Embryology 1 :The Genetics of Anterior-

Posterior Axis Determination

Embryos develop three axes: the anterior-Embryos develop three axes: the anterior-posterior axis (from head to tail), the posterior axis (from head to tail), the dorsal-ventral axis (from back to belly) dorsal-ventral axis (from back to belly) and the right-left axisand the right-left axis

Structures show a specific localization Structures show a specific localization (neural tube is a dorsal structure in the (neural tube is a dorsal structure in the embryo and the heart is on the left side of embryo and the heart is on the left side of an adult body)an adult body)

The specification of axes occurs The specification of axes occurs early early during embryonic developmentduring embryonic development

Major Axes in Body Plan Development

The specification of early embryonic cells is The specification of early embryonic cells is due to cytoplasmic determinants stored in due to cytoplasmic determinants stored in the oocytethe oocyte

The cell membranes that form during The cell membranes that form during cleavage establish the region of cytoplasmic cleavage establish the region of cytoplasmic determinants that direct different gene determinants that direct different gene expression in every blastomereexpression in every blastomere

Major Axes in Body Plan Development

DrosophilaDrosophila is easy to breed, cheap to rear is easy to breed, cheap to rear in the labin the lab

It is hardy and prolific (genetic studies are It is hardy and prolific (genetic studies are possible)possible)

A genome-wide approach has identified A genome-wide approach has identified the molecules for body plan developmentthe molecules for body plan development

Drosophila as a Model for Molecular Embryology

A/P Polarity in the Adult

■ The body comprises three thoracic and eight abdominal segments

■ Every segment is specific and has different appendages: T1 has only legs, T2 legs and wings, T3 legs and halteres

■ The body is organized accordingly to a “plan”

Drosophila Early Embryonic Development

■ The zygotic nucleus divides several times without formation of cell membranes■ The specification of the A/P and D/V axes is accomplished by interaction of molecules in the same single multinucleated cell

How do we get from an homogenous egg to a body that is a collection

of different segments?

The Question

The determination of the A/P axis is due The determination of the A/P axis is due to a cascade of interacting genesto a cascade of interacting genes

These genes act sequentially to first These genes act sequentially to first divide the embryo in several segmentsdivide the embryo in several segments

Segments acquire then their specific Segments acquire then their specific identityidentity

General Principles

Cascade of Genes Determining A/P AxisMaternal genes

Hunchback

Gap genes

Pair-rule

Segment polarity Homeotic genes

Bicoid and Hunchback at the Anterior TipOvary

Nurse Cells

Oocyte

Bicoid mRNA

Bicoid Protein

Embryo

A P

In the mother ovary the bicoid mRNA produced by accessory cells, the nurse cells, is deposited in the oocyte and it is tethered to the anterior tip by microtubules

The bicoid mRNA is translated into the corresponding protein which forms a gradient with the highest concentration at the anterior tip

The same gradient is also created for hunchback, another anterior determinant

Nanos and Caudal at the Posterior End

A P

Nanos protein

Similarly, nanos mRNA is given to the egg by the mother and it is bound to the posterior region of the unfertilized egg by interaction with the cytoskeletonA gradient for the corresponding protein is created as reported in the picture, with the highest concentration at the posterior endA similar gradient is also created for Caudal

Early A/P Determinants

Four gradients are present: an A/P gradient for Bicoid and Hunchback and a P/A gradient for Nanos a Caudal

All these proteins function as transcription factors that can activate the expression of the following genes in the cascade, the gap genes

Positional information has been Positional information has been generated: the presence of Bicoid and generated: the presence of Bicoid and Hunchback “label” the anterior end, Hunchback “label” the anterior end, Nanos and Caudal the posterior oneNanos and Caudal the posterior one

Embryos defective (mutant) for Bicoid Embryos defective (mutant) for Bicoid lack the anterior structures while lack the anterior structures while embryos mutant for Nanos lack the embryos mutant for Nanos lack the posterior partposterior part

Summary

THE GAP GENES

■The Gap Genes are expressed into broad regions of the embryo

■ Mutations in the gap genes produce embryos lacking a series of contiguous segments corresponding to the segments where the gap gene is expressed

giant

Kruppel

Knirps

How do we get from an embryo with smooth gradient of proteins to an embryo characterized

by proteins expressed in broad stripes?

The Question

Regulation of Gap Gene Expression

Stripe I requires high level of Hb protein

Stripe II requires high level of Caudal

Kr requires low levels of Hb

Stripe II requires low levels of Caudal

II

I II

The Pair-Rule Genes

The pair rule genes are expressed in a zebra-like pattern dividing the embryo in 15 sub-regions where a vertical band of nuclei express the gene and the next one does not

The stripe 2 of eve is repressed by both high levels of giant and Kruppel

Stripe 2

Even-skipped

The expression of eve stripe II is limited to the “valley” region between high levels of giant and Kruppel proteins since both are repressors

Bicoid and Hunchback activates the expression of eve in the same stripe

Regulation of eve stripe 2

We are half-the way done!

Drosophila Early Embryonic Development

■ The zygotic nucleus divides several times without formation of cell membranes■ The specification of the A/P and D/V axes is accomplished by interaction of molecules in the same single multinucleated cell

The Segment Polarity Genes

■ The segment polarity genes reinforce and maintain the periodicity generated by the pair-rule genes

■ They are expressed in 14 stripes along the A/P axis ■ Their expression is regulated by the pair-rule genes

Regulation of Segm-Polarity by Eve, Ftz

Fushi-tarazu (Ftz) and even-skipped (eve) are expressed in close, non overlapping regionsHigh levels of Ftz and Eve induce the expression of engrailed (en), a Segment-polarity gene expressed in 14 stripesConversely Eve and Ftz inhibit wingless (wg). The expression of wg will be confined in stripes running between the expression of Ftz and Eve where there is no Ftz or Eve

Maintenance of en and wg Expression by Reciprocal Interaction

Wg is a secreted protein. It binds to wg receptor on the adjacent en cells and it activates en.

En is a transcription factor and it activates the expression of hedgehog (Hh).

Hh is a secreted protein that binds to its own receptor on wg expressing cells and it activates the expression of wg.

En and wg establish a polarity in every segment

Homeotic genes

Colinearity between the position of the gene on the chromosome and the sequence of the segment they specify: Scr specifies T1 and precedes on the chromosome Antp that specifies T2Every homeotic gene represses the expression of the previous gene in the segment they specify. Ubx identifies T3 and represses Antp in T3

When Something Goes Wrong…

In Ubx mutants T3 becomes T2Ectopic expression of Antp in the head induces the formation of legs attached to the head

Normal fly head

Extra legs

Mammals and Drosophila

Every homeotic gene in Drosophila has its homolog in mammals (Scr corresponds to a5, b5 and c5 but the three genes in mammals have the same function as Scr in Drosophila)

Colinearity is also seen in mammals (panel B)

When Something Goes Wrong…

Lumbar vertebra

Thoracic vertebra

Extra rib

The function of Homeotic genes in mammals is the same as in flies: the KO of Hoxc8 in mouse causes an homeotic transformation: the first lumbar vertebra becomes a rib. A rib is associated with the thoracic vertebra anterior to it

RA can become a teratogen if present in large RA can become a teratogen if present in large amounts or at particular times during amounts or at particular times during developmentdevelopment

It is a secreted molecule involved in A/P axis It is a secreted molecule involved in A/P axis formation in mammals and in forming the jawsformation in mammals and in forming the jaws

It affects Hox gene expression in A/P axis It affects Hox gene expression in A/P axis determination and it inhibits neural crest cell determination and it inhibits neural crest cell migration from the cranial region of the neural migration from the cranial region of the neural tubetube

Retinoic acid as a teratogen

The role of cell-adhesion molecules in creating boundaries

between tissues

Cells do not sort randomly

Cells from the neural plate and from the epidermis were dissociated in alkaline solution. When cells were mixed together, they re-aggregated and they became spatially segregated: the neural cells are inside while the epidermal cells stay at the periphery

Boundaries between tissues can be created Boundaries between tissues can be created by different cell types having both different by different cell types having both different types and different amounts of cell-adhesion types and different amounts of cell-adhesion moleculesmolecules

The most common cell-adhesion molecules The most common cell-adhesion molecules are cadherinsare cadherins

Cadherins establish and maintain Cadherins establish and maintain intercellular connections and they are crucial intercellular connections and they are crucial in the spatial segregation of cell typesin the spatial segregation of cell types

How boundaries between tissues are established

Cadherins

Cadherins are Ca-dependent adhesion molecules

They are anchored to the cell by a complex of proteins called catenins

Catenins interact with actin cytoskeletonThey have an adhesive recognition site to bind to similar cadherins

Cadherins join cells together by binding to the same type of cadherins on another cell (homophilic interaction)

Cadherins

Cells with E-cadherins (Epidermal cells) stick together and they will sort out from cells containing N-cadherins (Neural Cells) in their membranes

Cadherins during gastrulation

During gastrulation the presumptive neural tube expresses N-cadherins while the presumptive epidermis expresses E-cadherins

These tissues separate: the cells expressing N-cadherins invaginate to form the neural tube while the cells expressing E-cadherins will form the epidermis

If the epidermis is experimentally manipulated to inactivate the E-cadherins the cells will not hold together. If the N-cadherins are inactivated in the cells of the presumptive neural tube, the neural tube will not form

EM consists of macromolecules secreted EM consists of macromolecules secreted by cells into their immediate environmentby cells into their immediate environment

Cell adhesion and cell migration is Cell adhesion and cell migration is mediated by the EMmediated by the EM

EM is made up of proteoglycans, collagen EM is made up of proteoglycans, collagen and specialized glycoproteins such as and specialized glycoproteins such as fibronectin and laminin fibronectin and laminin

The Extracellular Matrix as a Source of Developmental Signals

FN functions as an adhesion molecule linking FN functions as an adhesion molecule linking cells to one another or to other molecules cells to one another or to other molecules (collagen and proteoglycans)(collagen and proteoglycans)

FN has an important role in cell migrationFN has an important role in cell migration The roads over which migrating cells travel are The roads over which migrating cells travel are

paved with FNpaved with FN FN leads germs cells to the gonads and heart FN leads germs cells to the gonads and heart

cells to the midline of the embryocells to the midline of the embryo Cells expressing integrins can bind adhesion Cells expressing integrins can bind adhesion

moleculesmolecules

Fibronectin

INTEGRINSOn the extracellular side integrins bind to the sequence Arg-Gly-Asp found in adhesion molecules including fibronectins

On the intracellular side they bind Vinculin and a-Actinin, these proteins bind to Actin filaments

This dual binding allow cells to move by contracting Actin filaments against the EM