L2: Polarity and regional identity: generating neuronal diversity

What this lecture is about, now that we know how the neurogenic region emerges during embryonic development

• examine how this region is further polarized

• and regionalized

• leading to the generation of an great diversity of neuronal cell types for developed animals

What does the neuroectoderm anertior vs posterior half develop into?

• Anterior→ brain and relative strucutres (eyes)

• Posterior→ spinal cord (vertebreates) or nerve cord (insects and invertebrates) and complemtns of motor neurons for coordinating muscles groups in locomotion

How are these regional divisions made in vertebrates vs invertebrates: Drosophila

1. Neuroectoderm delaminate into neurolast cells

2. Stereotyped sequence of asymmetric cell divisions

3. give rise to population of neurons but in subdivisions

   • Anterior→ brain, in subdivisions: protocerebrum, deuterocerebrum, tritocerebrum

   • Posterior→ sub-oesophageal ganglion (homolgous to vertebrate brain stem) and (more posteriorly)→ thoracic and abdominal segments of the nerve cord (homoglous to spinal cord)

How are these regional divisions made in vertebrates vs invertebrates: Mouse

1. Neuroectoderm invaginates

2. forms neural tube alongside the notochord (a mesodermal strucutre)

3. Anterior neural tube→ brain, divided into

   • Prosencephalon (telecephalon and diencephalon)

   • mesencephalon

   • metencephalon

4. Posterior neural tube→ spinal cord→ further subdivided

Main difference between vert and invert

• Inverts→ neuroblasts→ specialised neurons

• Verts→ invagination first and then neural tube→ specialised neurons

From both of these sequences, the next question to ask is

• How does each neuroblast/progenitor of the neural tube know which neuronal cell tpye and neural strucutures to develop?

• how do they adapot a parituclar neural identiy and start expressing genes needed for developing a parituclar neural strucutre?

• We will look at

  • Anterior-Posterior axis

  • Dorsal-Ventral

A-P axis→ Forms a kind of cascade of TF differential distributions: First Wave

Maternal mRNA

• The egg itself is already polarised

• differential distribution of maternal mRNA

• these are translated into transciption factors

Why are TFs often referred to as morphogens

• when applied experimentally

• they can morph a part of the embryo into a particular tissue shape or type

A-P axis→ Forms a kind of cascade of TF differential distributions: First Wave→ How does the mRNA gradient activate different TFs

• Promoter of each TF gene is differentially sensitive to the concentration of activating TFs

• so the differential concentration of mRNA along the A-P axis will transcribe different TFs along when enough amount of activating TF protein is present

• But→ this ia a continuous gradient→ it needs to be binarissez so that we have switching on and off of the TF genes

How is the continuous gradient binarized

French Flag Model

• continuous gradient binarized into regions

• boundaries are defined by concentration threshold of each subsequent TF gene promoter to be activated

A-P axis→ Forms a kind of cascade of TF differential distributions: Second Wave

Gap genes

• Expression defines of large chunks of the A-P axis of the embryo

• Experimental knock out→ removes substational and continuous portion of body regions

  • visible morphologically towards the end of embryonic development

  • hence the name ‘gap’

• They are TFs themselves→ control the activation of the next wave…

A-P axis→ Forms a kind of cascade of TF differential distributions: Third Wave

Pair-rule genes

• expressed in alternaing pairs of body segments

A-P axis→ Forms a kind of cascade of TF differential distributions: Fourth Wave

• the combination of all or many of the TFs above control the expression of

  1. Segment polarity genes→ expressed in a narrow band of cells of every body segment

  2. Homeotic genes→ Hox genes are the most famous among the homeostic genes

What are Intermezzo Homeotic genes

• genes that drive homeosis

  • → the transformation of one organ into another

Famous examples from the history of homeotic genes

1. Ultrabithorax (Ubx)→ mutant in Drosophila= 2 sets of wings instead of 1

2. eyeless (ey) or vert homolog Pax6→ head and eye development and its induction ectopically

   • e.g tips of a fly leg or antennae

Hox genes features

• key actos in patterning of A-P body axis

• exist in all-freeliving animals

• Sequential order of Hox genes corresponds to the region of expression along A-P axis

  • 3’ to 5’ A→P

• Individual Hox genes are homolgous across animals and play similar toles in development

Experiments showing Hox genes conservation

• DNA sequence for a specific Hox gene in a mammal can rescue the function of its homologous gene in Drosophila when knocked into its place

Why are there four clusters of Hox genes in vertebrate genome

• experienced two round of whole-genome duplication

• duplication easrly in the evolution of the chordates

But why have we mainainted the 4 clusters?

• Clusters become specialised

• now need them for limbs→ patterning along limb axis

What are the various Hox gene clusters called in vertebrates

• Hoxa

• Hoxb

• Hoxc

• Hoxd

→ Each gene in the cluster gets a numeric suffix

Hox gene clusters in invertebrates?→ Drosophila

• Its only Hox gene cluster split into two throughout evolution:

  1. Antennapedia complex

  2. Bithorax complex

names reflect each cluster contains the homonious Hox gene

name itself derives from description of mutant phenotype

Role of Hox genes in patterning the A-P axis of the neural tube: (focusing on the mammalian hind brain)→ how does it work

• Hox gene knockout shows

  • single gene can ultimately control the positional identiity and peculiarities of an organ or body region

Quick summary of the regions of the hindbrain

Subdivided into 8 Rhombomeres

• each is a repeated subdivision→ a segment

• distinct cranial nerves emerge from

  • each rhombomere

  • or

  • a combination of one or two adjacent rhombomeres

How do Hox genes help form these specific rhomobmeres with their specific cranial nerves

• Genes from clusters Hoxa, Hoxb and Hoxd are expressed in particular pattern across rhombomeres

• Each rhombomere has a unique combination of Hox genes expressed

  • each code can be shown using Hox gene knockouts to observe which rhombomeres have changed

Examples of these codes

1. Hoxa→ expressed in r4

   • knowck out= loss of r4 and r5

   • abducens cranial nerve which normally emerges from r5 is lost entirely

   • facial nerve, from r4 and r5 neurons→ stronly affected

   • Cranial nerves from the other intact rhombomeres are themselves also intact

Why do we know Hox genes caudalize

• Hox gene knockouts of whole Hox gene clusters show that all posterior segments resemble most anterior segments of the body

Example of experiment of this 1

Beetle→ Tribolium

• removal of entire Hox gene cluster

• → all larval segments resemble the first, most anterior segment

Example of this 2

Zebrafish

• removal of whole pbx cluster (a group of Hox genes expressed i nthe hindbrain and its rhombomeres)

• result→ entire region normally showing the rhombomeres→ now acquires the characteristics of the first rhombomere

Therefore these 2 experiments show what about Hox genes

• they causalize (posteriorize) the animal

  • the absence results in an otherwise posterior body region

  • presenting the identity of more anterior region

What is the 3-step model of nervous system induction and patterning

1. First signal→ activates (neuralizes) the gastrula’s ectoderm→ neruoectoderm

2. Second signal→ stabilizes neural fate of a region of neuroectoderm

   • defines anterior (forebrain) and posterior (the rest)

3. Third signal→Hox genes→ transforms (caudalizes) a region of the neural tube

   • details the subregions→ midbrain, hindbrain and spinal cord

But how is the Hox gene expression itself regulated along the A-P axis? (2 main facotrs)

1. Retinoic acid (RA) gradient

2. FGF gradient (Fibroblast growth factor)

Where is RA released from

• the notochord→ a mesodermal structure

Distribution of RA release along the notochord

• Anterior→ little

• Increases more and more…

• Posterior→ highest level

What does RA do

1. small molecule→ diffuses into the developing neural tube

2. crosses cytoplasmic membranes

3. binds to an intracellular receptor (RAR)

4. activates Hox gene expression

How does the gradient cause different Hox genes to be expressed

• Different Hox gene promotoers are activated by different concnetrations of RA

• Experimental evidence:

  • High RA→ loss of anterior strucutres

    • shows→ high levels drive expression of Hox genes for posterior identity

  • Low RA-. permits expression of anterior identity

• Each Hox gene is activate at a different level of RA concentration→ therefore each region of the neural tube along the A-P axis will express one or more Hox genes

• The combinatorial code further assists in narrowing down the identity of each neural tube region in the A-P axis

What is FGF where where expressed

• secreted by the lateral mesoderm

  • (compared to RA emitted by the notochord , ventral to the neural tube)

FGF gradient what it does

• along the A-P axis

• role in regulating Hox gene expression in a dose-dependent manner

• combination further refines the posistional identity of every A-P region of the nerual tube

  • by differentially driving gene expression

Dorsal-Ventral axis is also patterned by→ protein gradients: Early development

Early in nerual tube development , when it invaginates

• notochord already expresses Shh (sonic hedgehog)

what does Shh do

• induces the formation of the floorplate

What is the floorplate

• the most ventral region of the neural tube

• it subsequentally releases Shh itself

So with the initial release and the induced release from floor plate of Shh

• Shh forms a gradiet from ventral upwards

Further in development→ once the neural tube closes what happens

• Most dorsal part forms the roof plate

What does the roofplate do

• secrets BMP and Wnt

• in a D-V gradient→ Dorsal down

Overall summary of D-V patterning in vertebrates neural tube

1. Neural tube invaginates alongside the notochrod

2. Notochord Shh induces floorplate

3. epidermal Bmp induces roof plate

4. Roofplate starts releasing Bmp 9autonomously

5. floorplate starts releasing Shh autonomsouly

6. Opposing gradient of Bmp and Shh confer unqiues positional identity to cells distriuted along the D-V axis

Overall we have a combination of D-V gradients

• Shh→ Ventral up

• BMP and Wnt→ Dorsal down

What this does:

• gradient specifies a uniques D-V positional identity for differentiating neurons of the neural tube

In addition, what does the interaction between the dorsal part of the nerual tube and epidermis cause (using the BMPs)

• induces differentiation and subsequent migration of the neural crest cells

What do these neural crest cells do

Differentiate into numerous cell tpyes:

• epidermal pigment cells

• sensory ganglia

• endocrine cells

• etc

How do sympathetic vs parasympathetic ganglia arise from the neural crest

• Sympathetic→ from the neural crest of the trunk but not the head

  • e.g posterior to rhombomere 4

• Parasympathetic→ arise from neural crest of the head (but not the trunk)

Experimental embryology and the D-V axis: role of the notochrod in inducing the floorplate formation has been determined

Procedure: ectopic tissue grafts

results:

• Notochord removal→ prevents formation of floorplate →necessity

• Notochord addition on the lateral side→ ectoptic floorplate→ sufficienty

  • complete with correspndinng complement of ventral motor neurons

Experient 2: deafult D-V identity of spinal cord studied with tissue explants

Procedure: tissue explant alone or with added Shh, compared to in vivo

Results:

• In vivo→ dorsal half of spinal cord expresses Pax7 gene

  • bottom third expressed Olig2 and Nkx2.2 genes

• Cultivated intermediate part of spinal chord alone→ neurons express dorsal marker genes→ e.g Pax7

• Addition of Ash→ shift identity to ventral→ observing then the expression of Olig2 and Nkx2.2 genes

Moto neuron diversification in the spial cord along the D-V axis

• DV BMP ad Shh gradients

• each row of cells receive a different combination of BMP and Shh morphogen concentraions

• neuron differentiation into different morot neuron types is visualized by labeling for post-mitotic transciption factors TFs

• each moto neuron specializes in targeting different muscle groups

D-V patterning genes are evolutionarily ancient→ how

Similar how Hox patterns work in mouse vs Drosophila (sp both A-P axis and D-V axis are conserved)

Examples of homolgous:

• Drosophila→ Msh, Ind, Vnd

  • Expressed in the neuroectoderm from droal to ventral

• Mouse→ Msx, Gsh1, Nkx2.2

  • expressed in neurla tube dorsal to ventral

(How these genes are really homolgous even though look like they are being expressed in the oppsoite direction)

Must consider the Dorsal-ventral inversion in vertebrates

• Neural tube is invaginated

• therefore→ bottom of the neural tube is embryonically the most dorsal part of the neuroectoderm

• the invagination of the neural tube reverses the relative position of the regions expressing these genes

  • therefore→ match with the absolute position of their homologues i nthe fly

• recall: invertebrates are D-V inverted relative to vertebrates

Therefore: genes and their spatial expression patterns are conserved between vertebrates and invertebrates

Overall

• D-V gradient along the spinal cord generate great diversity of motor neurons

  • as detected by the unique expression of myriad additional genes specific of each neuronal cell type

• Together with A-P axis partiationing

  • each spatial location of the neural tube acquired a unique identity needed for development of the complete organism