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The Genetics of Development

Life begins with two cells fusing to form a single cell, the TOTIPOTENT zygote. (A totipotent cell has the capacity to develop into any type of cell. A pluripotent cell can develop into many, but not all different types of cells. A multipotent cell can develop into multiple types of cells, but not as many types as a pluripotent cell.)

Zygote becomes organism via a series of orderly developmental events which includes the increase in cell number and diversification of cell structure and function.

How do the cells diversify?

VIA THE ACTIVITY OF GENES THAT TURN ON AND OFF AT VARIOUS TIMES DURING DEVELOPMENT, AND SERVE AS "SWITCHES" FOR EACH STAGE OF THE DEVELOPMENTAL PATHWAY.
How do two apparently similar zygotes develop into completely different creatures, from a six-legged bee to a four-legged lion? The basic instructions are carried on the genome, although it is important to remember that environmental conditions may also play a vital role in determining the fine points of phenotype in any given individual.

A few definitions...

  • cell fate - the developmental pathway to be undertaken by a specific cell.
  • developmental field - a group of cells which cooperates and signals to each other in order to determine final position of the dividing, developing cells of an embryo within that field.
  • fate refinement - cell division and "decision making" that results in the final diversity and fates of all the blastomeres of an embryo.
  • paracrine signaling - a molecule secreted by one cell binds to a receptor on or inside neighboring cells, thereby inducing a pathway for signal transduction to the cell(s) receiving the secreted molecule.
    Interesting sidelight: cell fates can be flexible or fixed relatively early in development, depending on species (recall the difference between determinate and indeterminate cleavage of DEUTEROSTOMES and PROTOSTOMES).

    The flexibility of various cells is of interest to modern medicine because of the regenerative capacity of some cells in some species. A salamander can regenerate an entire severed limb! Yet human nervous tissue, once destroyed, is gone forever. At least so far.

    By learning the mechanism of this retention of totipotency, 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

  • As we already know, protein activity/function can be regulated at many levels such as

  • In these ways, multicellular Eumetazoans and True Plants can effect protein activity specific to tissue function and, earlier, tissue development

  • The existence of epigenesis suggests that modification of gene structure (as distinguished from gene composition) can be inherited
  • Embryo development proceeds due to an orderly sequence of regulatory pathways.
  • The blastomeres of an embryo have predetermined fates which are directed by the activities of specific "instructions" in the genome.
  • Multicellular organisms have more genes and more proteins than unicellular organisms.

    TRANSCRIPT PROCESSING AND TISSUE-SPECIFIC REGULATION

    A preview of things to come: The specifics of the P Element.

    Remember the P element? The one that encodes its own transposase and is able to insert into an M cytotype Drosophila and cause hybrid dysgenesis? Well, there's more...

    In normal, wild type flies, the P element transposes only in germline cells--never in somatic cells. However, the P element is transcribed in all cells! So why is its product (transposase) found only in germline cells?

    As it turns out, it's all about introns and exons! The mRNA transcript is processed differently in the germline versus the somatic cells, like this. (The lower half of the diagram shows how Laski, Rio and Rubin tested this hypothesis by inserting a modified P element into germline cells and generating mutant flies in which P caused transposition and multiple mutations in somatic cells of flies inheriting the mutant P element.)

  • The totipotent zygote has the capacity to develop into any type of cell (and eventually into an entire organism), depending on which genes are turned on at any given time in its developmental mitotic series.
  • Regulatory proteins from mom establish polarity along the axes of the ovum. These proteins control transcription of genes that serve as "master switches."
  • Master regulators may be transcription factors. Others are involved in signaling between cells.
  • Some cells seem to determine their own fates. Others seem to communicate and cooperate to determine their fates.
  • The major regulator genes studied well in Drosophila do not seem to be significantly different from those in other animals. They are relatively primitive and conserved across species!

    TURNING POINTS: EARLY EMBRYONIC DECISIONS

    Cellular developmental "decisions" may be binary (somatic or germline? male or female?) and irreversible--or they may be more flexible and complex, such as:
  • orientation of major axes and planes of symmetry (dorsal/ventral; cephalic/caudal)
  • organization into metameres (segments)
  • organization into germ layers (endoderm, mesoderm and ectoderm) and functionality of each layer
  • morphogenesis: organs, tissues, systems, appendages, etc.

    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.

    In development, gene regulators tend to be molecules manufactured by the organism itself--not some substance (as in the lac operon) obtained from the environment.

    The concentration of this embryo-produced molecule will determine whether a particular pathway will be turned "on" or "off"--and which pathway will be chosen in a binary decision.

    (Example of the "default" pathway illustrated is the Mullerian system development seen in human embryos that do not have the sry in their genome turned on. These become females "by default.")

    SEXUAL DIMORPHISM AND SEX DETERMINATION

    As we already have seen, there are several different ways by which sex can be determined, and they don't always involve heteromorphic sex chromosomes. Sex determination seems to have arisen and been maintained many times in animal evolution--It's probably safe to guess that genetic recombination conferred an evolutionary advantage to the sexual ancestors.

    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. Remember that when we're talking about gene ratios, we are also talking about the ratios of the PRODUCTS OF THOSE GENES!

  • In the fruit fly, a "master regulatory switch" is essentially "thrown" to determine sex. If the switch is left "off" you get a MALE. If the switch is flipped "on"--you get a FEMALE. But how?

  • The Master Switch is turned on or off by the activity of a gene now known as "sex-lethal" (sxl) (named for a mutant form which is lethal if inherited in homozygous condition). The sxl gene is X-linked, and its product is part of the "sex switch" which directs development towards femaleness or maleness.

    In Drosophila, sex is determined via a complex series of genetically programmed developmental "choices" 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.)

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

    or "metamales" (X/A < 0.33). These are, of course, found primarily in the laboratory; wild type flies are generally male or female.

    How are these "switches" thrown? Let's have a look at the complex interaction of protein TRANSCRIPTION FACTORS (a TRANSCRIPTION FACTOR is a protein that actually binds to the DNA, affecting transcription (either promoting or repressing transcription, depending on what it is and where it attaches.) encoded on the X and A chromosomes. The

    STEP BY STEP SEX DETERMINATION IN DROSOPHILA

  • the proteins encoded by the X and the A genes are bHLH proteins capable of binding to one another to form DIMERS (two-part proteins). All three types can form:

  • The sxl gene has two promoters, an EARLY promoter and a LATE promoter. The EARLY is downstream from the LATE promoter.

  • Only the num proteins have a DNA-binding sites which make them usable as transcription factors, and only the num/num dimers will activate the EARLY promoters.

  • If there is a relatively high concentration of numerator bHLH proteins (due to a high X:A ratio), they form binary proteins WITH EACH OTHER. This high level of num/num proteins promotes transcription by binding at the EARLY promoter.

  • When transcribed from the early promoter, the SXL protein is a big, functional transcription factor whose function is to bind to ITS OWN mRNA TRANSCRIPT and direct the exon splicing of the newly made sxl mRNA transcripts.

  • When the SXL binds to a newly-made sxl mRNA transcript, the exon splicing that follows results in a new SXL protein (just like the one squatting on the transcript) being made.

  • If the SXL is not present to bind to the newly-made sxl transcript, the exons splice in a different way, and form an mRNA transcript with an early STOP codon.

  • The protein made from this, instead of being a big, functional SXL, is a wee little short thing. It cannot bind to the sxl transcript the way the large form of SXL can.

  • Thus, once any SXL protein is present, it directs the formation of more SXL protein via exon splicing, no matter the configuration of the primary mRNA transcript. This is what happens in female flies, in which SXL promotes its own manufacture.

  • In male flies, the num/num dimers are very scarce. Whatever num proteins might be transcribed are far more likely to become part of a num/den dimer, which does NOT act as a transcription factor.

  • Thus, in males, no SXL protein is ever made, since transcription never takes place from the early promoter.

  • In female flies, SXL protein is made and even when, later in development, the SXL gene is transcribed from the LATE promoter, the SXL protein still directs the formation of more SXL from the primary mRNA transcript.

  • So in effect,

    But WHAT DOES THE SXL PROTEIN DO to detemine sex?

  • Obviously, the SXL has to do more than merely continue promoting more copies of itself. It has another job.

  • That job is to promote the activation of female-determining genes. It does so with its already-famous RNA-binding ability!

  • There is another gene, known as tra (named for a mutant version, "transformer"). The SXL protein apparently can bind to the primary tra transcript, and only when SXL is so bound, is an active form of the TRA protein formed. (SXL is a transcription factor, you know!)

  • Wouldn't you know it. TRA is also an RNA binding protein (transcription factor). However, it has a specific attraction to the primary transcript of a third gene, dsx (named for a mutant form, "doublesex").

  • When TRA is bound to the dsx mRNA transcript, the resulting exon splicing encodes a protein transcription factor (DSX-F) that represses the expression of male-specific genes. Hence, the fly with this cascade intact becomes female.

  • If SXL isn't present, no TRA is ever made, and hence, the dsx transcript is spliced in a different way, leading to the production of a different form of DSX protein (DSX-M), which represses the expression of female-specific genes.

  • Isn't that just....elegant. Let's have a look at a more details depiction of the cascade.

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

    CELLULAR DIFFERENTIATION AND MORE BINARY DECISIONS: SOMATIC OR GERMLINE?

    In animals, this decision is final and irreversible. (Though not in plants, as you know.)

    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:

  • vimentin filaments
  • tubulin microtubules
  • actin microfilaments

    The cytoskeleton determines

    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:

    Hydrolysis of ATP yields the energy necessary to propel molecules from either "-" to "+" along a tubule via the action of a protein called kinesin.

    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. (I vote for "nutella" for the next organism.)

    Let's have a look...

    Fates of the blastomeres:

  • AB - most of the skin and most of the neurons.
  • P1 - most of the muscles; all of the GI tract; all GERMLINE CELLS

    In all organisms studied so far, the P(whatever) cell that ends up with all the P granules/polar granules or nuage or fudge frosting 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....BODY.

    In Drosophila, unusual among animals in that it forms a syncitium (multi-nucleate cell mass with no plasma membranes dividing them) until just after the 13th mitotic division, 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!

    MORPHOGENESIS: ESTABLISHING DIRECTIONALITY AND POLARITIES IN THE EMBRYO

    (As usual, we're relying on Our Friend Drosophila. And note that even though it's not spelled out here, most of what is known has been derived by the study of naturally occuring and induced mutations in the normal pathways of these developmental steps. The names of the genes are usually derived from mutant forms of the wild types.)

    Anterior/posterior axes are also determined by cytoskeletal transport of various factors.

    Let's follow the works...
    1. Two maternal effect (recall what these are!) genes, bicoid (bcd) and hunchback (hb) encode protein transcription factors known as BCD and HB-M, respectively.

    2. These proteins are initially focused at the anterior pole. BCD has a higher concentration and is more focused. HB-M is less concentrated, but is distributed farther back in the embryo.

    What about the HB-M protein? That one's a little bit more complicated.

  • Like bcd mRNA, hb-m mRNA is deposited in the oocyte by the mama fly as she's making eggs. At the time of fertilization, the transcripts are distributed uniformly throughout the oocyte.

  • A third gene, named "nanos" (nos) now comes into play. The product of this gene (NOS protein) prevents translation of HB-M.
  • The nos mRNA transcripts are laid down by mama fly. But this time, their concentration is greatest at the posterior of the embryo, at the "+" ends of the microtubules.

    (You can see it coming, can't you?)

  • End result: NOS represses the translation of hb-m at the posterior pole, but does so less and less as we travel towards the anterior pole (again, it's a simple matter of diffusion and concentration in the nuclei, since these are transcription factors with affinity for the nuclei), since NOS protein diffuses from posterior to anterior.
  • The shallower gradient of HB-M (relative to BCD) results from the initial distribution of nos transcripts, which are concentrated at the posterior pole and almost absent by the time you get to the middle of the embryo. Because there's no NOS at the anterior, HB-M can be readily produced there, but the farther you get from the anterior pole, the more NOS there is--so HB-M protein synthesis will be blocked.

    HOW IS THE ANCHORING ACCOMPLISHED?

    mRNA consists not only of a central protein-encoding region (the ORF), but also of flanking, untranscribed retions (UTR's) at the 5' end (these are the leaders) and at the 3' end (the trailers).

    Base sequences in the UTR's of the nos, bcd and hb-m transcripts have an affinity for particular proteins which can also bind to the "-" or "+" ends of the microtubules, effectively "gluing" them together.

    OKAY, WHAT ABOUT THE DORSAL-VENTRAL AXIS, THEN?

  • A gene known as "dorsal" (dl) encodes a protein (DL), which is present in both the nucleus and the cytoplasm.

  • When DL occurs in the nucleus, it is unbound and acts as an active transcription factor (more on this later).

  • When DL is in the cytoplasm, it is bound by the product of the "cactus" gene (cact), CACT protein. This causes it to be inactive and unable to bind to mRNA.

  • In the oocyte and the early embryo, dl and DL are both evenly distributed throughout the embryo.

  • After fertilization, a gradient of active DL protein begins to develop, and by about the 13th mitotic division active DL is most highly in the NUCLEI of cells at the midline of what will become the ventral side of the embryo, and in the CYTOPLASM of the more dorsal cells. The purple in the drawing shows the concentration of DL protein.

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

  • A maternal gene called "spaetzle" (spz) is responsible for manufacturing a ligand protein called SPZ. (Recall that a ligand is a molecule that binds to a specific receptor site in the plasma membrane.)
  • The SPZ ligand is deposited (during oogenesis) on the inside of the egg's vitelline membrane, just inside the egg shell (Remember, these are flies we're talking about here.), and is most highly concentrated at what will be the ventral midline.

  • BAM! Fertilization! Grow-grow-grow...

  • Just as the embryo is about to change from syncytium to multicellular beastie, the SPZ ligands are released from the vitelline membrane. pouf

  • The SPZ ligand has a high affinity for another protein named TOLL (encoded by toll), which happens to be a transmembrane receptor embedded in the plasma membrane of the oocyte (which still serves as the plasma membrane still surrounding the whole, syncytial embryo).

  • When the SPZ ligand is bound to the TOLL receptor, a signal transduction pathway lights up like a little Yule tree.

  • The result of this signal transduction (through two other proteins called TUB and PLL) causes the DL-CACT complex to be phosphorylated.

  • As you might expect, this causes DL and CACT to lose affinity for one another, and they separate.

  • Free and single again (and most highly concentrated at the ventral midline) the DL proteins are now able to enter the nuclei near the ventral midline.
  • Here, DL serves as a transcription factor to turn on genes that direct cells to take on the characteristics of "ventral" cells!
  • This doesn't happen dorsally, since there are no SPZ ligands in the dorsal areas to trigger the phosphorylation of DL-CACT. If you don't believe me, have a look HERE.

    THE BOTTOM LINE:

  • The A/P cardinal genes (i.e. genes that respond to A/P transcription factors) are several, and are know collectively 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 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).

    (Are you ready to make yourself a flow chart? It might not be a bad idea. But doing it yourself will be a better learning aid than my doing it for you. So consider it a practice assignment!)

  • At each level of enhancer/silencer interaction, the transcription factors of one type of gene affect the transcription/activity of the next gene down on the resolution scale!

    (Read all about the House of Weird Fruit Fly experiments that elucidated the gradient-dependent nature of gene expression and subsequent morphogenesis in your text. You might especially enjoy the section on The Fly With Two Butts.) (And you thought South Park was all make believe.)

    What We Learn from Homeotic Mutants

    In a homeotic mutant, one cell type follows the developmental pathway normally followed by another cell type (a process known as HOMEOSIS).

    In animals, homeosis results from mutations in Homeobox (Hox) genes: genes involved in embryo development and morphogenesis.

    Homeoboxes are usually about 180 base pairs long, and each encodes a protein domain (a Homeodomain) which acts as a transcription factor, binding directly to DNA and affecting gene expression downstream in the developmental pathway.

    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.

    More Fun with Gene Expression: a few notes of developmental interest.

    Recall the polytene c'somes of Drosophila. Using Giemsa stain, investigators were first able to note chromosome banding. (Note: early studies were done on frogs and Drosophila; later studies on Caenorhabditis elegans, the 1000-cell nematode with a handy 3.5 day life cycle.)

    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?

    Stay tuned. Research in this area is one of the most important and vital now being undertaken.

    Morphogenesis, Evolution and Heterochrony: Timing is Everything

    Divergent ontogenies result in a diversity of adult forms.

    Over evolutionary time, mutations that resulted in developmental changes via the timing of morphogenetic events have resulted in embryonic changes leading to changes in adult organisms, and perhaps contributed to speciation.

    HETEROCHRONY (from the Greek hetero meaning "other" and chronos meaning "time") describes a change in the timing of ontogenetic events between two taxa. These can be the result of relatively small genetic changes that may not even be alterations in DNA sequence, but in the timing of particular genes being expressed during development.

    Neoteny and Progenesis: Two Examples of Heterochrony

    When compared to an ancestor or related taxon, a taxon is said to exhibit
  • neoteny if its somatic development is delayed with respect to its (expected/ancestral) reproductive development

    or

  • progenesis if its germline/reproductive development is accelerated with respect to its (expected/ancestral) somatic development

    The difference between these two processes is subtle, but important to note when comparing a taxon's development to that of a closely related taxon, or to that of a hypothetical ancestor, as an evolutionary geneticist is wont to do.

    Are We Really Just Big, Baby Chimpanzees?
    A tale of Heterochrony and Allometric Growth

    Many animals undergo ISOMETRIC GROWTH as they mature from new hatchling to adult. This means that all the body parts grow at approximately the same rate, and the adult proportions are not significantly different from those of the juvenile. For example, see our pal Batrachoseps, one of the few salamanders that has a terrestrial (not a gilled, aquatic) larva:

    A heterochronic change can result from a mutation that causes the rate of one cell line of the body to develop at a rate different from that of other cell lines in the body. This can result in ALLOMETRIC GROWTH.

    In a species that exhibits ALLOMETRIC GROWTH (from the Greek allo meaning "different" and metr meaning "measure" (and also, interestingly "womb")), different cell lines/body parts grow at different rates (relative to an ancestral, isometrically growing form) during development from juvenile to adult.

    Humans are a good example of a species that undergoes allometric growth. The head, limbs, and body grow at different rates, resulting in a human adult with proportions completely different from those of the newborn baby:

    .

    Hold that thought.

    PAEDOMOPRHY is a result of HETEROCHRONY

    In animals, the body becomes reproductively mature at a very specific stage of somatic development. In some species, a heterochronic mutation can cause the organism to become reproductive relatively sooner than an ancestral species. As we already have seen, this can happen in one of two ways:

    Neoteny: Germ line cell development proceeds at the same rate as in an ancestral species, but somatic cell development is retarded as compared to the rate in that same ancestor. Progenesis: Somatic development proceeds at the same rate as in an ancestral species, but germ line cell development is accelerated as compared to the rate in that same ancestor.

    In either case, the resulting condition is known as PAEDOMORPHY: a reproductive adult that has the juvenile form of the ancestral species.

    Examples of paedomorphic organisms:
    1. The Common Mudpuppy (Necturus maculosus) is a salamander that retains its juvenile gills as an adult
    Most salamander species have aquatic larvae that lose their external gills when they reach adulthood: Juvenile Tiger Salamander (Ambystoma mabeei):

    Adult Tiger Salamander:

    The mudpuppy is paedomorphic with respect to other salamander species: It retains its external gills as a reproductive adult due to either neoteny or progenesis:

    2. Many domestic dog breeds (Canis lupus familiaris), derived from wolves (Canis lupus), exhibit paedomorphy with respect to adult wolves.

    3. Homo sapiens, whose prolonged brain development period and relatively flat face reflect a prolonged juvenile period, relative to that of our closest relatives, the chimpanzees (Pan paniscus and P. troglodytes)

    Let's face it:

    Although some humans tend to look more like our ancestors than others do.

    Remember:

  • Paedomorphy/osis: the CONDITION of an adult organism retaining juvenile features as an adult

  • Progenesis and Neoteny are two PROCESSES by which this state can occur.

    Paedomorphy isn't the only possible result of Heterochrony. Other phenotypic differences between closely related species also can be a result of differences in developmental timing. It is the time of onset of certain developmental characters that determines differences in phenotype.

    In any species:

    Color pattern differences in animals can be a result of heterochronic changes that affect pigment deposition during ontogeny, which might possibly explain