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Polarity and Segmentation
Chapter Two
Polarization
• Entire body plan is polarized– One end is different than the other
• Head vs. Tail– Anterior vs. Posterior
• Front vs. Back– Ventral vs. Dorsal
• Majority of neural tube = spinal cord• Anterior end = brain
• Vertebrate body is symmetrical but polarized (tail vs. head)
• How does polarization of the animal body originate during development?
• In vertebrates, the rostral portion of neural tube gives origin to brain structures
• Caudal (tail) areas of neural tube give origin to spinal cord
CNS SubdividedCNS = central nervous system• Spinal cord
– Derived from neural tube directly• Brain
– Specialized anterior section of neural tube• Brain has three primary divisions:
– Prosencephalon – forebrain– Mesencephalon – midbrain– Rhombencephalon – hindbrain
3 primary brain vesicles are further divided:
Prosencephalon1. Telencephalon:Cerebral hemispheres 2. Diencephalon:Thalamus, hypothalamus,
and optic vesiclesMesencephalon remainsRhombencephalon1. MetencephalonCerebellum2. Myelencephalon Medulla
• In Drosophila, development of the anterior portion of the nervous system also undergo a three-chamber pattern:
1) Protocerebrum2) Deutocerebrum3) Tritocerebrum
• The main difference here is that the ventral nerve cord is generated by delamination of epithelial cells that are fused together into interconnected ganglia
• So how do regional differences between rostral and caudal structures originate in vertebrates and insects?
Formation of the Anterior-Posterior Axis In Drosophila
• In Drosophila, the A-P axis is set by two molecules:
1) bicoid2) nanos
• These two molecules create an opposing gradient
• Opposing gradient separates Anterior from Posterior
• Triggers other genes to be on or off
Anterior Posterior
Anterior Posterior
A/P axis in Drosophila
Levels of development:1. Cytoplasm gradients of nanos and bicoid
1. Inherited from mother’s oocyte2. Gap genes3. “Pair rule” genes4. “Segment polarity” genes5. Homeotic genes
1. Trigger regional gene expression differences
A/P axis in Drosophila
Formation of the Anterior-Posterior Axis In Drosophila
Normal development of anterior region in nanos -/-
Anterior Posterior
Lack of anterior region in bicoid mutant
Development of anterior structures
following injection of bicoid protein in posterior region
Exact reverse for Nanos and posterior development
Once the A-P layout is defined, what factors determine the differentiation of each body
segment?
Now each segment must express unique genes in order to be different
In Drosophila, differentiation of each segment requires the expression of Hox homeobox genes
Homeobox genes are all transcription factors
Once A/P axis is laid out
Once the A-P layout is defined, what factors determine the differentiation of each body
segment?
Homeobox genes are arranged in a linear array on the chromosomeHomeobox genes at the 3’ end are expressed in more anterior locationsHomeobox genes control regional identity of body segment
Once the A-P layout is defined, what factors determine the differentiation of each body
segment?
Homeobox genes are conserved among animal species
Vertebrates:Have more hox genes
Complex interactions
Overlap of same functionStill exist in order 3’ to 5’
Homeobox genes
• Hox genes• Always transcription factors• Bind DNA directly:
– Through the homeobox domain• Activate the genes that directlycause specific regional identity• Deactivate other genes
Distaless is NOT a homeobox gene
• Insects have three pairs of legs• One pair on each thoracic segment• No legs on the abdominal segments• Distaless gene forms the legs• Distaless expression is suppressed in
abdominal segments of insect• By BT-X – a hox gene
– BT-X binds the DNA and repressed distaless
Example of Hox mutations:
wild type Antennapedia
Mutation in the antennapediacomplex result in the formation of a leg where an antenna was
supposed to exist
• XlHbox1 in Xenopus
Do Hox genes control vertebrate development as well?
Regional expression patternof XlHbox1
• Injection of an antibody against the XlHbox1 protein result in the enlargement of the hindbrain
Do Hox genes control vertebrate development as well?
Knock Out Mice
Specific genes deletion studies
Study the role of homeobox genes in segmental differentiation
Hox knockouts have allowed the study of regional differences in the developing
mice hindbrain
Segmentation of the hindbrain result in the formation of rhombomeres – similar to Drosophila’s segments
Rhombomere formation in the mouse is encoded by different combination of Hox homeobox genes.
Hox mutations affect the development of specific rhombomeres
Hox knockouts have allowed the study of regional differences in the developing
mice hindbrain
Deletion of Hoxa1 gene results in fusion of rhombomeres (R) 5 & 6 and reduction of R4
Deletion of Hoxb1 gene results the loss of motoneurons R5
Double mutants of Hoxb1 & Hoxa1 genes show a combined effect
Segmental Specification is Encoded by Combination of Factors
Segmental arrangement of the chick hindbrain rhombomeres (r1-r7) result in a steroptypic pattern of motoneuron localization in hindbrain
Early transcription factors, Eph family receptors, and homeobox genes establish the segmental specification of the hindbrain rhombomeres
Important to Note
• Vertebrate hindbrain segmentation• Occurs by exact same mechanism as
insect body segmentation• Express different hox genes
– Produce different regional identities• In insects get body segments• In vertebrates get different motor neurons
– In exactly to the correct brain region
Removing Hox genes:
Entire hindbrain looks like R1 segment
What controls Hox genes?
• Step backwards one• If Hox genes regulate all other genes• What regulates Hox genes?• In flies:
– Cytoplasmic gradients– Control Gap genes– Control Pair rule genes control Hox genes
• Same in vertebrates?
• Retinoic acid (RA) has been show to regulate Hox gene expression.
• RA crosses cell membrane and bind to cytoplasmic receptors.
• The RA-receptor complex can translocate to the nucleus and regulate gene expression after binding to RA response element (RARE)
What signal molecules pattern the Hox expression?
• RA levels are significantly higher in posterior regions of Xenopus embryos
• RA normally activates posterior identity
• Suppresses anterior identity
• Exposure of the developing embryo to RA results in malformations of anterior structures (head structures fail to develop)
There is a gradient of RA expression in the developing embryo
• Low RA – results in Hox genes normally expressed in the anterior portion
• High RA – results in Hox genes normally expressed in the posterior portion of the embryo
• Target deletion of RA receptors results in head structure formation
• RA normally regulates in more posterior regions
Retinoid Acid Controls Formation of A-P Axis and Hox expression
in vitro and in vivo
Heads vs. Tails?
• Spemann and others proved there were both head and tail organizers
• This meant that if you transplanted a small piece of tissue from head to anywhere it would still form into a head
• Same with piece of tail tissue• What are in these regions?
– Transcription factors that induce other genes
Heads vs. Tails?
• Nieuwkoop transplanted small piece of head tissue to different places along axis
• All pieces transformed into head tissue• However, if transplanted into caudal
regions – actually formed two tissue types:– Anterior and Posterior both developed
• New theory:– Activator = 1st signal– Transformer = 2nd signal
Activator-Transformer Hypothesis
Activator = gene that turns ectodermal cells into neural tissue
• Anterior is default state of neural tissueTransformer = gene that turns neural tissue
into posterior types• Posterior requires two signals:
– Neural positive– Posterior positive
Activator Genes
• Genes that induce neural tissue• Noggin, Chordin, Follistatin• All produce anterior structures• Therefore anterior must be default for
neural tissue• Remove these activator signals and get
NO neural tissue– Rather than posterior-like tissue
Transformer Genes
• Retinoic acid– Posteriorize embryos– Regulates hindbrain hox genes
• Wnt and beta-catenin– When wnt is inhibited a second head can form– Adding wnt posteriorizes neural tissue
• FGF– Induce posterior gene expression
• Blocking of BMP signal enables formation of neural tissue. In this case noggin, chordin, follistatin play the role of neural inducers that allow formation of anterior structures
• Formation of a retinoid acid-gradient enables polarization of the neural tissue. In this case RA is the transformer that promotes Hox gene expression and formation of caudal structures
The Activator-Transformer Hypothesis
Complexity
• Although anterior type seems to be “default”– Suppressing BMPs is necessary to form
anterior tissue• Suppressing BMPs is NOT sufficient to
form functional anterior tissue• If all you do is suppress BMPs you will
form anterior structures– Will not be fully functional or normal
Anterior Tissues
Anterior tissues require two signals:1. Inhibition of BMPs
1. Forms neural tissue2. Inhibition of wnt pathway
1. Anteriorizes the neural tissue completely• Inhibition of BMPs is necessary, but not
sufficient to form anterior structures• Inhibition of wnt pathway is necessary
but not sufficient
Role of wnt in Xenopus embryos
G = Suppressing BMPs and wnt pathwaysI = Suppressing BMPs alone
– Small head and cyclopia
Wnt inhibitors
• All expressed within organizer:• Cerberus
– Injection of Cerberus will cause ectopic head formation
• frzB– Injection forms larger than normal heads
• dkk1– Ectopic head formation
WntRemove wnt= extra head
Inhibit wnt= extra head
Remove wnt inhibitor= messed up head
Cooperation
• BMP inhibitors work together with wnt inhibitors to form head and brain
Wild Type
Double mutant:dkk and Noggin
FGF
• Third transformer is FGF• Both FGF and wnt signals suppress
expression of enzyme cyp26• cyp26 is an enzyme that breaks down
retinoic acid• Without cyp26 – RA builds up:
– Posteriorization of tissues• Exact mechanism/interaction of FGF and
wnt is unknown
Any Questions?
Read Chapter Two
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