection of iPS from other individuals. -Ethical consideration of working with human ES cells. -Positives: Puromycin selection allowed for cells to be selected that were nanog:GFP expressing. -Fibroblasts could be reprogrammed to contribute to the male germline in chimeric mice. -Closer to ES cells in gene expression and methylation patterns. -Oct-3/4, Sox2, Klf-4 and c- myc tend to be only activated in the iPS cells. -Regions of genome insertion heavily methylated. -Negatives: Low success rate. -Only one line was obtained where male chimeric mice contributed to viable progeny. -Of these progeny 20% develop tumours due to c-myc reactivation. -Cells need c-myc expression but reactivation a problem in tumour formation. -Re-programming still not well understood.

Question 37

Neuron axon development

Answer 37

a very important but complex type of cell migration

Question 38

What are the two types of extensions in neurons?

Answer 38

dendrites and the axon (can travel long distances to make the correct connections with other neurons or tissues such as muscles

Question 39

The central nerve cord (a precursor to the spinal cord in vertebrates) develops from a dorsal structure called the neural tube. The neural tube develops from the most dorsal aspect of the ectoderm called the neural plate. As illustrated in Figure 12.9, there are three major subtypes of neurons that develop from the neural tube of the three day old chick embryo. List these three sub-types, and briefly explain the function of each. [3 marks]

Answer 39

-Sensory receive signals and relay back to the spinal cord [1] -Commissural have axons that cross the mid-line to connect the left and right side of the body [1] -Motor neurons originate from the ventral root connect the spinal cord to the muscles [1]

Question 40

During axon outgrowth, the neuron uses a specialized cellular extension that directs growth and migration. Name this structure and briefly explain two type of cues that it can respond to mediate its direction of outgrowth and migration. [3 marks]

Answer 40

- Growth cone - Chemoattractant or Chemorepellents - Long-range vs. short-range

Question 41

There are several classes of molecules that mediate the response of a neuron to guidance cues. One is Robo receptors that respond to the Slit ligand and a second is the DCC receptor that responds to the Netrin ligand. Using a diagram to help explain your answer, explain how a commissural neuron can use these cues to cross the dorsal midline of the vertebrate nerve cord. [4 marks]

Answer 41

-Netrin – ligand for the DCC, is expressed at the midline and is a chemoattractant [1] guiding a commissural neuron towards the midline. -Slit also acts as a chemorepellant and is received by Robo 1 and 2, but remains low at a distance from the midline. -Once approaching the midline, the neuron expresses Robo3 which inhibits Robo repulsion [1], this allows the neuron to cross the mid-line. -Once the mid line is crossed, Robo3 inhibition is lost and Robo 1/2 mediate the repulsion of the neuron away from the midline [1]. -Non- commisural neurons use Slit and Robo1/2 to prevent crossing the midline [1].

Question 42

Why do the lungs develop into a highly branched structure?

Answer 42

to maximize surface area for gas exchange within this organ

Question 43

What does the tracheal system of the fly do?

Answer 43

a highly branched tubular network that mediates gas exchange between the environment and tissues

Question 44

In early mouse lung development, how does patterning occur?

Answer 44

an epithelial tissue originating from the endoderm increases cell division in specific regions to initiate branch outgrowth, a primary branch is formed, secondary branches can be generated from the primary branch to increase the complexity of the branching pattern

Question 45

In Drosophila three molecules, Branchless (Bnl), Breathless (Btl) and Sprouty (Spry) have been found to be critical for regulating tracheal branching. a) Describe the role of each of these molecules in the FGF pathway. Explain how these molecules are guiding primary branch formation. Use a diagram to explain your answer. [6 marks]

Answer 45

-Branchless Fibroblast Growth Factor (FGF) ligand [0.5] -Breathless FGF receptor [0.5] -Sprouty negatively regulates FGF signal transduction [0.5] by inhibiting or dampening the mitogen ­activated protein kinase (MAPK) pathway. -Primary Branch Formation: the gene encoding the FGF ligand Branchless (Bnl) is expressed in a dynamic fashion in clusters of nontracheal cells [0.5] at positions toward which branches extend [0.5] -the Bnl signal is transmitted and interpreted in tracheal cells via the FGF receptor Breathless which accumulates in cells competent to form a primary bud [0.5] -Bnl expression is under tight genetic control by the AP and DV genes, and thus identical in each embryo and hardwired [0.5] -expression is dynamic and changing through the branching process [0.5] [in the embryo, Bnl expression precisely placed based on the A/P D/V coordinate system] -the Bnl signal results in the formation of dynamic filopodial extension [0.5] from the basal side of those tracheal cells that are closest to the Bnl source [0.5] -regulates cell motility via cytoskeletal rearrangements [0.5] -cells transition from a polarized epithelium to one that become motile and invasive [0.5] -cells with the highest BTL receptor activity assume lead positions in the tip cells [0.5]

Question 46

Tissues in the fly that experience hypoxia due to low oxygen levels can regulate Branchless expression. Explain how this could help the organism adjust to the changing oxygen requirements of their tissues during tracheal development. [2 marks]

Answer 46

-in the larvae, the extension of terminal branches is variable and plastic and can be regulated by oxygen during larval development -cells experiencing oxygen deprivation induce the expression of Bnl guiding new terminal branches towards them [1] -the oxygen delivered by the newly formed terminal branches relieves hypoxia, which decreases Bnl expression and aborts further terminal sprouting [1]

Question 47

During mouse lung development, the lung epithelium interacts with signals from the surrounding mesenchyme. A branch initiates as a bud that grows into the mesenchyme tissue and then bifurcates to initiate two secondary branches. FGF-10, FGF Receptor 2 (FGFR2) and Sprouty2 (Spry2) regulate this branching process. Explain how FGF-10 expression in the lung mesenchyme can generate primary and secondary lung branching. Use a diagram to explain your answer. [4 marks]

Answer 47

-FGF10 is dynamically expressed [0.5] in the splanchnic mesoderm of the lung [0.5], and signals through FGFR2b to the lung epithelium [0.5] -Epithelial-mesenchymal crosstalk at the tip results in modulation of FGF signaling [0.5] -FGF10 levels are controlled in time and space in the distal mesenchyme as well as by modulating the response in the adjacent epithelium along the proximal-distal axis -Shh restricts FGF10 expression [0.5], the growing epithelial bud, which expresses high levels of Shh (independent of FGF10) [0.5], interacts with the chemotactic source FGF10 as it approaches [0.5], this interaction leads to a reduced expression in the immediate vicinity of the approaching tip [0.5] and a splitting and lateral displacement of the FGF10 source [0.5]. -Spry genes encode a family of cysteine-rich proteins [0.5] that antagonize receptor tyrosine kinase signaling [0.5] -spry2 is part of a negative feedback loop [0.5] by which increased FGF signaling in the most distal lung epithelium limits FGF signaling in responding cells, ultimately resulting in decreased cell proliferation [0.5] -TGFB-1 accumulates at sites of cleft formation [0.5] and along proximal airways. -TGFB-1 promotes synthesis of extracellular matrix [0.5] that, when deposited in the epithelial- mesenchymal interface, might prevent local branching [0.5] -FGF10 source bifurcates as the growing tip approaches causing two new sources of FGF10 and subsequent branching

Question 48

During mouse lung development, the lung epithelium interacts with signals from the surrounding mesenchyme. A branch initiates as a bud that grows into the mesenchyme tissue and then bifurcates to initiate two secondary branches. FGF-10, FGF Receptor 2 (FGFR2) and Sprouty2 (Spry2) regulate this branching process. What type of branching patterning would you expect to see if the FGFR2 receptor or Sprouty2 was genetically knocked down in the developing mouse lung. [2 marks]

Answer 48

-FGFR2 is required to respond to the FGF10 chemoattractant. -Lower activity should result in less out growth and reduced branching [1] -SPRY2 regulates the site of initiation and the number of branches in specific domains. -Less SPRY2 activity may result in ectopic outgrowth or excessive branching. [1]