Study Questions 5

Question 1

What are the relationships between epiblast, ectoderm and epidermis? When are they specified during mammalian development

Answer 1

Epiblast > ectoderm > epidermis
Epiblast – one of the layers in the laminal disk that give rise to embryo proper
• Forms ectoderm
• Made by inner cell mass
• Split by small clefts that combine to separate embryonic epiblast from amniotic ectoderm
Ectoderm – specification during beginning of blastulation, gastrulation
• Originate from embryonic epiblast
Epidermis – outer epithelial layer of embryo, derived from ectoderm

Question 2

What tissues are developing from ectoderm?

Answer 2

The surface ectoderm, neural crest and neural tube are developing from ectoderm.

Question 3

What are the four stages in the formation of neuroblast from a blastula cell?

Answer 3

There are 4 stages through which the cells of the epiblast/blastula need to pass through in order to become neural precursor cells or neuroblasts
· Competence/induction: the cells can become neuroblasts if they are exposed to the appropriate combination of signals
· Specification: cells have received the appropriate signals to become neuroblasts, but progression along the neural differentiation pathway can still be repressed by other signals
· Determination=commitment – neuroblasts have entered the neural differentiation pathway and will become neurons even in the presence of inhibitory signals
· Differentiation: the neuroblasts leave the mitotic cycle and express those genes characteristic of neurons
· Two sets of cells can contribute to the developing nervous system, cells within the neural tube and neural crest cells

Question 4

What is the neural plate and how is it formed? Why did we talk (again) about Spemann’s organizer transplant experiment in this context?

Answer 4

The formation of the neural plate is the first sign of a nervous system; it eventually folds inward and becomes the neural tube. It is formed by the thickening of the dorsal ectodermal cells, induced by the underlying mesodermal cells (proven by Spemann’s transplant experiment). The transplant had altered the fate of the overlying cells.
· Neural plate- when the ectoderm on the dorsal side of the embryo, anteriorly from the primitive node, thickens it forms the neural plate. The ectodermal cells in that area are transformed into specialized nervous tissue cells due to morphogens released from the underlying mesoderm
· Spemann’s organizer transplant experiment: (it is mentioned bc just as shown in the experiment, the mesoderm induces the neurlation process
· The underlying mesoderm acts on the dorsal ectoderm causing thickening of neuroblast cells anteriorly form the primitive node to form a neural plate
· Speman showed that excision of the mesodermal organizer and then grafting causes a secondary dorsal neurulation, which shows that mesoderm can induce ectoderm to neurolate
· Neurulation specification signaling by mesoderm is region specific. Grafting of early mesoderm neurolas causes dorsal neurulation at site with region specific expression

Question 5

Explain inhibition as a regulatory mechanism during development. Give two examples (one could be d-v specification in Drosophila; go back and check).

Answer 5

Inhibition as a regulatory mechanism: inhibitions of certain proteins is due to the context of their immediate envionrment and it is required for the induction of certain pathways
· Ex 1. Inhibition of bmp signaling is required for induction of neural plate.
· Bmp expressed in the ectoderm on the ventral side, allowing ectoderm to become epidermis
· Organizers on the dorsal side releases inhibotrs of BMP: noggin, chordin which diffuse into the ectoderm on the dorsal side and block the effects of BMP which allow for the neural tissue to form
· Ex. 2: gurken is only found on the dorsal side and it inhibits PIPE which prevents the movement of dorsal protein into the nucleus. Therefore due to the inhibition of the protein, dorsal protein can only ventralize the ventral side of the embryo.

Question 6

Define neurulation. List four stages of primary neurulation in chick embryo.

Answer 6

Neurulation is cells from the neural plate form the neural tube, and embryo known as neurula during the process. There are two ways of this occurring: primary and secondary neurulation (bird and mammal specific).
Stages of primary neurulation in chick embryo
1. Formation of neural plate through shaping and folding
2. Shaping of neural plate through elevation
3. Bending of neural plate to form groove through convergence
4. Neural groove form neural tube via closure

Question 7

7. What is happening with the neural plate cells during neurulation?

Answer 7

During neurulation, neural plate cells will deepen to form neural tube which will close up

1. Shape, fold, elevate, converge to become neural groove
2. Columnarization – microtubules within cells elongate to columnar shape
3. Wedging – apical actin filaments constrict which narrow apical area of cells

Question 8

What is the role of hinge points during neurulation?

Answer 8

The medial hinge point cells and dorsolateral hinge points help the neural plate bend to form the neural tube

1. MHP anchor to notochord and change shape
2. Dorsolateral hinge points become wedge shaped to help convergence of neural fold to neural groove
3. Both hinges are induced to become wedge shaped to act as pivot that direct rotation of cells around it

Question 9

9. What are the two most important proteins that force the detachment of a neural tube from the surface ectoderm? (When did we mention e-cadherin before? Check blastocyst formation.)

Answer 9

Cadherin
● Neural tube express E-cadherin
● Neural tube and groove express isoform N-cadherin
○ Production of this isoform reduces binding affinity between neural tube and surface ectoderm
Flashback = E-cadherin (in mammals)
● Adhesion molecule aiding compaction in mouse embryo of blastomeres to form compact ball

Question 10

10. What is the difference between primary and secondary neurulation? (It’s about the position in an embryo where they happen.)

Answer 10

Neurulation is the formation of the neural tube from the neural plate.
Primary neurulation – occurs in anterior human, chick embryo
1. Shaping and folding = form neural plate
2. Elevation = shape neural plate
3. Convergence – bend the neural plate to form neural groove
4. Closure – close neural groove to form neural tube
Secondary neurulation – condensation (coalescing) of mesenchymal cells
1. Mesenchymal cells form medullary cord that becomes hollow to form neural tube
2. Occur in posterior embryo of chicken and human

Question 11

11. What are the three primary vesicles formed during early brain development? Five secondary vesicles?

Answer 11

Primary: prosencephalon (forebrain), mesencephalon (midbrain), rhombencephalon (hindbrain)
Secondary: telencephalon (forebrain), diencephalon (forebrain), mesencephalon (midbrain), metencephalon (hindbrain), mylencephalon (hindbrain)

Question 12

Where and how is the occlusion of the neural tube formed? What is the outcome of the temporary occlusion?

Answer 12

● Early brain forms occlusion of neural tube to increase brain cavity size without increasing cell number
● Occlusion occurs at the interface of presumptive brain and spinal cord
● Build of of positive fluid pressure
● Neural folds close region between presumptive brain and spinal cord
● Surrounding dorsal tissue push inward to constrict neural tube at base of brain
● Outcome: separate presumptive brain region from future spinal cord to allow buildup of fluid pressure to increase cavity size (allows expansion of future brain region)

Question 13

What are the two major paracrine factors involved in dorso-ventral polarization of the neural tube? Where do they originate? How do they establish d-v axis?

Answer 13

The two major paracrine factors are Sonic Hedgehog and Bone Morphogenic Protein (BMP) and they are both primary transcription factors that vary their concentration to differentiate dorsal from ventral. Sonic Hedgehog originates from notochord and induces medial hinge cells to become floor plate of neural tube. BMP originates from dorsal ectoderm and establishes dorsal axis through induction of roof plate formation.

Question 14

Outline the experiment that showed the role of notochord as the d-v axis organizer.

Answer 14

● Removal of notochord = no formation of floor plate and motor neurons
● Transplant notochord and folding neural plate to another region of neural tube cause another floor plate and set of motor neurons to form
normal: during development, the floor plate (red) develops above the mesodermal notochord (n) and motor neurons (yellow) differentiate in adjacent ventrolateral regions of the neural tube
- grafting a donor notochord (n’) alongside the folding neural plate results in formation of an additional floor plate and a third column of motor neurons
- removing the notochord from beneath the neural plate results in the permanent absence of both floor plate and motor neurons in the region of the extirpation. Pax6 expression (blue) extends through the ventral region of the cord

Question 15

What is the origin of neural crest cells? Draw the diagram showing their creation.

Answer 15

The origin of neural crest cells is neural fold cells or cells of most dorso-lateral cells of neural tube, derived from ectoderm
● Neuroectoderm + non-neuroectoderm induce formation of neural plate border
● Neural plate border elevates causing plate to invaginate and roll into neural tube
● Neural crest cells (dorso-lateral cells) delaminate from fold or dorsal neural tube by pinching off

Question 16

16. What are the derivatives of the neural crest cells? (You HAVE to know this.)

Answer 16

●	Peripheral nervous system (neuron, glia) (PNS, SNS)
●	Endocrine, paraendocrine derivatives
●	Pigment cells (melanocyte)
●	Facial cartilage, bone
●	Connective tissue (head, neck)

Question 17

List two neural crest specifiers. What is their role?

Answer 17

FoxD3 and SOX9 give instructions to neural crest cells as they leave neural tube (based off their location), during migration, and at the site where they will differentiate.
● FoxD3 – specification of ectodermal cells as neural cells
● SOX9 – autosomal determinant of male sex

Question 18

Outline the experiment that showed the pluripotency of a single neural crest cell.

Answer 18

● Inject dye into single neural crest cell
● After division, label found in different daughter cells in many locations
● Conclusion: single neural crest cell can become sensory neuron, melanocyte, adrenomedullary cell, glia = pluripotent

Question 19

Explain in general terms how is a neural crest cell specified and differentiated into a particular neural system cell.

Answer 19

Neural crest cell is specified through receiving information from specifiers such as FoxD3 and SOX9, at the point of exiting the neural tube (information given depends on exit location), migration path and final destination. This information influences their gene expression which will result in a certain neural system cell
Process:
● Progenitor cells differentiate depending on instructive signals that they receive when they leave neural tube (neural tube exit position specific) à become precursors committed to particular path
● Precursors get exposed to instructions during migration and at sites of differentiation. This will influence their patterns of gene expression.

Question 20

Similar to the above: how do you understand differentiation of the neural crest cell (or any other cell)? Are genes lost during the course of differentiation, i.e. does the mechanism of differentiation involve the selective loss of genes (or something else is going on)?

Answer 20

No genes are lost during the course of differentiation. The mechanism of differentiation just causes expression of certain genes and repression of others which causes a variety of differentiation cell results.

Question 21

21. How do we group neural crest cells based on location (place where they pinch off AND where they end up)?

Answer 21

Classified based on which tissue they enter along anterior-posterior axis – grouped into 4 overlapping anatomical regions
● Cranial neural crest – migrate into pharyngeal arch and face – NCC become cartilage, bone, cranial neuron, glia, face connective tissue
● Trunk neural crest – somite 6 to tail – NCC become sensory neurons or melanocytes depending on path
● Vagal, sacral neural crest – somite 1-7, posterior somite 38 respectively – form parasympathetic ganglia of gut
● Cardiac neural crest – subregion of cranial – somite 1-3 – NCC become face structure, connective tissue of artery/aorta

Question 22

What are the two pathways for the trunk neural crest cell migration? What is the destiny of cells that take each of the pathways?

Answer 22

Ventral pathway – early NCC go through anterior sclerotome
● To become sensory and sympathetic neurons, adrenomedullary cells, Schwann cells
Dorsolateral pathway – late NCC follow dorsal pathway
● Become melanocytes

Question 23

What are the roles of ECM in migration of neural crest cells?

Answer 23

The roles of ECM in migration of neural crest cells is for trunk neural crest migration. Migration depends on ECM component condition in addition to chemotactic factors.
● Anterior sclerotome has ECM molecules that permit neural crest migration (fibronectin, laminin, tenascin, specific collagen, proteoglycan)
● Ephrin and semaphorin obstruct NCC migration (also components of ECM)

Question 24

What are the cranial neural crest cells going to form? (in general)

Answer 24

Osteoblast and head

Question 25

Define (1-2 sentences) ossification.

Answer 25

Intramembranous ossification is the formation of bone from mesenchyme. Cranial neural crest-derived mesenchymal cells condense into nodules and then pre=osteoblast progenitors (low BMP). Pre-osteoblast progenitors become chondrocyte-like osteoblasts by downregulating transcription factor Runx2 and then become osteoblasts which are bone precursor cells that secrete osteoid matrix that binds Ca.

Question 26

26. What are cranial placodes? What is their position in respect to the neural plate? To the neural crest? List two of them.

Answer 26

Cranial placodes are local, temporary thickening of ectoderm in the head and neck. They become sensory neurons that form distal portions of ganglia associated with hearing, balance, taste, and smell. They are in the proximal part of ganglia formed from cranial crest cells and they are laterally arranged around the neural plate. Examples: olfactory, lens, trigeminal, optic