Development

Purves et al., Chapters 22-23, one figure from Chapter 11

Pep talk

The overall theme relates to "plasticity." In that regard, learning and memory are considered to be continuations of development, so the boundary line between development and memory is not clear.
Dogma is that invertebrate nervous systems are hard-wired with little plasticity or learning (though there are lots of exceptions) and that vertebrate adaptability relies on rewiring, alterations, and learning.

Prof Schreiweis taught a course in embryology (BL A344, Fall, 5 credits, lecture plus lab). He retired summer 2012. Traditionally, embryology, specifically comparative embryology, has been fundamental in organizing life in biology.
Developmental biology is a very different field, and workers in developmental biology, Lewis, Weichaus, and Nusslein-Volhard -won the1995 Nobel Prize.
Prof Ogilvie teaches developmental biology (BL A460, Spring, 3 credits, lecture; BL A493-36, lab)

Famous molecules

Fig. 22.5, p. 486
Signal transduction, refer back to Chapter 7
Here are several of the ligand-receptor pairs covered in this figure:
wnt-frizzled
shh-patched
fgf-rtk
The entire cascades for these pathways (and others) are really fundamental in modern biology. To a limited extent, find coverage in my signal transduction course outline.

Stem cells

Box 22A, pp 479-480
Stem cells (instead of presenting what is in the box, I will talk about my work)

Because cells lose their pluripotency, researchers have focussed on their discovery that embryonic stem cells are better at differentiating into cells that can repair cell damaged areas such as in the case of spinal cord injury; the issue is very controversial because it may encourage practitioners to create and destroy human embryos for no other purpose than to harvest stem cells. Of note, there may be "left-overs" (it is hard to find a diplomatic euphemism) from in vitro fertilization after a couple has had all the children they want (that might go to "waste"). For this reason, for humans, only the use of some 60 cell lines that are already in culture was dictated in the US by President Bush.

Several colleagues and I collaborated to cure blindness in a mouse mutant with cells that started as embryonic and were induced to become precursors of nerve cells; identified by green fluorescent protein, here is a cell that has been put into the retina and is beginning to show a neuron-like phenotype.

Recent paper

SSHall, Diseases in a dish, Scientific American, March 2011, 40-45. Take skin of elderly person who has ALS in the family, make stem cells, turn them to neurons, watch ALS develop, test drugs
Time Line
1998 James Thompson (University of Wisconsin - Madison)
2001 (Aug) George W Bush restrictions
Harvard, Columbia, Stanford - labs with private funding
2002 TMJessell, HWichterle et al (Columbia) how to get embryonic stem cells to be motor neurons
2006 SYamanaka (Kyoto) how to get stem cells from skin2009 Obama relax restrictions
2010 court banned NIH support

Review

Know from earlier this semester:
No regeneration of neurons in the (mammalian) CNS. Interesting regeneration in olfactory and taste receptors.
Hubel and Wiesel (1981 Nobel) (and others since) - need for patterned vision during critical period to maintain visual cortical binocularity and feature (contrast) detectors (this will come up in chapter 24)
Wiring in cerebellum is disrupted in mutants (Chapter 19)

Drosophila Embryology

Drosophila is a model for understanding development, generally
Order of action: maternal genes, zygotic genes, homeotic genes

Fig. 22.4, p. 483
a lot has to do with segmentation

Fig. 22.4 again
Maternal means that the gene was transcribed in the mother; that is how bcd (bicoid) was deployed
zygotic genes have the order of action as shown in Fig.
gap such as kr (kruppel), pair-rule such as h (hairy), and segment polarity such as wg (wingless)

TRANSPARENCY
Imaginal discs are structures in larvae destined to become structures in the adult (entomologists call the adult the "imago")

Fig. 22.4 still
Homeotic mutants - with names like "antennapedia" - (with leg where antenna should be)
(i.e. often transplanting something which should be in one segment to another)
Homeotic gene has homeobox (["box" is in DNA] 183 bp of DNA) which codes for DNA binding protein with 61 amino acid homeodomain (["domain" is in protein] helix turn helix)

The sevenless signalling pathway

Fig. not in 5th edition
(I put more information below than is in the book)
How do > 750 ommatidia with some 19 cells develop?
(receptors (R1-6, R7 & R8, cone cells, bristles, pigment cells)
Development in the eye imaginal disk.
In sev (sevenless) mutants, the R7 precursor becomes cone cell.
(I wrote the paper that introduced sevenless, see here)
Sevenless is a receptor tyrosine kinase, and signalling involves ras = small G protein.
Sequential addition of receptor cells in Drosophila eye: R8, R2 & R5, R3 & R4, R1& R8, R7
Boss = bride of sevenless is 7 transmembrane domain ligand

Fig. 22.5 C p. 486
sevenless is receptor tyrosine kinsae -
2 transmembrane subunits, 2 extracellular subunits
expressed everywhere except R2 R5 and R8
It is a topic of intense present interest how this signals across membrane
Drk = downstream of receptor tyrosine kinase
which is a small SH adaptor protein, SH = src homology
src = oncogene of Roux sarcoma virus
Sos = son of sevenless, a GNRP (guanine nucleotide releasing protein) to exchange GTP for GDP on ras
ras = rat sarcoma [viral ras oncogene of normal protooncogene]
other steps -> signalling to nucleus
MAPK = mitogen activated protein kinase
alias ERK = extracellular signal regulated kinase

Embryology

Fig. 22.1AB, p. 478
Neural plate forms from ectoderm -> neural groove -> neural tube to make CNS

Fig. 22.1C, D
One area remains outside CNS - neural crest gives rise to PNS structures like sensory ganglia

Fig 22.2 p. 481
Different cells migrate to make (1) sensory ganglia, (2) autonomic ganglia, (3) adrenal, or (4) non-neural tissues like melanocytes

Fig. 22.11C, p. 499
Factors on how neural crest progenitors turn into specific PNS types
(more on factors later)

Brain subdivisions

Fig. 22.3AB, p. 482
Prosencephalon -> telencephalon and diencephalon
Mesencephalon
Rhombencephalon ->Metencephalon and myelencephalon
Note, "optic vesicle" signifies that retina is outgrowth of CNS

Fig. 11.4, p. 233
Induction from optic vesicle makes lens form from ectoderm

Histogenesis

Fig. 22.7A p. 491
Cell divisions in monolayer with nuclear migration (mitosis near neural tube lumen (ventricle) and have S-phase near pial surface)

Fig. 22.12A, p. 502
Fig. 22.13, p. 504
Then cell migrates out along tracks made by radial glia
Recall Weaver mutant mouse in which cerebellar granule cells are missing:
Bergman glia screwed up - granule cells not migrate, die

Fig. 22.8, p. 493
then each layer (e.g. V) migrates past previous (e.g. VI)

Axon pathfinding

Retinotectal projection in frog


Fig. 23.7B
Background was that Weiss had proposed the resonnance principle which goes something like this -- that growing and connecting axon induces the cell type in the postsynaptic cell.
Then Roger Sperry did an important experiment (1981 Nobel prize, though not for this)
turn frog eye upside - down and projection reverses
(would jump in the wrong direction)
note - advantage of amphibian system - regeneration of optic nerve in adult
this work being in the adult
Sperry proposed "neurobiotaxis" gradients
- recently shown retinoic acid gradient in zebrafish
Jacobson and Hunt - specified after stage 28, first AP laid down, then DV, implying that something about position of eye in head picks up information specifying DV, AP
First neuroblasts which develop undistinguished neurites
Pathfinding complex - growth cones
growth cones secrete protease, express growth associated protein GAP43
feel way with filopodia

Work since then

Fig. 23.2C, p. 510
These are drawings from Ramon y Cajal
There are these growth cones (enlargements) at the tip of an extending axon which extend and retract filopodia, feeling their way along.

Fig. 23.2B, p. 510
growth cone, confocal microscopy
SEM growth cone

netrin elicits axon growth from explant from spinal cord
Although, netrins (Sanscrit "to guide") serve as chemoattractants, Sperry's neurobiotaxis idea was overly simplistic:

Fig. 23.4, p. 514
Growth cone with integrin follows laminin and stops when laminin runs out
Axons stick to eachother and to growth cones with cadherins and CAM's (cell adhesion molecules) like Ng-CAM (neuro-glial) and N-CAM (neuronal)

Synaptogenesis

Fig. (not in 5th edition)
Synaptogenesis at neuromuscular junction
agrin & its receptor cause aggregation of AChR

Trophic factors

Inductive interaction is important - like trophic effect of nerve on muscle (in polio, nerve disease leads to wasting away of muscle)

Fig. 23. 10, p. 525
This would also work in reverse, those nerves deprived of muscle vanish, or if extra limb, there are more spinal motor neurons.
Thus, there are too many nerves at first, then those which do not connect degenerate.
This would rely on programmed cell death (apoptosis), not really emphasized in chapter.

Rita Levi-Montalchini 1986 Nobel Prize "discoveries of growth factors"
NGF (nerve growth factor) is from targets like glands.

Fig. 23.13, p. 529
Here is a dorsal root ganglion (somatosensory ganglion) without (A) and with (B) NGF making it obvious, from the neurite outgrowth in B, why it is named NGF (work of Rita Levi-Montalchini)
Take-up makes sympathetic (and other nerve cells, like certain sensory nerves) survive.
Antibody to NGF kills sympathetic nervous system.
Oddly, one good source of NGF is male salivary gland.
Cytokines include:
Neurotrophins like NGF, BDNF (brain derived), NT-3, NT-4/5
Hematopoietic factors (like interleukins)
Growth factors like EGF, FGF, TGF, IGF

Fig. 23.16, p. 533
Trk ("track") receptors (with tyrosine kinase activity)
TrkA for NGF, TrkB for BNDF, TrkC for NT-3

There is a box on retinoic acid (Box c, Chapter 22). I am and have been very interested in retinoic acid and have written a lecture on retinoic acid and its relation to steroid and other hormone signalling for my last semester's signal transduction course, but will not talk about it much here. (The figure referenced from Alberts et al. is from Molecular Biology of the Cell Third Edition.)

Exam questions from 2005 - 2012 related to this outline

In contrast with signalling via the heterotrimeric G protein, signalling by Wnt, FGF, BMP, shh and RA were posited as going in a fairly direct pathway into the nucleus. Say something about the effect these ligands have in the nucleus.

ultimately they control transcription into mRNA of specific genes

"Inside the larva are these pieces of tissue that are determined to become adult structures but are not yet differentiated. Say something about them (e.g., What are they called? When do they differentiate?).

imaginal disks have cell proliferation in larvae then they differentiate into adult structures in the pupa case

The genes involved in homeotic mutants-- answer either (1) What shape is the critical domain of the protein they encode? or (2) What is the function of that protein?

helix turn helix, bind DNA to activate transcription

Sevenless is a receptor tyrosine kinase. Name another protein in the sevenless signal transduction pathway.

boss, downstream of receptor kinase, sos, ras, MAP kinase,

There are 3 layers (suffix ...derm). From which is the neural plate derived?

ectoderm

Give one of the fates of neural crest.

sensory ganglia, autonomic ganglia, adrenal medulla, non-neural cells like melanocytes

In a classic example of induction, what does an out-pocketing of the diencephalon make the overlying ectoderm turn into?

lens and cornea of the eye

Your lab is the best in the world in all histological techniques including the Golgi technique. And yet, NSF and NIH resoundingly reject your grant proposal to look for differences in growth cones in the hypothalamus in male vs female rats at the age when males continue to get heavier but females do not. What is the reviewers' criticism?

there is no axon pathfinding in the adult mammalian brain

"The chick spinal cord generates an excess of neurons prior to the differentiation and innervation of the limb. Normally some of these neurons are lost..." Answer either (1) What is the evidence for the first statement? or (2) Say something about this process of loss of neurons.

more neurons if supernumerary limb bud, less if ablated limb bud; apoptosis

A cell from the mouse inner cell mass is treated for 4 days with retinoic acid after 4 days without retinoic acid. This protocol converted the cell from what kind of cell to what kind of cell?

from an embryonic stem cell to a neuronal precursor

Why are the first genes to guide Drosophila embryology referred to as "maternal genes?"

the mRNA is active in, and the protein laid down by, the mother, not the zygote

Say something about the receptor in the MAP kinase (or the sevenless) signaling pathway.

transmenbrane receptor tyrosine kinase

Somehow a cell is told to become R7 in every ommatidium (facet) of the Drosophila compound eye. Answer either (1) What cell tells that cell to become R7? Or (2) What is the ligand on that cell that sends the signal?

R8, boss

For retinoic acid, [answer either] (1) What would happen if you treated embryonic stem cells with retinoic acid? Or (2) What would be required for it to activate transcription of specific genes?

they would become neural stem cells, a retinoic acid receptor (RAR), and response element (RARE) and dimerization with another hormone-receptor complex

In "diseases in a dish," how did S. S. Hall propose to get motor neurons from an elderly donor, one from a family with familial ALS, to test drugs on?

skin cells would be used to get neurons and only then would they begin

Why did they decide to call bicoid a "maternal gene?"

mRNA expressed in maternal cells where protein is made (and put into egg)

If you tried Sperry's experiment, turning the eye up-side-down and letting it reconnect to the tectum, before stage 28, how would the frog respond to a moving fly presented to the side of its visual field?

it would flick its tongue to the correct place

Dogma has it that there is no regeneration of neurons in the mammalian central nervous system. Describe how post-mitotic cells exit from the cell cycle.

arrest in G1

What does the neural crest become?

sensory neurons, sympathetic nervous system, adrenal medulla, melanocytes

What finding led to the conclusion that apoptosis reduced the number of spinal motor neurons to the right number to innervate a limb?

there were enough precursors of cells to innervate two limb buds

In all of our coverage of neuroanatomy of the spinal cord, with terms like "ventral horn" and "dorsal columns," there was no mention of floor plate and pMN. Why not?

these are in the embryonic neural tube

What kind of a molecule is a chemokine receptor?

g protein coupled

"Olig-2 is expressed in oligodendrocyte precursors and it is a basic helix-loop-helix factor." What does such a factor do in the cell?

regulate transcription

What is the difference at birth vs. at maturity of how many spinal motor neurons can innervate one end plate?

at maturity, only one, more earlier

In the time since the stem cell debate was in the forefront the federal administrative scrutiny, what advancements have lessened the ethical dilemma?
 
there are sources of stem cells other than embryonic
 
In what fundamental way are bicoid and kruppel different in the locations where their genes act?
 
bicoid mother ("maternal"), kruppel embryo ("zygotic")
 
Say something about the comparison of ras with a heterotrimeric G protein.
 
ras is a G protein, smaller, monomeric
 
Several signaling ligands were introduced to you: retinoic acid, wingless, fibroblast growth factor, bone morphogenetic factor and sonic hedgehog. Which pathway most closely approximates the one initiated by bride of sevenless and why?
 
fgf b/c it is membrane receptor tyrosine kinase signaling through ras and MAPK
 
In between ectoderm and the neural groove are cells whose fate is very different from those of the neural groove. In what way?
 
neural crest will become components of peripheral n.s.
 
Here is a list of cell types: sensory neuron, melanocyte, adrenergic sympathetic neuron, cholinergic sympathetic neuron, and chromaffin cell. Answer either: (1) What is the common ancestor? Or (2) How is it that they assumed different fates?
 
neural crest progenitor, "growth factors" (NGF, LIF, glucocorticoids, etc.)
 
Embryologists tell us that the retina and optic >nerve< are part of the central nervous system. So you were told earlier. Now add some details that justify that position.
 
optic vesicle becomes optic cup, outpocket from diencephalon
 
Sperry proposed neurobiotaxis for how each optic nerve found the right address in the tectum. What had been the previous thinking?
 
weiss, resonance, connection from eye assigned function of central cell
 
Several times, the neuromuscular junction was used as a model system in development and "learning." Where did agrin fit into the discussion?
 
agrin mediates the localization of acetylcholine receptors to the end plate
 
In a comparison of spinal motor neurons on the normal side vs the side where a second limb bud had been transplanted, how does apoptosis apply?
 
there would be zillions of spinal motor neurons, decreased by apoptosis on the basis of limited target
 
Describe the geometry of the TrkA receptor for nerve growth factor.
 
it is a single membrane passing enzyme that dimerizes on binding the ligand
 
Receptor molecules on the axonal growth cone are inhibited by well-studied molecules derived from damage to (what?) in CNS injury.

oligodendrocytes

"Alpha and gamma secretases act on p75ntr to yield an ECD and an ICD." Translate.

these enzymes cut in the membrane neighborhood to give intracellular and extracellular domains

Fibroblast growth factor (FGF) is the ligand in a pathway with ras and MAPK signaling to the nucleus. Describe the receptor, pick one (1) location (in the cell), (2) structure, or (3) specific biochemical function.

1 membrane, passes one time, dimerizes, phosphorylates tyrosine

A mouse embryonic stem cell can be converted to a neural stem cell that expresses neuron-specific proteins by what important developmental ligand?

retinoic acid

With respect to anterior vs posterior (as opposed to segmentation), why is bicoid referred to as "maternal" (as opposed to zygotic)?

laid down into egg by mother as opposed as involving expression after the egg is fertilized

"Bride of sevenless (Boss) is the ligand for sevenless (sev). Answer either: (1) What cell's expression of Boss "tells" the R7 precursor to become R7? Or (2) What happens to this precursor if sev is mutant?

1 R8, 2 becomes cone cell

A sympathetic progenitor can become an adrenergic neuron or a cholinergic neuron." How is this determined?

with NGF vs CNTF

An explant of developing spinal cord is placed near some tissue that is a source of netrin. What is observed?

neurites going toward source

In the cerebellum, what cell type is "inside" the Purkinje nuclear layer? (Its terminals are "outside" [i.e. toward the meninges] the Purkinje nuclear layer.)

granule cell

"In the neural tube, cells exit the cell cycle to become post-mitotic neuroblasts." Address either (1) At what stage are they arrested in the cell cycle? Or (2) At what surface are these new cells found?

G1, pialRemoving or adding a supernumerary limb bud speaks to a general mechanism to regulate the number of spinal motor neurons. How is this number regulated?

too many precursors, apoptosis prunes numbers

Ligands such as shh, RA, FGF, BMP and Wnt eventually control transcrption. Nane one receptor corresponding to any of these ligands.

patched, retinoic acid binding protein, receptor tyrosine kinase, receptor serine kinase, frixxled

R8 tells a cell to become R7. Name one famous protein in this cascade.

boss, sev, sos, ras, MAPK, (others)

What is the precursor of sensory ganglia, autonomic ganglia, adrenal medulla or melanocytes (depending on which factors are acting)?

neural crest

The optic vesicle, an outpocket of the diencephalon, eventually forms the retina. What do these induce the overlying ectoderm to form?

lens and anterior portion of eye

"The S stage occurs near the pial surface." Translate.

synthesis of DNA in the cell cycle (between mitoses) is when nuclei are on the outer part of the neural tube

In Weaver mutant mice, granule cells are missing, but it is the fault of what other cell type?

(Bergman) glia

Rotating the frog eye up-side-down gives opposite results if done in the adult vs. a few days before Harrison stage 28. What is the behavior in the adult after early rotation?

animal will flick its tongue (to catch an insect) in the correct direction

Filopodia protrude from what important structural specialization in axon path finding?

growth cone

What are integrins and cadherins used for in nervous system development?

contact guidance

What was changed in the adult spinal cord if a limb bud had been ablated earlier?

number of spinal motor neurons for that limb is lower

What is apoptosis and why is it so important in development?

programmed cell death. If too many cells are made, extras must be eliminated.

"Cytokines include trophic factors, hematopoietic factors and growth factors." In which category is NGF?

Despite ist name (nerve growth factor), it is a trophic factor.

The patched receptor acts in concert with the smoothened protein to mediate the response to what famous developmental ligand?

shh

Larvae of holometabolous insects have tissues determined to become adult structures. What are these called?

imaginal disks

Name something that neural crest gives rise to.

sensory ganglia, autonomic ganglia, adrenal, melanocytes

What process is agrin involved in in muscle cell development?

aggregation of ACh receptors

In the sevenless signal transduction cascade, what is the name of the small GTP binding protein?

ras

Contradicting Weiss's resonance principle, how did Sperry explain proper connection of ganglion cells to tectum?

neurobiotaxis directs axon tips to correct places in tectum

Why is bicoid referred to as a maternal gene?

the mRNA is transcribed in mother

What is the output neuron of the cerebellar cortex?

Purkinje

On what kind of a molecule would you find a domain such as a homeodomain?

protein

An antibody to nerve growth factor (NGF) causes what part of the nervous system to be lost?

sympathetic n.s.

In axon path-finding, what is the bulbous knob with extensions at the tip?

growth cone

Name a neurotrophin that uses the Trk ("track") receptor.

NGF (and others)

Shh and Wnt are two important secreted ligands used in developmental signalling. Tell me the receptor (for one of them).

patched frizzled

The word "pluripotent" is used in reference to what type of cell?

embryonic stem cells, neural precursors

How does Sperry's notion of "neurobiotaxis" explain the poor visual performance of a frog whose eye has been inverted?

upside down eyes axons grew to addresses in tectum as if the eye did not know it was upside down

The optic vesicle is an outpocket from the diencephalon. What does the optic vesicle induce in the overlying ectoderm?

lens

The structure that will eventually fold in to make the neural tube is called the neural plate. From what major embryonic layer is the neural plate partitioned?

ectoderm

What is it that gives rise to sensory and autonomic ganglia, adrenal neurosecretory precursors and melanocytes?

neural crest

"Receptor tyrosine kinase." What does that mean? Give an example in developmental signalling.

it is a membrane receptor protein with enzymatic activity to phosphorylate itself on tyrosine residues, sevenless

A sympathetic progenitor can either become cholinergic or adrenergic. What determines which pathway?

NGF vs CNTF

What is the difference in distribution of the nicotinic acetylcholine receptor in a muscle cell before vs. after a neuromuscular junction is formed?

before distributed, after under junction

What effect does transplantation of a supernumerary limb bud in the chick embryo have on spinal motor neurons?

extra ones formed

What is the difference in appearance of an explanted dorsal root ganglion with vs. without NGF in the medium?

with has neurites growing out

Ligands such as shh, RA, FGF, BMP and Wnt eventually control transcrption. Nane one receptor corresponding to any of these ligands.

patched, retinoic acid binding protein, receptor tyrosine kinase, receptor serine kinase, frixxled

R8 tells a cell to become R7. Name one famous protein in this cascade.

boss, sev, sos, ras, MAPK, (others)

What is the precursor of sensory ganglia, autonomic ganglia, adrenal medulla or melanocytes (depending on which factors are acting)?

neural crest

The optic vesicle, an outpocket of the diencephalon, eventually forms the retina. What do these induce the overlying ectoderm to form?

lens and anterior portion of eye

"The S stage occurs near the pial surface." Translate.

synthesis of DNA in the cell cycle (between mitoses) is when nuclei are on the outer part of the neural tube

In Weaver mutant mice, granule cells are missing, but it is the fault of what other cell type?

(Bergman) glia

Rotating the frog eye up-side-down gives opposite results if done in the adult vs. a few days before Harrison stage 28. What is the behavior in the adult after early rotation?

animal will flick its tongue (to catch an insect) in the correct direction

Filopodia protrude from what important structural specialization in axon path finding?

growth cone

What are integrins and cadherins used for in nervous system development?

contact guidance

What was changed in the adult spinal cord if a limb bud had been ablated earlier?

number of spinal motor neurons for that limb is lower

What is apoptosis and why is it so important in development?

programmed cell death. If too many cells are made, extras must be eliminated.

"Cytokines include trophic factors, hematopoietic factors and growth factors." In which category is NGF?

Despite ist name (nerve growth factor), it is a trophic factor.

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