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