C2006/F2402 '04 -- Outline for "Lecture #26" -- Introduction to Development

The outline below is posted for those who are interested in the topic of development. There is NO lecture #26. Some of this material may be discussed briefly at the end of lecture 25 but this material will NOT be on the final. If you find this interesting, I suggest taking a course in development, which we hope to have next spring. I am happy to answer questions on this topic, but to repeat, there will be NO questions on this topic on the final.

(c) 2004 Dr. Deborah Mowshowitz,  Columbia University, New York, NY. Last Update: 04/28/04 12:43 PM .

I. Introduction to Animal Development -- the issues

    A. The question --  How do we build a multicellular (3D) organism with specialized cells in the first place? 

    B. What's the problem? DNA is linear; how can it be used to create a 3D organism? Has linear written instructions not blueprints or pictures.

    C. The basic spatial solution: Most animals start convert egg (with instructions in text-only format) to multicellular 3D organism in about the same way, as described below. We'll look at the overall pattern, and then consider the underlying mechanisms at the molecular level (how do you turn the appropriate genes on and off). 
 

II. Steps of Early Embryonic Development -- see Purves ch. 43-- overall animal scheme to Gastrula Stage

    A. Fertilized Egg 

1. Egg is often very big, contains lots of stuff. Contains nutrients (yolk) in addition to stockpiles of usual cellular contents. Frog egg has enough ribosomes to last until tadpole hatches! (Mammalian eggs are relatively small.) 

2. Generally has extra copies of all the materials for division stockpiled except has no extra copies of DNA and chromosomal proteins (histones, etc.). (DOES have DNA -- just no extra copies.)

3. Has all the regulatory stuff (proteins and mRNAs) needed to get started and turn on the genes in the right order. (How do we know this? Dolly and all the other clones. See below.) Where did the regulatory stuff come from? Some was made in the mom and stuffed into the egg. Some was made in the egg itself. (So who came first....?)

    B. Cleavage -- no increase in total volume -- see Purves fig. 43.7 (40.6 in 5th ed) or Becker 17-27 & 17-29.

1. Blastomeres (cells of early embryo); ---> smaller as cleavage proceeds.

2. Make mostly DNA (& histones) -- all else already in excess and just divided up.

3. Very short cell cycle -- G1/S switch is in override -- need signal to go from G2 to M only. (Regulation of the cell cycle will be discussed in detail in next lecture.)

    C. Morula = solid ball

    D. Blastula =  hollow ball with cavity (blastocoel) . Note distinction between somatic cells and germ cells is set up early and maintained throughout. For pictures of blastulas, see Purves 43.8 & 43.9 (40.7 & 40.8).

1. How formed -- By invagination. Sheets of cells move, crawl over and in = major rearrangements. Blastomeres move relative to one another. Details depend on amount of yolk -- see pictures in Purves 43.11, 43.13 & 43.16 (40.10-40.11, & 40.14)

2. Three primary germ layers (2 in some very simple organisms) -- See Purves Table 43.1 (40.1) for fates of each layer.

• Ectoderm --> epidermis, & nervous system  -- both CNS (central nervous system = brain and spinal cord)  & PNS (peripheral nervous system = all nervous tissue outside CNS)

• Mesoderm --> connective tissue (skeleton, blood), muscle, some organs (heart, kidney)

• Endoderm --> linings: gut, lungs, bladder; some other organs (liver, pancreas)

Note: germ layers (3 basic layers of embryo) are not the same as germ cells (cells that will eventually --> eggs and sperm)

3. FYI: There are two major types of organisms; depending on fate of blastopore -- which end of Anterior/Posterior axis it becomes.

III. Regulative vs. mosaic development

    A. There are 2 major styles of development

1. Mosaic development (also called determinate)

a. Instructions are (mostly) built in. 

b. What happens is largely independent of environment.

c. Common in protostomes (worms, flies -- many of the model organisms used to study development)

d. Typical type of determinate instructions in human terms:  Go 10 feet and then turn left.

2. Regulative development (also called indeterminate)

a. Instructions come (mostly) from outside. 

b. What happens is highly dependent on environment and/or who (what cells) you meet

c. Instructions come from contact w/other cells, diffusible signals from other cells, etc.

d. Common in deuterostomes (vertebrates, sea urchins)

e. Typical type of regulative instructions in human terms:  Go until you meet the lampost (or girl in red dress, or until air is not polluted) and then turn left.

    B. How to distinguish the two types of development? By observation and experimental manipulations.  Either way, you try to figure out: How rigidly is the pattern of development set? For interesting examples of these approaches, see Purves Chapter 43.

    C. Practical Implications of fact that human development is regulative -- Separating early cells of the embryo in vertebrates allows for:

1. Natural occurrence of "identical" (really monozygotic) twins, triplets etc. See Purves fig. 43.10 (40.9). Cells of early embryo accidentally separate into two or more groups and each group forms a complete individual.

2. Cloning on purpose from blastomeres. You can separate blastomeres, and implant each one in a separate foster mother. You get multiple identical individuals i.e. clones -- this has been done in monkeys. (Note: This is not what people usually mean when they talk about "cloning" of animals. They mean what is sometimes called "nuclear transfer" -- creation of a new identical (?) individual by transfer of a nucleus from an  adult cell into the cytoplasm of an egg. This is discussed below.)

3. Prenatal diagnosis at 8 cell stage in humans using blastomeres (& reimplanting the rest if ok) You do fertilization in vitro, then let development go to 8 cell stage (also in vitro).  Remove one cell and use its DNA to test for genetic defects. If DNA is ok, implant remaining 7 cells in uterus and they will develop into a normal human. If DNA is not ok, discard embryo and try again. (Question to think about: If human development were mosaic, what would happen if you implanted an embryo with 7 cells instead of 8?)

4. Isolation of ES (embryonic stem) cells. Cells from early embryo are all equivalent and capable of forming any cell type (as natural twinning shows). These cells can be isolated and grown under certain conditions, so that they multiply and are maintained in this unspecialized state indefinitely. These are called ES cells. Their genes can be manipulated in culture (genes can be added, deleted, etc.) and/or the fate of the cells can be manipulated by addition of growth factors, implantation into other embryos etc. ES cells of animals have been used extensively for research purposes. The current big question is, "What are the proper uses (if any) of human ES cells?" Human ES cells have great potential for repair of damaged tissues, but they must be obtained from destruction of human embryos. See Purves 16.6.

    D. Issue here is similar to nature vs nurture or genes vs environment -- But it's a false dichotomy

1. Development is never 100% mosaic or regulative. In all organisms, some developmental changes in cell behavior are triggered by external signals and some are "built in." 

2. Always get progressive restriction of gene expression without (usually) any loss of DNA. Somehow genes are turned off more or less permanently with time as cells become more and more specialized.

3. Differences: Restriction is more rigid, less reversible and less susceptible to outside influences in cases if mosaic development; in other words determination (irreversible choice of cell fate) occurs earlier and there is less embryonic induction (choice directed by external signals).

IV. Development -- Post Gastrula 

The rest of the cycle includes organ formation, metamorphosis (transition of sexually immature organism into sexually mature adult), production of eggs and sperm and restart of cycle. All this is fascinating but will not be covered in lecture; the parts of Purves chapters 43 & 16 that we will not discuss in class are highly recommended.

V. How does all this work at molecular level? How does adult make egg that is ready to go; how does egg make adult? In other words, what does it take to go through the cycle?

    A. Cells must selectively do the following :

1. Grow & Divide. Cleavage is a special case of this.

2. Die (by apoptosis = programmed cell death as vs. necrosis.)

a. In apoptosis, cell shrivels up and dies without releasing contents or affecting neighbors. Apoptosis = cell suicide in response to certain types of damage or developmental signal; requires active gene expression by apoptotic cell. Needed during development to resorb tadpole tail, remove webs between fingers, etc. See Becker fig. 10-27 & 28 [Box 10B]. 

b. In necrosis, cell swells and explodes in response to certain other types of damage, releasing contents and triggering inflammatory response.

c. Note: Both NK cells and cytotoxic T cells kill virus infected cells by inducing apoptosis. The process may be triggered by juxtacrine signaling or by entry of proteolytic enzymes into holes made by perforin.

3. Move (as in gastrulation)

4. Specialize  -- turn some genes on, and others off, so that cell makes a specific set of proteins characteristic of that cell type. Rest of discussion of development will focus on how this works.

    B. Cells must do all of the above without changing their DNA. (Except for lymphocytes)

1. How do we know this? Even if there are no physical differences between the DNA of an egg and the DNA of a specialized somatic cell, how do we know all the DNA in the somatic cell is functional?

a. With plants, one cell (any one) can start over and regenerate a whole plant. Therefore all DNA in all cells of plant must be functional. 

b. With animals, you might think all DNA is not functional because generally one somatic cell will NOT  --> whole organism, and state of any particular type of cell (kidney, liver, brain) is stable in organism and in vitro. But 

(1). Regeneration shows some specialized cells can switch fates.

(2). Cloning by nuclear transfer (as in Dolly & other organisms) shows some adult nuclei are "totipotent" --  they can "start over" and provide all the genetic information necessary to build and run all the cell types of an adult. Important point: the material in the cytoplasm of an egg is required to reprogram the nucleus -- to alter state of chromatin -- so nucleus can start over. See Purves 16.4 (15.4) & Purves 16.5.

This is what people usually mean when they talk about cloning a person or animal -- an adult nucleus is put into a anucleated egg, and the added genetic material drives development of a new complete organism. This procedure -- nuclear transfer -- works, but with current technology it is very inefficient. Most transferred nuclei do not successfully produce an adult, probably because most nuclei are not reprogrammed successfully.  The new adult is a "clone" of the adult that contributed the nucleus.  However, the clone and the original will not be identical, just as "identical" twins are not exactly the same. (Also the clone and its parent may not have identical mitochondrial DNA.) Should it be legal to clone your pet? Yourself? Your child?

2. What can we Conclude about Changes in Genes during Differentiation? 

a. Which genes are transcribed must change, not the genes.  During differentiation a stable pattern of transcription is set up and maintained in cells (& their mitotic offspring). Exception: lymphocytes -- in these cells only, genes change as well as gene expression. (In other words, how lymphocytes specialize is NOT the norm.)

b. The pattern of transcription must be stable under normal conditions so (for example) liver cells do not change into kidney cells.

c. The pattern of transcription is not fixed irreversibly (see results above), although we don't fully understand the conditions needed to establish or change it.  

d. Possible ways of establishing/maintaining a fixed pattern -- DNA and/or proteins of chromatin can be covalently modified by methylation, acetylation of histones, etc.  

(1). These modifications do correlate with changes in gene expression in many cases.

(2). Many of these modifications are stable -- once chromatin is modified, replication by mitosis --> daughter cells with same modifications.

(3). However, it is not clear if these modifications trigger changes in gene activity or changes in gene activity (due to other signals) result in these modifications. What is cause, and what is effect here? 

(4). Note: Some organisms survive and develop perfectly well without methylating their DNA.

    C. How are the differential patterns of transcription set up? Current Basic Explanations --  have a mix of mosaic & regulative development.

1. Regulative interactions = (Embryonic) Induction -- some signals come from outside

a. Usual signaling mechanisms: GF's, TK receptors, G prots. 2nd messengers, cascades, etc. (In problems, ignore any questions on instructive vs. permissive differences).

b. Close cell contact required because signals are either paracrines or juxtacrines (on surface of signaling cell).

c.  Signals and receptors are evolutionarily conserved -- Have signal from mouse mesoderm that normally induces production of hair in mouse ectoderm.  Same signal --> chicken ectoderm to make feathers (chicken equiv. to hair).

2. Mosaic features -- some signals/materials that determine cell fate come from inside the cell

a. Materials arranged asymmetrically in egg

(1). Materials ("cytoplasmic determinants") arranged asymmetrically in egg by mother --  Mother's genes determine what's in egg & where. Position of determinants sets fate of cells in that part of the egg. (Case like this: determination of location of germ cells by germ plasm.)

(2). Often have gradients of mRNA (to be translated after fertilization) or protein in the egg.

(3). Material distributed in a gradient (from one side of egg or embryo to another) is often called a "morphogen" = a diffusible substance that has different effects on development at dif. concentrations. (See bicoid in text.)

b. After cleavage, different cells have different concentrations of cytoplasmic determinants, morphogens, etc. 

c. Cells with different amounts of a particular material  -->  turn on different genes of embryo   -->  different fates.

3. What do external/internal signals do? Start a sequence of sequential gene activations

a. Different TF genes turned on in dif. cells and/or different TF's activated/inactivated --> dif. active TF's

b. Each active TF (or combo) turns on a different sets of genes (all those with enhancer for that TF)

c. Genes turned on by one TF can --> new TF's or make proteins that activate/inactive old TF's  --> turn on and/or turn off diff. genes, and so on.

4. What changes as cells divide & development progresses?

a. Original cytoplasmic factors are redistributed, diluted, etc. --> different  internal signals (Purves, fig. 15.8 in 5th ed)

b. New signals are received from outside.

c. In response to signals, new proteins are made, old ones modified   -->  new cytoplasmic/nuclear factors (See Purves 43.6 for nice example)

d. So total collection of active TF's, splicing factors etc. constantly changes.

e. Get progressive restriction of gene expression without (usually) any loss of DNA. More and more genes are turned off more or less permanently with time -- chromatin is altered in some semi-permanent way.

5. Reach stable end result (differentiated state) -- set of TF's and genes that are "on" is self reinforcing; all other genes stay "off."  

6. Adult Stem Cells --  Many adult cells are so specialized they cannot divide. When these cells wear out, they are sometimes replaced by new cells derived from adult stem cells. The stem cells are special cells that are held in reserve (in a partially differentiated state) and used for replenishing the supply of other specialized cells in the adult. When stem cells divide, one daughter cell becomes terminally specialized while the other remains a stem cell. The potential of adult stem cells seems to be much greater than was previously suspected -- it looks like many types of stem cells are able to give rise to a large variety of differentiated cells. This raises many new possibilities for repair.