Developmental Exam I

How can you birthdate a neuron?

add a molecule that gets added during replication or use antibodies to tag PCNA

tagging PCNA vs molecule replication

PCNA is only good short term and antibodies are species specific and molecule replication used to be radioactive

Lineage Analysis vs Birthdating

birthdating tags new neurons while lineage analysis provides a family tree

How does lineage analysis with a retrovirus work?

a cell is infected with a modified retrovirus which replenishes with division tagging new cells

Pros and Cons of retrovirus

a pro is that it contains a reporter gene but it cannot replicate

Retrovirus Examples

Horseradish peroxidase and B-Galactosidase

How does lineage analysis with Cre-LoxP work?

cre is an enzyme that causes recombination at a floxed DNA site

what are major challenges to studying gene expression?

* multiple time points to be studied
* Neuroplasticity
* diverse cell populations intermingle

what methods can find DNA anatomically?

Immunohistochemistry

What can you learn using immunohistochemistry?

where protein is expressed using primary and secondary antibodies

Pros and cons of immunohistochemistry

it provides spatial information but antibodies have to be made and are species specific

what methods can find DNA molecularly?

Western blot and Elisa

what can you learn using western blot?

the quantity of protein expressed

Pros and Cons of western blot

It comes in a large quantity but gives no spatial info and needs antibodies

How is Elisa used?

there is a 96 well plate lined with antibodies where proteins are read

What method can find mRNA anatomically?

In Situ Hybridization

what does ISH tell you?

where mRNA is expressed

Pros and Cons of In Situ Hybridization

It provides spatial info and has a wide range of probes but the probes are specific

What methods can find mRNA molecularly?

Northern Blot and qRT-PCR

What is northern blot used for?

to figure out if mRNA is present or not

Pros and Cons of Northern Blot

the cost is low and its fast, but there is no spatial info

what does qRT-PCR tell you

the specific quantity of mRNA

Pros and cons of qRT-PCR

it is fast and can measure a lot but there is no spatial info and it is hard to be specific

what is a primary antibody?

It finds a proteins

what is a secondary antibody?

it finds the primary and reveals it

Carnegie Stage 1

fertilization of the egg

Carnegie Stage 2

first cleavage through morula

Carnegie Stage 3

unimplanted blastocyte

what does the inner cell mass become?

the embryo

what does the outer cell mass become?

the placenta

when do the inner and outer cell mass form?

Carnegie Stage 3

Carnegie Stage 4

shedding of zona pellucida

Carnegie Stage 5

implantation is complete

Carnegie Stage 6

Gastrulation begins and the primitive steak appears

When do the 3 germ layers form?

Carnegie Stage 6

What are the 3 germ layers?

endoderm, mesoderm, ectoderm

Carnegie Stage 7

cell migration defining gastrulation

what does the endoderm turn into?

insides and organs

what does the mesoderm turn into?

muscles

what does the ectoderm turn into?

skin and nervous system

Carnegie Stage 8

appearance of neural plate and neural groove

Carnegie Stage 9

somites appear on either side of the neural groove

what are somites?

precursors of dermal tissue, muscles, vertebrae

Carnegie Stage 10

formation of neural tube

Carnegie Stage 11

more somites grow

Carnegie Stage 12

neural tube closes

Carnegie Stage 13

30 pair of somites

Carnegie Stage 14

rapid brain growth with definition of forebrain, hindbrain, and midbrain

Carnegie Stage 15

cerebral hemispheres can be discerned

what are the three primary brain vesicles?

prosencephalon, mesencephalon, and rhombencephalon

which of the primary brain vesicles subdivide further?

prosencephalon and rhombencephalon

what does the prosencephalon divide into?

the telencephalon and diencephalon

what does the rhombencephalon divide into?

metencephalon and myelencephalon

What does the telencephalon form?

cerebrum

what does the diencephalon form?

eye cups and thalamus

what does the metencephalon form?

pons and cerebrum

what does the mesencephalon form?

midbrain

what does the myelencephalon form?

medulla oblongata

What defects are caused by the failure to close the anterior/cranial neuropore?

iniencephaly, encephalocele, and anencephaly

characteristics of iiencephaly?

“stargazing” posture with a stillbirth or premature death

Characteristics of encephalocele?

herniated meninges and brain tissue

Characteristics of anencephaly

total or partial absence of brain

What failed to close in craniorachischisis?

middle portion

Characteristics of craniorachischisis?

anencephaly along with lesion on spinal cord with “stargazing” look

what failed to close in spina bifida?

posterior/caudal neuropore

Characteristics of spina bifida

herniation of meninges and spinal cord

mutation

change in DNA sequence

null mutation

complete failure to express protein coded by mutated gene

hypomorph

wild type gene expressed at a low level

wild type

standard, unmutated gene

strain

a reproductively isolated group of animals

inbred strain

over 20 consecutive generations of sibling mating to produce homozygosity

isogenic

strain where every individual is genetically identical

What is forward genetics?

identifying a gene then finding the phenotype

What is reverse genetics?

selecting a gene to then find a phenotype

Forward vs Reverse genetics

forward genetics has no bias of previous knowledge and can exploit spontaneous mutations while reverse genetics must inactivate a known gene and the mutation may not be visible

What committee oversees institutional use of animals

Institutional Animal Care and Use Committees (IACUC

Theiler Stage 1

fertilization

Theiler Stage 7

blastocyst sheds zona pellucida and implants

Theiler Stage 11

appearance of neural plate and neural groove

Theiler Stage 12

portion of neural tube closes

Theiler Stage 14

anterior portion of tube closes

Theiler Stage 15

posterior neuropore forms

Theiler Stage 16

posterior neuropore closes

Theiler Stage 18

rapid brain growth

Danio rerio stage 1

early blastula

Danio rerio stage 2

midblastula

Danio rerio stage 3

early gastrula

Danio rerio stage 4

midgastrula

Danio rerio stage 5

early segmentation stage

Danio rerio stage 6

mid-segmentation stage

Danio rerio stage 7

pharyngula stage

Drosophila melanogaster formation

embryo → 1st instar → 2nd instar → 3rd instar → prepupa → pupa

Bownes Stage 1-3

unusual pattern of cleavage

Bownes Stage 4

syncytial blastoderm

Bownes Stage 5

cellular blastoderm

Bownes Stage 6

gastrulation

Bownes Stage 7

germ band elongates

Bownes Stage 9

neuroblasts form

Bownes Stage 11

delamindation of neuroblasts