Principles of Experimental Embryology

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1 Biology 4361 Developmental Biology Principles of Experimental Embryology June 16, 2008

2 Overview What forces affect embryonic development? The embryonic environment: external and internal How do forces within the embryo cause the differentiation of cells? Differentiation definition Specification, commitment, and determination concepts Types of specification: autonomous, syncytial, and conditional Morphogens and morphogen gradients Stem cells and commitment How do cells organize themselves into tissues and organs?

3 The Embryonic Environment What is the embryonic environment? External influences: light temperature humidity predators competitors intraspecific signals 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 (e.g. endocrine), that changes the developmental pathway.

4 Differentiation Differentiation development of cellular specialization How does the intraembryonic environment direct cellular differentiation? Differentiation is a process preceded by the commitment of cell to certain fate(s) Commitment is also a staged process: specification determination

environment (functional definition) reversible 2. Determination.

5 Commitment Stages Stages of cell commitment: 1. Specification. Capable of differentiating autonomously when placed in a neutral environment; not when placed in non neutral environment (functional definition) reversible 2. Determination. Capable of differentiating autonomously even when placed into another embryonic region. (functional definition) essentially irreversible undifferentiated differentiated specification determination

6 Specification Types I. Autonomous specification II. Syncytial Specification III. Conditional Specification

7 Autonomous Specification Cells are specified by differential distribution of cytoplasmic components during cleavage of the egg and early embryo. proteins RNA

8 Autonomous Specification Tunicate (sea squirt) blastomere separation

split, each dissociated blastomere pair forms original structures Each

9 Autonomous Specification 2 Tunicate (sea squirt) Blastomeres are committed at a very early stage in mosaic development dissociated blastomeres If split, each dissociated blastomere pair forms original structures Each blastomere contains positional information in the form of specific proteins and genes

10 Autonomous (Mosaic) Development differentiated cell mitosis later embryo early embryo

11 Syncytial Specification Syncytium nuclear division without cell division; results in cytoplasm with many nuclei

12 Drosophila Cleavage nuclei & cytoplasm form syncytial blastoderm FELICE FARBER

13 Syncytial Specification through Morphogen Gradients Drosophila egg Maternal messages: bicoid anterior determinant nanos posterior determinant U Irion & D St Johnson

14 Syncytial Specification through Morphogen Gradients Maternal messages: bicoid anterior nanos posterior Bicoid & Nanos proteins = morphogens Each morphogen establishes a gradient throughout the embryo (like a diffusion gradient) 1:0 10:1 1:1 1:5 1) each region has a distinct Bicoid:Nanos ratio 2) Bicoid:Nanos determines anterior posterior identity

15 Syncytial Specification through Morphogen Gradients Maternal messages: bicoid anterior nanos posterior Each morphogen establishes a gradient throughout the embryo (like a diffusion gradient) Cells establish identity depending on their position in multiple gradients

16 Bicoid Protein = Head

17 Bicoid Manipulation = morphogen gradient

18 Conditional Specification

can change fates to compensate for missing parts =

19 Conditional Specification Cell fate depends on interactions with neighboring cells Embryonic cells can change fates to compensate for missing parts = Regulation Conditional specification produces Regulative Development

20 Conditional Specification differentiated cell mitosis later embryo early embryo?

Experiment: Transplant embryonic cells that would produce proximal leg

21 Conditional Specification Legs and antennae: structurally related; different morphologies. Experiment: Transplant embryonic cells that would produce proximal leg (close to the body) to an area that would ordinarily produce antenna tip. proximal leg antenna claws distal antenna? I. Duncan claw

22 Morphogen Gradients Conditional Specification Cell commitment and differentiation are programmed by various morphogen gradients. e.g. cells respond to protein concentration by turning different colors

23 Morphogen Gradients Conditional Specification Cell commitment and differentiation are programmed by various morphogen gradients. Transplants of flag cells shows that they retain their identity (nationality), but grow according to the cells around them.

24 Conditional Specification: Leg Antenna Transplant I. Duncan Transplanted leg cells keep leg identity but modify development from their original location (proximal to the body), to that of their new location (the distal most point). A morphogen gradient started at the body (source) specifies proximal structures. As the morphogen concentration decreases more distal structures form. Therefore, while transplanted leg cells kept their identity, they were conditioned by the low morphogen concentration at the tip (sink) to form the most distal leg structures claws.

25 Stem Cells and Commitment * * Totipotent* ability to make all cell types; embryo and trophoblast (fetal portion of the placenta). Pluripotent* uncommitted; makes many types of cells. Multipotent* committed; makes several different types of cells *all stem cells regenerate copies of themselves

26 Stem Cell Derived Blood Cells

27 Overview How do forces outside the embryo affect its development? How do forces within the embryo cause the differentiation of cells? How do cells organize themselves into tissues and organs? Morphogenesis and Cell Adhesion

28 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 cells reach their destinations? How do organ and their cells grow, and how is their growth coordinated throughout development? How do organs achieve polarity?

29 Cell Interactions Cells interact with each other either through paracrine signaling at some distance, or through direct contact. Cell membrane protein components bind cells together; e.g. Cadherin Calcium dependent adhesion multiple forms

30 Cadherin Mediated Cell Adhesion Different cells have different cadherins. Different cadherins have different affinities for each other. Thus, cell types can segregate themselves based on membrane components. actin microfilament system = anchoring and movement NOTE Ca 2+ dependent binding Ca 2+ can control both strength and reversibility of binding

Principles of Experimental Embryology

Biology 4361 Developmental Biology Principles of Experimental Embryology September 19, 2006 Major Research Questions How do forces outside the embryo affect its development? (Environmental Developmental