Signals in frog embryos How can we identify developmental signaling pathways? How do other vertebrate embryos develop?

Signals in frog embryos

How can we identify developmental signaling pathways?

How do other vertebrate embryos develop?

Developmental biology jargon

Cell fate – the normal future identity of a cell (muscle, skin, etc.). A fate map represents future cell fates.

Specification – the process by which a cell acquires a particular fate. Specified cells could still switch to some other fate in response to extracellular signals. A specification map represents the identities that cells have acquired at a particular stage.

Determination – the process by which cells fix their fate stably. Determined cells do not change fate even if they receive new signals.

Differentiation – the process by which cells actually change to take on a particular identity (e.g. express muscle-specific genes).

~64-cell stage late blastula gastrulation

Mesoderm induction and patterning

What experimental results demonstrate that a particular signal induces a response?

What experimental results demonstrate that a particular signal induces a response?

Location – The signal must be present in the right place and at the right time.

Sufficiency – The signal must be able to induce the response (perhaps outside normal context).

Necessity – When the signal is removed or blocked, the response is also blocked.

Using these logical criteria to discover and test signals that might regulate a response.

Location – Find molecules that are present in the right place and at the right time.

Separate mRNA or proteins from different places, look for differences.

Sufficiency – Identify potential signaling molecules that can induce the response (perhaps outside its normal context).

Inject mRNAs or proteins corresponding to putative signals. Use gain-of-function mutations or transgenes.

Necessity – Remove or block the signal, test whether the response still occurs or not.

Loss-of-function mutationsInhibitors of particular pathwaysRNA interference

~64-cell stage late blastula gastrulation

Mesoderm induction and patterning

Vg1 mRNA

TGF- receptors activate gene regulatory proteins at the membrane

The TGF- family includesVg1, nodal, Xnr, activin, BMPs.

Some of these are localized to the vegetal region.

The TGF- family includesVg1, nodal, Xnr, activin, BMPs.

Some of these are localized to the vegetal region.

Maternal VegT (transcription factor, vegetally localized) activates zygotic Xnr (Xenopus Nodal-related) genes on vegetal side.

(VegT also specifies endoderm.)

Activin can induce different mesodermal cell types at different concentrations

Activin can induce different genes at different concentrations

General mesoderm

Dorsal mesoderm (organizer)

TGF- receptors activate gene regulatory proteins at the membrane

The TGF- family includesVg1, nodal, Xnr, activin, BMPs.

Some of these are localized to the vegetal region.

Dominant negative activin receptor

Another important pathway in inducing dorsal mesoderm-catenin immunolocalizations:

dorsal side ventral side

-catenin is a transcription factor

Wnt signaling pathway

-

+ -

-

-

-

-

+

+

+

+

+

Inject dominant-negative GSK3 protein at 2-cell stage

Current model of frog mesoderm induction

(=Xnr)

~64-cell stage late blastula gastrulation

Mesoderm induction and patterning

noggin expression in blastula

Dorsal side

Noggin protein injection rescues UV-irradiated embryos

[noggin]

none

low

high

medium

very high

noggin expression in dorsal mesoderm

(also acts at later stages after gastrulation has begun)

Model for mesoderm patterning

Antagonize BMP-4

Antagonizes Xwnt-8

TGF- receptors activate gene regulatory proteins at the membrane

The TGF- family includesVg1, nodal, Xnr, activin, BMPs.

BMP4 acts in ventral mesoderm.

~64-cell stage late blastula gastrulation

Mesoderm identities

Brachyury expression in blastula

Maternal patterning signals set up zygotic gene expression

Brachyury and Goosecoid are transcription factors

Activin can induce different genes at different concentrations

General mesoderm

Dorsal mesoderm (organizer)

Injection of goosecoid mRNA into ventral vegetal cells of 16-cell embryo

Control embryos (not injected)

How do other embryos develop?

Chick gastrulation:the squashed frog model

Chick gastrulation (equivalent to organizer in frogs)