The Genetics of Development

• Fate refinement can occur via asymmetric divisions of a particular intermediate cell, and the daughter cells of that intermediate cell can inherit different regulatory instructions. Thus, the various descendants of a single progenitor cell can become committed to diverse fates.

Protostomes vs. Deuterostomes: Determinate vs. Indeterminate Cleavage

Recall the phylogeny of the Bilateria, and the differences between two major lineages, the protostomes (e.g., Annelida, Mollusca, Arthropoda) and deuterostomes (e.g., Echinodermata, Hemichordata, Chordata).

In both lineages, many species retain pluripotent and multipotent cells into adulthood, and can regenerate lost body parts.

The developmental flexibility of various cells is of interest in medical science because of the the potential for understanding the genetic basis for this regeneration. A salamander can regenerate an entire severed limb! Yet human nervous tissue, once destroyed, is gone forever. Can we change this? We have yet to see.

By learning the mechanism of this retained of pluripotency, researchers hope to find ways to stimulate regeneration in human tissues that are not normally capable of regeneration. (e.g., The Miami Project to Cure Paralysis)

Gene Regulation During Development

• gene structure modification
• regulation at the transcriptional level
• mRNA processing (e.g., exon shuffling)
• translational control
• post-translational control

• structural
• metabolic enzymes
• regulatory proteins that act to control gene expression

Transcript Processing and Tissue-specific Regulation

• Which genes are important in development?
• When is each gene active, and in what part of the animal?
• How is the expression of each gene regulated?
• How do gene products affect development at the molecular level?

The student of ontogeny has the job of discovering each species "genetic toolkit" for development.

The typical animal genome contains between 12,000 - 30,000 genes. These include

• housekeeping genes that control the products driving daily processes such as cellular metabolism and biosynthesis of macromolecules and nutrients

• genes that code for funcitional proteins such as immunoglobins, transport proteins, antibodies, etc.

• various classes of genes involved in regulating development

It's this last group that will be our focus for now.

Homeotic Genes and their Mutants

Metamerism is a symplesiomorphy linking all coelomate bilaterians, even if the segments of the body are no longer visible in the adult. In the primitive form, each body segment bears a single pair of appendages, and in the most primitive animals, the segments and appendages are not highly differentiated along the length of the animal, even if the animals have highly derived behaviors.

Changes in the identies of the segments and their associated appendages are caused by homeotic mutations:

• loss-of-function homeotic mutation: inactivates a homeotic gene in a pathway where it is normally active
• gain-of-function homeotic mutation: activates a homeotic gene in a pathway where it is normally inactive

The homeotic genes that control segment identity have been found at eight loci, and are known as Hox genes. Complete loss of function at these loci is lethal in homozygous condition, but heterozygotes can be viable.

Hox Genes: Where are they, and what do they do?

• Bithorax complex - three Hox genes
  The mutant for which it's named:
  

• Antennapedia complex - five Hox genes
  The mutant for which it's named:
  

Hox genes are expressed only in specific, spatially restricted domains of the embryo, as shown HERE. Gene expression can be visualized this way by showing either

• expression of mRNA transcripts
  These are visualized by hybridizing them in situ with labeled complementary nucleic acids. Pictures are taken under a light microscope.
• expression of Hox proteins
  These are visualized by binding the Hox proteins to labeled antibodies with affinity for those specific proteins. Pictures are taken under a fluorescence microscope.

Note: cDNA or complementary DNA is artificially produced DNA that is manufactured (with reverse transcriptase, which can make RNA --> DNA) from an mRNA transcript of a gene of interest. This can be used either for:

• localizing mRNA transcripts
  The cDNA of a gene of interest can be transcribed in vitro with labeled nucleotides. This new RNA probe is complementary to the mRNA of the gene of interest in the living organism. When injected into the embryo, it can hybridize with the mRNA transcripts, allowing localization of where they are being produced OR by...

• localizing expressed proteins
  The cDNA can be inserted into bacteria, which will now manufacture the protein of interest. The proteins are injected into a vertebrate host, which will produce antibodies against it. Antibodies injected into embryos will bind to the proteins of interest where they are being expressed. Special dyes can be used to reveal these locations.
  
  These procedures are outlined HERE.

The precise location of gene expression yields information about how segment identity is achieved by the embryo.

Hox Genes Establish Segment Identity

• All segments will still grow appendages
• But all appendages will be antennae!
• In a wild type fly, the hindwings should become clublike halteres
• But on segments in which wings form, the haltere primordia become forewings.

Why were halteres retained? Were they adaptive? Let me tell you.

In Search of the Origin

The homeobox encodes a protein domain, the homeodomain, 60 amino acids in length, and very similar in aa sequence among the eight Hox genes.

The Hox genes were discovered to form the helix-turn-helix shape known already to be typical of transcription factors in the lac operon as well as in genes controlling yeast mating type determination. The various Hox proteins bind directly to the DNA regulatory elements, either activating or repressing the expression of genes.

The Hox genes encode proteins that affect the transcription of other genes.

The Origin is Found, and it's Common

There has been some addition of complexity over that time, probably due to additional duplications of Hox complexes, or even entire chromsomes. In mice and other vertebrates, there are four Hox complexes on four different chromsomes, and each complex includes 9-11 genes, for a total of 39 Hox genes in vertebrates.

As in fruit flies, Hox gene mutations cause not the failure of a segment or appendage to form, but rather it changes the identity of the segment affected by the mutation.

Early Embryonic Decisions: Maternal Effect Genes

Surprise: Not all of an embryo's early developmental events are encoded by its own genome. As we know from our brief introduction to maternal effect in shell coiling direction in Limnaea, there are properties of the ovum itself that can act to affect an individual's development and phenotype even before its own genome starts working.

Genes whose products are provided by the female parent to her eggs are called maternal-effect genes. Their effects on the offspring depend solely on the genotype of the mother. Reciprocal crosses made in the laboratory can reveal whether a gene product is

• maternally required - active in the ovum, and encoded by the mother's genome
• zygotically required - active in the zygote, and encoded by the offspring's genome

Major Classes of Developmental Genes

• maternal effect genes - set up the anteroposterior axis polarity and the dorsoventral axis polarity
• gap genes - affect the development of a contiguous block of segments
• 
  pair-rule genes - control the proper development of adjacent segments (pairs)
• segment polarity genes - affect individual segments' polarity
• Hox genes - affect the identity of a particular segment, and determines what that segment will look like and what appendages it will bear

As always, the function of each of these gene types was determined by examining the appearance of mutants for each class of these genes.

The resolution of developmental fields becomes more fine as development proceeds:

This suggests that the expression of one set of genes might control the expression of genes farther down the developmental pathway. The products of these genes exert regulation of gene expression either as transcription factors (direct control), or as participants in a signal transduction pathway, which are often involved in gene activation or repression.

Which end is Front? Determination of the Anteroposterior Axis
As usual, most of what is known has been derived by the study of induced mutations. The names of the genes are usually derived from mutant appearance of the wild type traits.

How are the maternal mRNA transcripts anchored in their proper locations?
Specific base sequences in the UTR leader of the nos, bcd and hb-m mRNA transcripts have an affinity for particular proteins which can also bind to the "-" or "+" ends of cytoplasmic microtubules, effectively "gluing" them together.

Which End is Up? Establishment of the Dorsoventral Axis

Maternal effect gene products are also the triggers for the cascade of gene activity that determines which side of the embryo will be dorsal, and which will be ventral.

To understand what happens next, we have backflash to the unfertilized ovum.

Summary:

• Positional information is established by maternally deposited mRNA in the oocyte cytoplasm.

• Diffusion gradients in the syncytium are essential to the establishment of A/P and D/V axis polarities.

• Maternal effect transcript proteins act as transcription factors, affecting the expression of genes farther along in the developmental cascade due to variation in their concentration along the axes of the embryo.

• Genetic factors that determine cell fate via their concentration in the cell are known as morphogens.

• Genes that establish cell fates by responding to morphogen concentration are known as cardinal genes.

• Cardinal genes are the embryo's own genes--not maternal effect gene transcripts.

• The A/P cardinal genes (i.e. genes that respond to A/P transcription factors) are collectively known as gap genes (after the mutant form).

• The different gap genes respond differently to differences in the A/P factors' concentrations. This means that even though every gap gene is present in every cell, each will be expressed differently in any location that differs in A/P concentration from another location.

• Thus, not only is the correct number of segments formed, but each segment has its own characteristics, due to differences in gene expression initially set in motion by the A/P and D/V factors.

• The gap genes' differential expression results in cellular regions which will become different developmental fields.

• The products of the gap genes are also transcription factors, but these act at the next level of refinement, by affecting the transcription/translation of the pair rule genes. These are either acted upon directly by the products of the gap genes (primary pair-rule genes) or are acted upon by the products of primary pair-rule genes (segment polarity genes).

• The transcription factor products of the gap genes apparently also target Hox genes, influencing the transcription of Hox proteins, Like this.

It appears that in many developmental systems enhancer/silencer interactions between DNA and transcription factors from genes upstream in the developmental pathway are responsible for the orderly control of embryo development.

It is now known that other pattern-formation genes also operate on the basis of concentration gradients. Maternal effect and gap proteins control pair rule gene stripe formation, and the presence of Hox proteins can repress the formation of appendages in inappropriate locations.

Recall how conserved early developmental genes and cascades are across species, and then note the amazing similarity between the Drosophila CACT-DL system we discussed above, and a mammalian system that performs a similar function in determining dorsal and ventral body regions.

Fun in Plants
A more recently discovered gene family encodes the MADS-box transcription factors. Most notably, MADS-box transcription factors are responsible for flower development in anthophytes. Mutations in these genes can thus have profound evolutionary consequences.

Turning Points: Early Embryonic Decisions

In many cases, these developmental pathways are determined by regulating the activity of some critical enzyme at some point in its manufacture or activity cycle. One gene's product at the beginning of the developmental cascade can set the entire process in motion, or turn it off, and these are called master switch genes.

The concentration of this master switch product can determine whether a particular pathway will be turned "on" or "off"--and which pathway will be chosen in a binary decision.

Sex Determination

Sex determination in Drosophila
In the fruit fly, each cell independently makes the decision to become male or female, and this depends on the number of specific X-linked and autosomal genes it has.

The ratio of the genes is proportional to the ratio of the gene products, which have RNA binding capability.

• If the switch is left "off", then the embryo develops as a male (the default).
• If the switch is flipped "on", the embryo develops as a male.

How does this work?

This is, in turn, determined by the genic balance ratio (X:A) between

1. female determining x-linked genes (X - numerators)

2. male-determining autosomal genes (A - denominators)

(The Y chromosome, though present, plays a lesser role.)

• If X/A > 1.0 fly will develop as a female

• If X/A < 0.5 fly will develop as a male

Other ratios (found in the laboratory) result in inviable "metafemales" (X/A > 1.5)

or "metamales" (X/A < 0.33).

Sex Determination in Drosophila, Step by Step
Understanding how the switch is thrown requires a closer look at the protein products of these X-linked and autosomal) genes.

• sxl-affecting elements on the X chromosome are called numerator genes, since they act on the "X" part of the X:A ratio.

• sxl-affecting elements on autosomes are called denominator genes because they act on the "A" part of the X:A ratio.

• Some of elements encode bHLH (basic helix-loop-helix) proteins.
  • some bHLH proteins can bind to DNA promoters
  • all of the bHLH proteins can bind to each other to form dimers of three different possible types:
    • num/num dimers
    • num/den dimers
    • den/den dimers

What does the SXL protein do to promote femaleness?

In females, the manufacture of SXL occurs at a high rate only at the beginning of sex determination, and eventually the gene is transcribed from the late promoter, as in the male. But the SXL is already present, and continues to direct the activity of the developmentally downstream tra (and, indirectly, ds-x) for as long as its lifespan allows. This is enough to set the female embryo on the path to becoming female.

In effect,

• The female sxl is the "on" position, resulting in a shunt to a new pathway (female sex).

• The male sxl is in the "off" position, proceeds down the default pathway (male sex).

Here's a nice overview.

All mammals undergo relatively similar sex determination, as we learned in a previous lecture.

Another Binary Decision: Somatic or Germline?

Somatic cells are those that make up the body, and germline cells are those that give rise to gametes. In animals, a cell's "decision" to become somatic or germline is final and irreversible. (Though not in plants, as should recall.)

As strange as it might seem, it is the cytoskeleton that's intimately involved in this very early decision. From right to left, we see:

The cytoskeleton determines

• cleavage plane location
• cell shape
• directed transport of molecules and organelles

The cytoskeleton is the transit system for subcellular molecules and organelles. The filaments are polar (directional) and so able to direct wee things in specific directions.

Therefore, the orientation of the tubules and filaments themselves can be used to carry molecules and organelles to specific locations within the cells.

In microtubules, for example, the "-" ends are pointed towards the center of the cell (microtubule organizing center, or MTOC) and the "+" ends radiate outwards towards the periphery of the cell. Like so:

Here's a very close and person look at the obvious polarity of actin fibers:

In various species so far studied, regulatory molecules are carried along cytoskeletal filaments and delivered to specific cells that will become germ cells. (These have been variously named "P granules" in the nematode Caenorhabditis elegans, "polar granules" in Drosophila and "nuage" in frogs.

Let's have a look...

Fates of the blastomeres:

In all organisms studied so far, the P (whatever) cell that ends up with all the P granules/polar granules or nuage gets to be the cell that gives rise to the germ cells. This is the only evolutionarily important progenitor cell in the entire organism. The rest of it is just....soma.

The polar granules are actually anchored in the oocyte (by the mom fly) to what will become the posterior pole of the embryo. At mitosis #9, the P granules are enclosed by plasma membranes into pole cells, which will eventually become the germ line of the new fly.

Note that artificial disruption of the P-granules' compartmentalization into the germline progenitor cells (i.e., leaving P-granules in somatic progenitor cells) causes lethal developmental problems. There is a purpose to all this!

The Caenorhabditis elegans Project was begun by Sydney Brenner in 1963; many participants contributed, and now there exists a FATE MAP for every cell of the adult organism (i.e., which progenitor cells gave rise to which)

What was determined from studies of C. elegans development?

• germ layer cells don't necessarily all come from the same progenitor cell.

• developmental cell death is "programmed" and different in each sex.

• bilateral symmetry is NOT a natural outcome of the 1-->2-->4-->8 cell division process. Rather, some groups of cells must actually move about and migrate to produce bilateral symmetry.

The means by which cells communicate and migrate is a very hot area of research. Among the hottest areas are the Wnt Genes, a family of highly conserved genes that regulate signaling between cells during development.

Mosaics and Chimeras

• differences a gene's proximity to heterochromatin (silenced or not)
• silencing vs. non-silencing of genes via methylation or other inactivation
• variation in DNA sequence due to somatic mutation

An animal consisting of more than one genetically-distinct population of cells can be either a

• mosaic - the genetically different cell types all arise from a single zygote via somatic mutation during embryogenesis (very common, at least in mammals;

• chimera - the genetically different cell types originate from more than one zygote (rare in nature; usually known only in the laboratory). (It's named for the composite beast of mythology.)

Many individual mammals--including humans--are mosaics. An early somatic mutation can cause two different cell lines to proliferate during embryogenesis, and the earlier this mutation occurs, the greater the proportion of cells with the new, mutant genotype.
For example, a normal female mammal embryo (46 XX) might have one cell undergo a nondisjunction of the X chromosome resulting in some cells with a genotype of 47 XXX. The earlier this happens during ontogeny, the greater the proportion of cells with 47 XXX.

In a case such as duplication of the X chromsome, little or no damage will be done to the individual because of Barr Body formation. But in some cases, a potentially harmful genotype in a cell line can arise. If this happens early in embryo development, a larger proportion of cells will carry this deleterious genotype, and the individual may manifest signs of a genetic disorder. On the other hand, a sufficiently large population of normal cells can rescue an embryo from a lethal condition.
For example, women with Turner syndrome can have one of three different genotypes, and one of these is causes Mosaic Turner Syndrome. Because not all cells of the body lack the X chromosome, the symptoms of this condition are less pronounced.

As a reward for sitting through all that, let's explore the developmental wonder known as heterochrony.