Molecular Genetics of Pattern Formation II Flashcards
Fruit fly as experimental system
Short gen time, low cost, genetic screen, conserved pathways
House mouse as experimental sysetm
complex-more similar to humans
manipulable genetically
short generation time
kocnouts
delete any gene via embryonic stem cells
knckins
replace one version of gene with another by ES cells
transgeneics
add extra copies of any gene (no removing DNA)
ICM cells
Totipotent-retain totipotency in vitro-called ES cells
How to make knockout/knock in?
Homologous recomb
grw ES cells in vitro, gernate DNA construct with DNA change in lab, introduce construct to ES cells, select cells that underwent homo recombination
How to do transgeneics
get fert eggs from fallopian tubes
DNA injected into princely
DNA integrates randomly and then transmitted to offspring
Homeotic mutations tell us
that there are genes in genome that control patterning or germination of structures
Homeobox proteins facts
sequence of genes that are responsible for homeotic mutations
TFs form protein complexes in vivo to determine specificity
encodes protein domain called homeodomain
-recognize TAAT rich binding site in many promoters/enhacers
Homeobox genes encode Tf’s that result in cascade expression necessary for development of body structure
bind in sequence specific manner
How many protein domain in homeobox tfs
homeodomain is only one protein domain in homeobox tf
- -other proteins interact with nonhomeodomain region of TF to form protein complex that then regulate DNA binding sepcificty/trx specificty
- other proteins influence DNA binding specificity by protein protein interaction in vivo and trx activity of homeobox domain
2 classes of homeobox tfs
hoxgenes-mutation results in homeotic transformations
homeobox tf-play role in dev of structures and particular cell types
consistent expansion in number of homeobox gnes
true in evolution from fly to mouse/human
Hox genes
pattern body axis
expressed in specific embryonic domains and function in generation of that particular
positioned along chromosome in same order in which they are expressed along anterior-posterior axis in developing fly
conserved
hox clusters
4 present in mouse/human
conservation and expansion in number-evolution
orthologs vs paralogs
homologous gene between species
homologous gene within speices
P-A axis is along what DNA directionality
5’ to 3’…?
sclerotome
myotome
dermatome
skelton
skeletal muscles
dermis
Spatial colinearly
genes furter 3’ have more anterior expression boundaries
temperotal colinearilty
3; hx genes are expressed earlier in development than 5’ gene
Hox code
different groups of cells express distinct patters of ox genes-depending on thier position along the anterior-posterior axis
knockouts of hox genes
anterior transpformations
-single mutants aren’t as bad as double
gain of function hox mutations
lead to posterior transformations
-too many posterior hox genes expressed in anterior
transcriptional mech regulating hox
all ox coexpressed along same set of cells along A-P axis
- each HOX gene sits in specific gap
- if misexpresseed in another gap-it would make its gene there
Formation of head
controlled by homeobox tf OTD
-otd expressed in anterior blastocyst-region of embryo that will give rise to anterior brain structures
in mouse/human otx1/2
null otd, otx1-/-, otx2-/-
otd-/-=no anterior brain region develop
otx1-brain structures smaller
otx2-early CNS not formed
not having 1 is better than not having 2
sufficient and ncessary for brain creation
-on top of transitional cascade
Ectopic expression
misimpression of old or txx in non neural tissues will generate neural structures
how redudant is otx1 and otd?
functiaally equiblivalent-resuce otx-/- genotype
-otd can regulate same set of downstream genes as otx
how are motor neurons genreated
cascade of homeobox tfs
- allow us to garnet motor neurons in culture-rescue some disease phenotypes
- these motor neurons are created by neuronal stem cells that a rea create though cascade of homeobox tfs
what is tf code for embryonic stem cells
oct, sox, nanog-covnert other cells to iPS cells
synpolydactily and hox genes
hox genes are important in patterning of limb
- mutation in hoxd13=limb patterning malformations
- missense mutations affected 3d configuration of ox gene
