Principles of Experimental Embryology

Biology 4361 – Developmental Biology

September 19, 2006

Major Research Questions

How do forces outside the embryo affect its development? (Environmental Developmental Biology)

How do forces within the embryo cause the differentiation of cells?(Specification, Determination, Committment)

How do cells organize themselves into tissues and organs? (Morphogenesis and Cell Adhesion)

The Embryonic Environment

What is the “embryonic environment”?

Internal influences (e.g. intrauterine):

- chemicals (e.g. maternal hormones, caffeine, nicotine)

- competitors (e.g. litter-mates)

Environmental regulation pathway:

- external stimulation triggers signaling event in embryo

- signal stimulates an embryonic pathway, usually hormonal, that changes the developmental pathway.

Organisms are connected to their environment

External influences:

- light

- temperature

- humidity

- predators

- competitors

- intraspecific signals

Environmental Regulation of Development

Light, temperature & moisture = environmental signals that produce changes in embryonic hormones.

Hormones induce changes in color, reproduction, hibernation, behavior

Environmentally-produced changes in hormones during development can affect the adult phenotype; e.g.

Bicyclus anynana

seasonal polyphenism - common in butterflies

Nemoria bistriariawet season dry season wet seasondry season

Seasonal Polyphenism

Nemoria arizonaria larvae

early spring – oak catkin (flower) summer – oak twig

Environmental signal – tannins in oak leaves (probably)

- developmental response – change in cuticle morphology

Anthropogenic Effects

pepper moths

Industrial melanism

(“anthro” – man)

Environmental signals – UV radiationUV = mutagenic, damaging to cell and tissue development

- lower wavelengths have more energy; cause more damage- developmental stages are the most sensitive!

melanin

Mycosporine-like amino acid = sunscreen

HO HO

NOH

COOH

NH

COOH

OCH3

R

UV exposure can induce protective mechanisms

northern leopard frog

Bonellia viridis (marine echiuroid worm)

♂♀

Environmental Signals - Intraspecific

- larvae settles on sand/rock surface = ♀- larvae settles on proboscis = ♂

10 cm

2 mm

Therefore, signal (most likely chemical) to become ♂ is from ♀

Environmental Signals - Temperature

- sex determined by the egg temperature during weeks 2 & 3

≤ 30° C = female

≥ 34° C = male

Temperature-dependent sex determination in alligators

Differentiation

How does the intraembryonic environment direct cellular differentiation?

Differentiation – development of cellular specialization

Differentiation is a process

- preceded by several identifiable steps

- commitment

- specification

- determination

Commitment

undifferentiated differentiated

specification determination

1. Specification – capable of differentiating autonomously when placed in a neutral environment.- reversible

2. Determination – capable of differentiating autonomously evenwhen placed into another region of the embryo.- essentially irreversible

Stages of cell commitment:

Specification Types

I. Autonomous specification

II. Syncytial Specification

III. Conditional Specification

Autonomous Specification

-Cells are specification by differential distribution of cytoplasmiccomponents during cleavage of the egg and early embryo.- proteins- RNA

Autonomous SpecificationTunicate (sea squirt)

blastomere separation

Autonomous SpecificationTunicate (sea squirt)

- blastomeres are committed at a very early stage in mosaic development

- each dissociated blastomerepair forms original structures

-each blastomere contains positional information in the form of specific proteins and genes

dissociated blastomeres

Mosaic Developmentdifferentiated cell

mitosis

early embryo

later embryo

Syncytial Specification

Syncytium – nuclear division without cell division;results in cytoplasm with many nuclei

DrosophilaCleavage

nuclei & cytoplasm formsyncytial blastoderm

FELICE FARBER

Syncytial Specification throughMorphogen Gradients

Drosophila egg

bicoid – anterior determinant

nanos – posterior determinant

Maternal messages:

U Irion & D St Johnson

Syncytial Specification throughMorphogen Gradients

bicoid – anteriornanos – posterior

Maternal messages:

Each morphogenestablishes a gradientthroughout the embryo (like a diffusion gradient)

Bicoid & Nanos proteins = morphogens

1:0 10:1 1:1 1:5

- each region will have a distinct Bicoid:Nanos ratio

- Bicoid:Nanos determines anterior-posterior identity

Syncytial Specification throughMorphogen Gradients

Cells establish identity depending on their position in multiple gradients

bicoid – anteriornanos – posterior

Maternal messages:

Each morphogenestablishes a gradientthroughout the embryo (like a diffusion gradient)

Bicoid Protein = Head

Bicoid Manipulation

= morphogen gradient

Conditional Specification

Conditional Specification

Cell fate depends on interactions with neighboring cells

Embryonic cells can change fates to compensate for missing parts = regulation

Conditional specificationproduces Regulative Development

Conditional Specificationdifferentiated cell

mitosis

early embryo

later embryo

Morphogen Gradients in Conditional Specification

Cells respond to protein concentration by turning different colors.

Morphogen Gradients in Conditional Specification

Cell commitment and differentiation are programmed by various morphogen gradients.

Transplants of flag “cells”shows that they retain theiridentity (nationality), but grow according to the cellsaround them.

Conditional Specification: Leg-Antenna Transplant

Drosophila legs and antennae are structurally-related, but obviously have different morphologically.

Consider “leg” and “antenna” to be like the national identities in the flag analogy.

Experiment: Transplant cells that would ordinarily produce the proximal (close to the body) leg to an area that would ordinarily produce the tip of the antenna.

proximal portion of legdistal portion of antenna

clawsI. Duncan

The resulting structure would be an antenna with a claw at the end.

The transplanted leg cells have kept their “leg” identity, but have modified theirdevelopment from their original location (proximal to the body), to that of their new location (the distal-most point).

In this example of conditional specification, a morphogen gradient that started at the body (source) would specify proximal structures. As the morphogenconcentration decreased more distal structures would be formed. Therefore, while the leg cells kept their leg identity, they were “conditioned” by the very low morphogen concentration at the tip (sink) to form the most distal leg structures – claws.

I. Duncan

Conditional Specification: Leg-Antenna Transplant

Stem Cells and Commitment

Pluripotent – makes many types of cells.

Totipotent – ability to make all cell types.

Stem Cell Derived Blood Cells

Morphogenesis and Cell Adhesion

How are tissues formed from populations of cells?

How are organs constructed from tissues?

How do organs form in particular locations and how do migrating cell reach their destinations?

How do organ and their cells grow, and how is their growth coordinated throughout development?

How do organs achieve polarity?

Cell Interactions

Cell membrane protein components bind cells together; e.g.

cadherins catenins

Cells interact with each other either through paracrine signaling at some distance, or through direct contact.

Cadherin-mediated Cell Adhesion

actinmicrofilamentsystem =anchoring andmovement

Different cellshave different cadherins.

Different cadherinshave different affinities for each other.

Thus, cell types can segregate themselves based on membrane components.

NOTE - Ca2+ -dependentbinding:

Ca2+ can control both strength andreversibility ofbinding