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

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. FATE: what a cell will become at a later time--its future! DIFFERENTIATION: the actual elaboration of a cell’s fate

Determination can be assayed by transplantation experiments Late gastrula Early gastrula Transplant nervous system to epidermis and vice versa Cells alter their fate to match that of neighbors i.e.not determined Cells retain their original fates, regardless of neighbors i.e.determined (see also Figure 7.16)

Cloning: the ultimate transplant experiment Dolly (and Bonnie) Figure 2.1

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

How does cloning work? See Fig. 2.1 for how cloning works in sheep

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

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

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

Methylation of DNA Figure 2.17

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

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

Genomic imprinting occurs in most mammals * * *

“A Silent Struggle”, New York Times, March 14, 2006 Igf2rIgf2

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

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

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

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

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

DNA within cells is not naked!

DNA is wrapped around histone proteins as chromatin Figure 2.3

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

Post-translational modification of histones plays a key role in regulating gene expression X chromosome inactivation

myoD

By altering methylation, cells can be reprogrammed "Fibroblast-like" mouse cells  Treat with 5-azacytidine to reduce DNA methylation Fat cells Muscle cells Bone cells Experiment #1

Chromatin transferred from myoblasts can transform untreated fat cells. Experiment #2 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

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

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

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

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

Remember that Wingless is expressed by specific cells within each segment

Anterior Posterior Wg En Engrailed is a transcription factor expressed by cells posterior to those that express Wingless. It defines the posterior boundary of each segment. See also Fig. 6.33

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 boundary Segment boundary ON en More engrailed protein en gene

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 boundary Segment boundary en gene OFF Permanently OFF Polycomb group proteins turn off engrailed in cells it’s not needed.