Induction: information from neighbors influences cell fate inducer responder

If induction tells cells what to do, how do they remember these instructions for a lifetime?? DETERMINATION

And usually there’s no turning back. FATE: what a cell will become at a later time--its future! DIFFERENTIATION: the actual elaboration of a cell’s fate DETERMINATION: internal and self-perpetuating changes that distinguish a cell & its descendents from other cells and commit them to a specialized course In other words, a cell is set to become a certain kind of cell before it actually becomes that type of cell. And usually there’s no turning back. This concept differs from fate in that for a time a cell’s fate can often be changed by changing environment; it differs from DIFFERENTIATION because this is overt cell specialization.

Determination can be assayed by transplantation experiments Early gastrula Cells alter their fate to match that of neighbors i.e. not determined Transplant nervous system to epidermis and vice versa Late gastrula Cells retain their original fates, regardless of neighbors i.e. determined (see also Figure 10.18)

Cloning: the ultimate transplant experiment Dolly (and Bonnie) Figure 4.8 Dolly (and Bonnie)

The ultimate transplant experiment Cloning: The ultimate transplant experiment Freddy, a cloned leopard frog (Rana pipiens) Figure 4.5

How does cloning work?

Cloning success drops dramatically as the age of the nuclear donor increases Figure 4.6

DETERMINATION results from heritable changes in gene expression

All cells have the same genes yet different cells make different mRNAs Non-base change modifications of DNA result in selective gene expression

Evidence for cytoplasmic contributions to selective gene expression

DNA Methylation is used in mammals to keep unneeded genes inactive CH 3 CH 3 CH 3 Bone-specific genes are methylated 3 CH 3 CH Skin-specific Gene genes are not expression methylated CH 3 CH 3 CH 3 Muscle-specific Skin cells genes are methylated 3 CH

Methylation of DNA Figures\Chapter05\DevBio7e05181.jpg Figure 5.19

DNA Methylation is used in mammals to keep unneeded genes inactive CH 3 CH 3 CH 3 Bone-specific genes are methylated 3 CH 3 CH Skin-specific Gene genes are not expression methylated CH 3 CH 3 CH 3 Muscle-specific Skin cells genes are methylated 3 CH

Imprinting results from pre-programmed differences in DNA methylation of selected genes in the sperm and egg H19 function?

Genomic imprinting occurs in most mammals * * *

Igf2r Igf2 “A Silent Struggle”, New York Times, March 14, 2006

Imprinted genes affect fetal growth Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r) growth growth

What happens if dad’s copy of Igf2 is deleted? Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r) growth growth

copy of Igf2 isn’t imprinted? What happens if mom’s copy of Igf2 isn’t imprinted? Beckwith-Weidemann syndrome Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r) overgrowth growth

What if mom’s copy of Igf2r is deleted? Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r) growth growth

What happens if mom’s copy of Igf2 is deleted? Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r) growth growth

DNA within cells is not naked!

DNA is wrapped around histone proteins as chromatin Figure 5.1

Inactive genes are thought to be wrapped up in an inactive "chromatin conformation" Bone-specific genes are wrapped up and inaccessible Skin-specific mRNA genes are not Gene wrapped up and expression so are accessible Skin cells Muscle-specific genes are wrapped up and inaccessible

X chromosome inactivation: Silencing one of the two X’s in female cells Xist RNA is associated with the silenced X chromosome Read this in the text again

Post-translational modification of histones plays a key role in regulating gene expression X chromosome inactivation (See also Figure 5.25)

myoD

By altering methylation, cells can be reprogrammed Experiment #1 By altering methylation, cells can be reprogrammed Bone cells Muscle cells Normally fibroblast cells would become muscle cells? "Fibroblast-like" Fat cells mouse cells Treat with 5-azacytidine to reduce DNA methylation

Experiment #2 Chromatin transferred from myoblasts can transform untreated fat cells. Transformation frequency suggests only one or few of the genes have this ability

Ultimately a single gene was identified- myoD -whose expression could convert any other cell type into muscle cells

MyoD is expressed at the right place and time to be a master regulator of muscle development Somites Experiment #3

Does the myoD transcription factor bind DNA sites Experiment #4 Does the myoD transcription factor bind DNA sites within the promoters of the expected gene types? Bone-specific genes lack myoD binding sites OFF muscle-specific mRNA myoD ON Muscle-specific genes have myoD binding sites myoD site Fat-specific genes lack myoD binding sites OFF

Experiment #5 Scientists "knocked out" the myoD gene to test its predicted role in muscle development Eliminate gene encoding myoD Does mouse develop muscles?

!!

MyoD and related transcription factors Experiment #6 MyoD and related transcription factors work together to regulate muscle cell fate

How do cells remember their decisions? Cellular memory and the engrailed gene

Remember that Wingless is expressed by specific cells within each segment

Engrailed is a transcription factor expressed by cells posterior to those that express Wingless. It defines the posterior boundary of each segment. Anterior Posterior Wg En

Here’s the real thing: engrailed

Mechanism #1: cell-cell signaling Engrailed expression requires Wingless signaling by the neighboring cells.

Mechanism #2: autoregulation Later on, engrailed becomes autoregulatory ie. it turns on its own synthesis Segment Segment boundary boundary en ON More engrailed en gene protein

Mechanism #3: specialized chromatin proteins In cells where engrailed is turned off, a set of specific chromatin proteins bind to it and wrap it up in a permanently inactive state. Segment Segment boundary boundary OFF en gene Polycomb group proteins turn off engrailed in cells it’s not needed. en gene Permanently OFF