Homeobox Genes Flashcards
The important question of “how do we develop our body plan” has been extensively studied in which two model organisms?
Drosophila Melanogaster (fruit fly) and Mus musculus (house moue)
One advantage of using the fruit fly is that they have a short generation time. How long does it take for the fruit fly to go from egg to adult?
14 days
One advantage of using the mouse is that they are very amenable to genetic manipulations such as
Gen knockouts and knock ins, and transgenics
Deleting a gene via embryonic stem (ES) cells
Gene knockout
Replacing one version of a gene with another via embryonic stem cells
Gene knockin
Adding extra copis of any gene
Transgenics
In early mouse development, the group of cells in the center of the blastocyst is called the
Inner cell mass (ICM)
Pluripotent for the embryo
ICM cells
Cells in the outer epithelium are committed to generate part of the
Placenta
Can be isolated and grown in cultures
ICM cells
These mice ICM cells retain their pluripotency in vitro and are called
Embryonic stem (ES) cells
Enable a mouse to be generated that has a specific DNA change that we want to study
The pluripotent ES cells
If you delete a gene in the ES cells then a mouse can be generated that contains all the genes in the genome except the specific one that was deleted. These mice are called
Kock-outs
If you introduce more subtle specific changes into the DNA such as replacing a wild type sequence with a specific mutation, then you get mice called
Knock-ins
The insertion of DNA into the genome. DNA is not deleted, rather, extra copies of a gene are added. Mice that undergo this process are called
Transgenics
How are transgenics made?
DNA injected into nuclei of fertilized egg
Mutations where one structure is replaced with another or is duplicated
Homeotic Mutations
Homeotic mutations were known to exist since the 1930’s, but the genes responsible for these defects were not known until the development of
DNA recombinant technology (1980’s)
Cloning of genes responsible for homeotic mutations was first achieved in
Drosophila
Sequence homology was observed between genes responsible for different homeotic mutations. This DNA sequence was termed the
Homeobox
The homeobox is 180 bp in length and encodes a protein called a homeobox transcription factor. The part of the protein that binds DNA is called the
Homeodomain
Encode transcription factors that typically initiate a cascade of gene expression necessary for the development of a body structure or cell type
Homeobox genes
Homeobox proteins are transcription factors that bind DNA in a sequence specific manner. All homeobox proteins bind
AT rich elements in promoters and enhancers
The homeodomain amino acid sequence is responsible for binding and recognizing the AT rich binding site found in many
Promoters and Enhancers
The homeodomain protein sequence is comprised of 3 alpha helices. Which helix makes direct contact with the DNA?
The 3rd helix
All homeobox transcription factors bind
AT rich DNA sequences
In vitro, homeobox proteins demonstrate little or no specificity. However, in vivo, homeodomain transcription factors form protein complexes to determine
Specificity (very important in vivo or legs would be arms)
Only makes up a small portion of the homeobox transcription factor
Homeodomian
Other proteins bind to different parts of homeobox transcription factors to modify function and help determine
Specificity
There are many genes that contain homeobox sequences. These genes are broken into which two classes?
- ) Hox genes
2. ) Genes that when mutated, do not result in structural change
Genes that when mutated result in homeotic transformations
Hox Genes
The second class of homeobox sequences are not physically
Clustered together
Control the patterning of cell types, and mutations in these genes often result in the loss or gain of parts of cell types
Second class genes
Clustered into 4 physically linked groups in humans
Hox genes
In the evolution from drosophilia to mouse to human, there is a consistent increase in the number of
Homeobox genes (i.e. from 1 to 4)
Function in patterning the body axis
Hox genes
Expressed in specific embryonic domains and function in the generation of that particular body part
Hox genes
Hox genes are positioned on the chromosome in the same order in which they are expressed. Where are the following genes expressed?
- ) Genes positioned 5’ in the Hox cluster
- ) Genes positioned 3’ in the Hox cluster
- ) Posterior domain
2. ) Anterior domain
How many Hox clusters are present in mice and humans?
4 (Hox a, b, c, and d)
Homologous genes between species
Orthologs
Homologous genes within a species
Paralogs
Expressed in the developing neural tube as well as the flanking somites
Mouse Hox genes
Body segments containing the same internal structures
-seen in embryonic mouse
Somites
Somite expression is always shifted
Posteriorly
Hox genes further 3’ have more anterior expression boundaries. This is called
Spacial co-linearity
3’ Hox genes are also expressed earlier in development than 5’ genes. This is called
Temporal co-linearity
Different groups of cells express distinct patterns of Hox genes depending on their position along the anterior-posterior (A-P) axis. This is called the
Hox code
Dictates the development of different structures along the A-P axis
Hox code
Hox genes are transcription factors, so they will determine which downstream genes get turned
On
These downstream genes will in turn determine the developmental structures that will be
Derived
Loss of function Hox mutations (knock-outs) lead to
Anterior transformations
Gain of function Hox mutations lead to
Posterior transformations
Have less severe phenotype than double or triple mutants
-an example of redundancy or overlapping function
Single mutants
Function as transcription factors so they either initiate or repress downstream gene expression
Hox genes
One function of Hoxa2 and Hoxb2 is to initiate gene expression responsible for the generation of
Cervical vertebrae C2
Hoxa3, Hoxb3 and Hoxd3 are expressed in a more posterior compartment and one of their functions is to initiate gene expression responsible for the generation of
Cervical vertebrae C3
Hoxa2, Hoxb2, Hoxa3, Hoxb3 and Hoxd3 are all co-expressed in the same set of cells along the A-P axis. What would happen if a deletion knocked out Hoxa3, b3, and d3?
A second C2 vertebrea would form instead of a C3
Initiates the expression of genes responsible for the generation of C4 vertebrae
Hoxd4
Every Hox gene works together with the Hox genes located
Downstream in the cluster
In a transgenic mouse with misexpression of Hoxd in a more anterior structure, we would see
2 C4 vertebrae and no C3
Hox gene expression extends as far anterior as the
Hindbrain
Formation of Drosophila head structures is controlled by a homeobox transcription factor called
-NOT part of the Hox cluster
Orthodenticle (Otd)
No hox genes are expressed in the
Anterior head structures
Mice and humans have 2 Otd analogs called
Otx1 and Otx2
Drosophila Otd is expressed in anterior blastocyst in a region of the embryo that will give rise to
Anterior brain structures
Drosophila null mutants for Otd result in embryos without
Head and brain structures
Mice with Otx1 knockouts survive embryogenesis, but have
Smaller brains
Results in lethality and the deletion of forebrain structures during early mouse development, which is similar to the Otd Drosophila phenotype
Otx2 knockout
These studies demonstrate that Otx or Otd transcription factors are essential for regulating gene expression necessary to generate
Anterior (head/forebrain) brain structures
Turn on a series of downstream genes, which then function together to generate head/forebrain structures
Otc and Otx1/2
Situated at the top of a transcriptional cascade and NO other transcription factor in the genome is capable of performing this function
Otd and Otx1/2
Overexpression of Otd or Otx results in a
Larger brain
When mouse Otx1 was expressed in drosophila in place of Otd, the result was
Larger brains (shows functional redundancy)
What hapened when fly Otd was inserted into a mouse in place of Otx1?
The brain size was equal to wild type
Fly Otd is functionally equivalent to
Mouse Otx1
Caused by diseases such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, progressive muscular atrophy or by injury to the spinal cord
Motor neuron damage
Can generate mature cell types in vitro
ES cells
Basic science has identified a cascade of transcription factors required for
Motor neuron generation
Researchers have used this knowledge to generate motor neurons in culture from
ES cells
A transcription factor code has been identified for generating embryonic stem cells. These genes can convert other cell types into ES cells. What are the genes?
OCT4 and NANOG (homeobox TF’s), SOX2, C-MYC, and KLF4
The forced expression of these genes in skin fibroblasts converts them to
Induced pluripotent stem cells (iPS cells)
Have been implicated in human disease
Homeobox genes
Important for patterning the vertebrate limbs
Hox genes
Mutations in HOXD13 in both humans and mice result in digit malformations known as
Synpolydactyly
Observed in individuals with hand malformations
Missense mutations in HOX13D (3rd alpha-helix)
Protein modeling studies indicated that these amino acid changes affect the 3-D configuration of the
Hox gene
Protein-DNA binding assays demonstrate that the the missense mutations affect
DNA binding
