Study Guide- Neural Development 2

 What surgical experiments indicate that the dorsal lip mesoderm is sufficient to specify neural identity in
neighboring ectoderm? What experiment shows it is necessary?
Sufficient: Scientists took cells from the dorsal lip of the blastopore from an albino embryo (the albino is so that
you can track these cells) and transplanted it into the ventral portion of the blastopore. What happens is that
the ventral cells adjacent to the transplanted dorsal lip cells become dorsal like (with neural cells being
developed), meaning that the transplanted cells are signaling to the nearby ventral cells to become dorsal.
Another important point is that the development of dorsal cells in the ventral region of the blastopore is coming
from ventral cells; that is the dorsal lip mesoderm is sufficient to specify neural identity in the neighboring
cells.

Necessary: For this, scientists can isolate the dorsal ectoderm (animal cap) and grow it in a petri dish ex vivo
without the dorsal lip or the rest of the blastopore. If you remove and grow the dorsal ectoderm in a petri dish
before the dorsal lip cells can migrate and form an adjacent mesoderm next to the dorsal ectoderm, the dorsal
ectoderm cells become epidermis cells. However, if you remove and grow the dorsal ectoderm in a petri dish
after the dorsal lip cells migrate and form an adjacent mesoderm (and presumably provides a signal to the dorsal
ectoderm), the dorsal ectoderm cells become neural tissue. This means that the dorsal lip cells which migrate and
become the dorsal mesoderm is necessary to provide some signal to specify neural identity in the neighboring
ectoderm.

 Outline the molecular mechanism by which the dorsal lip of the blastopore triggers neural development in nearby
ectoderm. What is the default state of ectodermal cells, and how is that shown by dissociation of the ectodermal
animal cap? Are the ectodermal cells signaling to each other and, if so, what is the signaling doing? What kind
of molecules are secreted by the dorsal lip cells, and how do they work?
The dorsal lip of the blastopore triggers neural development in the nearby ectoderm by producing an
inhibitor of an inhibitor: the ectoderm cells by default wants to become neurons but produce BMP (bone
morphogenetic protein) which inhibits neighboring ectodermal cells from becoming neurons. The dorsal lip
mesoderm cells secrete inhibitors of BMP, which allows the ectodermal cells to become epidermis cells.

Ectodermal cells of the animal cap signal to each other through BMP to inhibit their default neuronal
specification and cause them to become epidermis cells. This was done through a couple experiments:

1) if you take an intact animal cap (ectodermal cells) and grow them in a petri dish, they become epidermis.
However, if you dissociate the cells into a single cell suspension and grow them as individual cells without
making contact to each other in a dish, they become neurons. This means that there is some signaling molecule
that ectodermal cells are signaling to each other to inhibit their neuronal formation, which turns out to be BMP 4.
If you take the dissociated cells and add BMP4, the dissociated ectodermal cells now become epidermis.

2) you can also take an in tact animal cap without dissociating it, which normally forms an epidermis, and add
a dominant negative receptor for BMP (explained in the next study guide question), and the in tact animal
cap now becomes neurons. So, if you remove BMP from the in tact animal cap, you get neurons.

 What is a dominant negative receptor, and how does it work?

A dominant negative receptor is basically a receptor for a ligand that works to inhibit that ligand-receptor
signaling. Most receptors consist of dimers (consisting of two transmembrane proteins), and a dominant negative
receptor works by inserting a truncated form of one of the transmembrane proteins. The truncated protein is
missing the intracellular kinase domain, which doesn’t allow it to signal even when a ligand is bound. So how
this works is that a dimer can form between a normal transmembrane protein and a truncated protein to
form a non-functional receptor (since it’s missing an intracellular kinase domain for one of the proteins), which
makes a “decoy” receptor that is non-functional even when a ligand is bound. This is the “negative” part of the
dominant negative receptor. The receptor is also dominant because you insert so much of the truncated protein
that statistically it tends to form a dimer with the functional protein, “poisoning” the normal receptors in the sense
that you have a cell containing 100% normal dimers to having mostly non-functional dimers with the insertion of
a non-functional truncated protein. This also reduces the ligand signal by reducing the function of normal
receptors.
 What bioassay was used to discover Noggin? How did it work?
A bioassay screening mRNA was used to identify an mRNA that makes the Noggin protein, which is a protein
made by the dorsal lip mesoderm that is sufficient in inducing neural tissue in ventral cells. This was done by
“encouraging” an embryo to develop mostly dorsal cells by adding Lithium to the embryo, OR “encouraging”
an embryo to develop mostly ventral cells by using UV Light (blocks cytoplasmic determinants and causes
embryo cells to develop mostly ventral cells). If you take the mostly dorsalized embryo (from adding
Lithium), and extract the mRNA, and then add all the mRNA you extracted back into a ventralized embryo (by
UV light), you can now cause the ventralized embryo to develop dorsal cells. This means that the dorsal cells
are producing some mRNA that is sufficient to dorsalize ventral cells. Additionally, you can isolate mRNA
from the dorsal lip of the blastopore and add it to a ventralized embryo (UV light exposed) and have it develop
dorsal cells, again showing that there is some mRNA produced by the dorsal lip that is sufficient to dorsalize
ventral cells. You can then narrow what mRNA was doing this, and eventually get to Noggin.

Other experiments identified things like Chordin (can also dorsalize ventral cells) and Follistatin (inhibitor of
BMP signaling). This was done by screening proteins that are uniquely expressed by the dorsal lip of the
blastopore and seeing if any of these proteins can specify neuronal fate.
*Noggin, Chordin, and Follistatin all bind BMPs or the BMP receptor in extracellular space, blocking BMP from
binding to their receptors and block signaling in the ectoderm from turning them into epidermis and thus making
neurons.

 What surgical evidence suggests that anterior and posterior mesoderm are producing different signals? What are
some examples of secreted molecules that help change anterior-posterior identities in the nervous system?
Another series of transplantation studies were done to show that the anterior and posterior mesoderm are
producing different signals. In the pregastrula, you can transplant the dorsal lip, anterior mesoderm, (at
this stage the cells will eventually become anterior; remember the cells from the animal cap loop around the
dorsal lip and migrate anterior) to the ventral region and induces head growth. However, if you transplant the
dorsal lip later in development during the gastrula phase, posterior mesoderm, to the ventral embryo you get
growth of spinal cord. This means that the anterior and posterior mesoderms are producing different signals to
specify different central nervous system regions.
Some examples of secreted molecules that specify anterior-posterior identities include:
Posterior Cues: FGF, Wnts, Retinoic Acid
Anterior Cues: Inhibitors of BMPs and Wnts such as Cerberus

 What is the notochord? Is it neuronal, or mesodermal? What evidence suggests that it produces a ventralizing
signal, with what effect on the adjacent neural tube? What is thought to be the critical signaling protein notochord
produces, and how was it shown to be necessary?
The notochord is a structure that provides signaling molecules to specify the dorsal-ventral axis of the spinal
cord, and is dorsal mesodermal and not neuronal. If you experimentally remove the notochord early enough,
the spinal cord loses floor plate development and motor neurons, which are ventral. You can also move the
notochord and transplant it somewhere else, and you move the motor neurons and floor plate to the region
nearest to the notochord. So the notochord is necessary and sufficient to specify ventral identities in the
adjacent neural tube.
The critical signaling protein that the notochord produces is Sonic Hedgehog (Shh) that specifies ventral
identities. You can take neural tube cells and add Shh and cause the neural tube cells to express ventral genes,
showing that Shh is sufficient. You can then take a neural tube and grow it next to a notorchord, which will
induce ventral signaling genes in the neural tube because of Shh produced by the notochord. However, if you
add antiserum against Shh, the neural tube can no longer express ventral genes, showing that Shh is also
necessary.