1995 Nobel Prizes Lewis, Wieschaus, Nussein-Volhard
Thomas Hunt Morgan started his ground breaking work on the fruit fly, Drosophila, at Columbia University in 1910. He chose Drosophila as a model genetic system because of its short life cycle (about 2 weeks), ease of culturing in the lab, and high rate of reproduction. One of the first mutations that Morgan identified was white eyed flies. This famous mutation led to the discovery of sex linked inheritance and with the identification of other sex linked genes the discovery of crossing over. Work by Morgan, his colleagues, and his students led to the understanding that chromosomes carry genetic factors (genes) and that genes are ordered linearly on each chromosome, defining a linkage group. Morgan was awarded the Nobel Prize in Physiology or Medicine in 1933. Morgan moved from Columbia University to Caltech in 1928 and took the chairmanship of the biology department. Ed Lewis became a graduate student and later a faculty member in this intellectually inspiring environment of Morgan's Biology Department. He became fascinated with fly mutations that caused homeotic transformations. This class of mutation causes the transformation of one body into that of another like the picture of the four winged fly shown above. In this mutation the halteres of the third thoracic segment are transformed into wings that are normally only associated with the second thoracic segment. He collected and studied homeotic mutations for over thirty years before publishing a review in 1978 that put forth his ideas on the genetic mechanisms of body segmentation and the specification of segmental identities. He described the homeotic selector genes of the Antennapedia Complex (lab, Pb, Dfd, Scr, and Ant) and the Bithorax Complex (Ubx abdA, and AbdB) that were "master" regulators of segmental identity in the fly. The 3' to 5' linearly arrangement of these genes along the chromosome parallels their expression pattern along the anterior to posterior axis of the fly and specifics the unique identity of each segment. Even more surprising, this physical arrangement on the chromosome and parallel expression along the anterior-posterior axis is conserved in humans (mouse). Truly, he had discovered the molecular "Rosetta Stone" for developmental biology and evolution.

Eric Wieschaus and Christiane Nusslein-Volhard collaborated at the EMBL in Heidelberg on genetic experiments designed to identify the genes regulating the early development of Drosophila. Their approach was to apply saturation mutagenesis to the fly to try and identify every gene involved in early anterior-posterior pattern formation. They looked at larval cuticle patterns from thousands and thousands of fly mutations and were spectacularly successful in identifying the major classes of genes involved in generating the segmental body plan of the fly. The major classes were: Anterior group, Posterior group, and Terminal group (maternal genes) and Gap, Pairrule, Segment polarity, and Homeotic (zygotic genes). The early patterning of the embryo is specified by a paltry 50 or so genes!

The work of Lewis, Wieschaus, and Nusslein-Volhard provided satisfying molecular framework that, for the first time ever, explained animal pattern formation during embryogenesis. Even more amazing and of greater scientific impact, many of the molecular pathways controlling pattern formation in flies are shared among all metazoans, including humans! Wieschaus, Nusslein-Volhard, and Lewis shared the 1995 Nobel Prize in Medicine or Physiology for their ground breaking work on early embryonic development in Drosophila.

Fly eggs are centrolecital and cleave superficially with one of the fastest cleavage cycle times known, about every 8 minutes. Males mate with female flies, depositing sperm that is stored in the female spermatheca. Mature eggs travel through the spermatheca and are fertilized by one sperm that travels down the micropile, a thin channel just big enough for one sperm. This is the fly physical block to polyspermy. The male and female pronuclei fuse to produce the zygotic nucleus and the egg begins to develop. One unusual feature of fly cleavage is that the zygotic nuclei divide (karyokinesis) without undergoing cell division (cytokinesis). Thus the free nuclei exist and migrate within the egg cytoplasm with only a single plasma membrane surrounding the embryo. At the 10th cell cycle most of the nuclei migrate to the periphery of the egg just under the plasma membrane forming the syncytial blastoderm. A few cells migrate to the posterior pole where they cellularize to form the pole cells, the future germ cell primordia. The rate of karyokinesis slows over the next 3 cycles to 25 minutes per cycle. .

At the 13th cell cycle the plasma membrane invaginates around each of the superficial nuclei forming the individual blastomeres of the cellular blastoderm. The 14th cell cycle represents the time of the mid blastula transition and is indicated by asynchronous cell divisions ranging from 75-125 minutes. (See a movie of fly development 7 Mb) or go to the WWW site for FlyMove.

A fate map of the fly cellular blastoderm is shown at right. Notice the mesoderm at the ventral midline and endoderm at the termini. The pole cells will invaginate with the posterior midgut endoderm. Gastrulation begins shortly after the midblastula transition at the 14th cell cycle.
The figures at right (A-E) show the fate maps at the start of gastrulation and the basic cell movements that give rise to three germ layers. The first indication of gastrulation is the formation of the ventral furrow (B) and the invagination of the mesoderm. (C). Invagination of the anterior and posterior midgut endoderm is show in (C) and (D). Notice the pole cells migrating inward with the posterior midgut invagination. The pole cells form the germ cell primordial. In (E) you can see the neuroectoderm now lies at the ventral midline (dark blue) and the embryo is covered by dorsally derived ecotoderm, now epidermis. The scanning electron micrographs at far right show stages roughly corresponding to the illustrations. (E) corresponds to the 3rd picture with each of the 14 segments labeled at the extended germ band stage.

The last scanning electron micrograph at bottom shows a newly hatched larva showing the unique pattern of denticle bands associated with each segment. It was these laval denticle bands that Weischaus and Nusslein-Volhard scored as a phenotypic correlate of axial patterning mutations.

The figure at left illustrates that the each embryonic segment (or unsegmented region) can be fate mapped onto the adult fly. Notice that although each thoracic segment looks similar in the embryo they look quite different in the adult.
The axial patterning of the embryo starts during oogenesis with the expression of maternal genes in the ovarian nurse cells and follicle cells. Maternal genes specify the anterior, posterior, and terminal regions of the early embryo by causing cytoplasmic determinants to be localized or activated in these specific regions. The anterior determinant is the bicoid mRNA that is localized to the anterior pole of the egg and translated on fertilization to produce a gradient of the anterior morphogen, Bicoid protein. The bicoid protein ensures the activation the GAP gene hunchback that specifies the anterior pattern elements. Nanos mRNA is localized to the posterior pole of the egg and again is translated on fertilization to produce a gradient of the posterior morphogen, Nanos protein. Nanos acts to specify the posterior pattern elements by inhibiting the translation of maternal hunchback and ensuring the expression of the other important posterior morphogen, Caudal. The termini morphogen Torso is actually everywhere in the egg, but only activated at the termini by the a signal, Torso-like, from the anterior and posterior most follicle cells.
The broad gradients of the anterior morphogens, Bicoid and Hunchback, and posterior morphogens, Nanos and Caudal are then used to regulate the initial expression of the 9 zygotic GAP genes that are expressed in broad bands about 3 segments wide along the length of the embryo. GAP genes get their name from the fact that mutations in GAP genes lead to a missing gap in a set of adjacent segments. The broad bands of GAP gene expression and the original anterior and posterior morphogen gradients are used to specify the expression of the 8 Pair Rule genes that are each expressed in 7 bands along the anterior posterior axis of the embryo. This is the first expression pattern that correlates with the 14 segments that will develop in the embryo. The 7 bands of Pair Rule gene expression are associated with every other segment. Thus mutations in pair rule genes lead to defects in every other segment. Up to this stage pattern formation occurs in the syncytial blastoderm where morphogens can freely diffuse the length of the embryo and directly effect the zygotic nuclei. In fact, most of the genes involved in patterning up to this stage encode transcription factors. The Pair Rule and GAP genes regulate the expression of the Segment Polarity genes that are expressed in 14 stripes correlated with the 14 segments. Segment polarity genes are involved in specifying the anterior posterior pattern across each segment. Mutations in segment polarity lead to defects in pattern elements within each of the 14 segments. Finally, all the earlier genes, Cytoplasmic Polarity, GAP, Pair Rule, and Segment Polarity, provide regulatory information for the segmentally specific pattern of expression of the homeotic genes. The homeotic genes specify the unique identity of each of the 14 segments. Mutations in the homeotic genes lead to transformations in segment identity.
Cytoplasmic Polarity: Maternal Effect Genes of the Anterior, Posterior and Terminal Group.

The maternal genes expressed during oogenesis (see animation) obviously play a key role in early patterning. The relationship of the ovarian nurse cells and the follicle cells to the developing oocyte is shown at left. It is the ovarian nurse cells that export bicoid, nanos, hunchback, caudal and torso mRNA into the developing oocyte. Several other genes of the anterior group and posterior group are involved in localization of bicoid mRNA to the anterior pole and the transport and localization of nanos mRNA to the posterior pole. Maternal hunchback, caudal and torso mRNAs are not localized, but found uniformly in the mature egg.

When the egg is fertilized the localized bicoid and nanos mRNA are translated into protein that diffuse towards the opposite poles and set up the morphogen gradients that will regulate the expression of hunchback and caudal This in turn will lead to the regulated expression of the zygotic GAP, Pair Rule, Segment Polarity and Homeotic genes along the anterior-posterior axis of the embryo and specify the Acron, Head, Thorax, Abdomen, and Telson pattern.
In the figure at left you can see the anteriorly localized maternal bicoid mRNA in the mature egg. Fertilization triggers the translation of the localized bicoid mRNA and formation of the Bicoid protein gradient. Remember, the bicoid mRNA is the cytoplasmic determinant while the Bicoid protein is the morphogen.
The experiments shown below prove that bicoid mRNA is the anterior cytoplasmic determinant. First, the bcd- embryo gives rise to an embryo with a defective Te-Ab-Te that is missing anterior pattern elements (Acron-Head-Thorax). Injecting bicoid mRNA into the anterior end of a bcd- embryo rescues the phenotype. Adding bicoid mRNA to the middle of a bcd- embryo causes a Head to develop in the middle! Adding bicoid mRNA to the posterior end of a wild-type embryo causes a mirror image duplication of the anterior pattern (A-H-T-Ab-T-H-A). These experiments prove that bicoid mRNA and thus Bicoid protein are both necessary and sufficient to specify anterior pattern. Bicoid was the first "morphogen" regulating embryonic development to be identified.
Experiments were also done to test the role of the bicoid protein gradient. Was the gradient of bicoid protein really important for specifying the allocation of anterior structures? To test this, experiments were designed to over express bicoid protein in otherwise wild type embryos. When six extra copies bicoid were expressed the gradient of bicoid protein was significantly increased. This lead to posterior expansion of hunchback expression and the development of embryos that allocated too much of the embryonic field to anterior structures. This is an important and elegant proof that the gradient of bicoid protein is truly acting as the long sought after "morphogen" gradient suggested by many years of classical embryonic studies.
The figure at left is a summary of the anterior group and the role of bicoid.
Nanos mRNA is the posterior cytoplasmic determinant and Nanos protein is the posterior morphogen. Other posterior group genes (oskar, staufen) are involved in the transport and localization of Nanos mRNA. Just as with Bicoid the ovarian nurse cells express and export Nanos mRNA to the oocyte. At the end of oogenesis nanos mRNA is localized to the posterior pole while hunchback mRNA distributed throughout the mature egg. Fertilization triggers translation of Nanos protein and establishment of the nanos gradient. Nanos acts to limit the posterior influence of hunchback by repressing hunchback mRNA translation and destabilizing the mRNA. Caudal mRNA is translated into protein in the posterior region of the embryo, specifying abdomenal structures by activating the posterior GAP genes knirps and giant. Notice that embryos missing maternal Nanos mRNA lack any abdominal structures but still have terminal structures.
These two figures, A and B to left, illustrate the concentrations of cytoplasmic polarity (maternal effect) mRNAs in the mature egg and the subsequence morphogen gradients after fertilization. Again, notice that bicoid and nanos mRNA are localized to the anterior and posterior poles respectively, while hunchback and caudal mRNAs are distributed throughout the egg. Fertilization triggers the translation of maternal mRNAs and the establishment of the anterior and posterior morphogen gradients of Bicoid and Nanos proteins. Bicoid proteins represses the anterior expression of Caudal, while Nanos protein represses the posterior expression of Hunchback. At the early cleavage stage you now see the four protein gradients that determine the allocation of anterior and posterior structures.

Notice that there is much more information in the four protein gradients than in the initial state of the unfertilized egg. The antagonistic gradients that develop in the fertilized egg will develop based on the size of the fertilized egg. Thus there is an ability to "size regulate" to ensure the proper allocation of cells to each pattern domain (anterior vs. posterior). The unique combination of protein concentrations along the anterior-posterior axis will now be used to specify a unique pattern of GAP gene expression.

The terminal group genes are maternal expressed in both the nurse cells and the follicle cells. The nurse cell produces Torso mRNA and export it to the developing oocyte. It distributes throughout the oocyte in an "inactive" state. The anterior and posterior most follicle cells express Torso-like, a maternal protein that activates Torso just in the terminal regions of the egg. Fertilization leads to the production of a Torso signal gradient that inactivates a transcriptional repressor, Groucho protein, of the GAP genes huckebein and tailless. Thus these two GAP genes are expressed only at the termini.

Why are the two terminal structures, acon and telson, unique if the terminal signal is the same? That seems to depend on exposure to Bicoid. Bicoid transforms the default terminal structure, the telson, into the anterior specific structure, the acron.

This last figure is a summary of the Terminal Group genes. Torso is the terminal group morphogen even though it is uniformly expressed in the embryo. The anterior and posterior most follicles express Torso-like and activate Torso just in the termini. Activated Torso generates a graded morphogen signal that represses Groucho in the terminal zones. Groucho is normally active throughout the embryo and represses the terminal group GAP genes huckabein and tailless.

Notice that as development proceeds the embryonic field expresses a more complex variety of morphogen gradients that can be used to further specify even more complex domains of gene expression. More complex systems can be self generated from less complex ones by relying on the biophysical properties of the embryonic field and complex feedback and feed forward gene regulatory networks.

For more information on maternal effect genes regulating cytoplasmic polarity see the Interactive Fly