We must not hide from ourselves the fact that the causal investigation of organisms is one of the most

difficult, if not the most difficult problem which the human intellect has attempted to solve, since every new cause ascertained only gives rise to fresh questions regarding the cause of this cause. Wilhelm Roux

Genes that control development are phylogenetically conservative.

 Most genes code for protein products; a few are RNA transcripts.  Names of genes are written italicized in lower case, while the protein product in Roman type (e.g. thewingless gene codes for the Wingless protein).  The genome of a cell contains in its DNA sequence the information to make many thousands of different protein and RNA molecules.  Both embryonic and differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism.

How does a fertilized egg cell know what kind of organ/organism to become? How does a cell determine which gene product to synthesize at any given time?

1. ENHANCERS ARE MODULAR. There are

various DNA elements that regulate temporal and spatial gene expression, and these can be mixed and matched. Whether the gene is transcribed or not will depend on the combination of transcription factors present.

Does this also mean that enhancers can direct expression of any gene sequence, or when placed in new locations?

Enhancers can direct the expression of any gene sequence. The -galactosidase gene from E. coli (the lacZ gene) can be used as a reporter gene and placed onto an enhancer that normally directs a particular mouse gene to become expressed in muscles. If the resulting transgene is injected into a newly fertilized mouse egg and gets incorporated into its DNA, the -galactosidase gene will be expressed in the muscle cells. Similarly, if the gene for the reporter gene green fluorescent protein is placed on the enhancer of genes encoding the crystallin proteins of the eye lens, GFP expression is seen solely in the lens.

Activation of regulatory genes such as Pax6 in new places Using Drosophila embryos, GAL4 transcription factor was placed next to an enhancer for genes normally expressed in the developing antennae. Therefore, GAL4 should also be expressed in antennal tissue. A second transgenic fly, placed the cDNA for the Drosophila Pax6 gene downstream from a sequence composed of five GAL4binding sites. In flies in which the Pax6 gene was expressed in the incipient antennal cells, part of the antennae gave rise to eyes. It appears that in Drosophila, Pax6 not only activates those genes that are necessary for the construction of eyes, but also represses those genes that are used to construct other organs.

How are enhancers detected?

The enhancer trap technique. (A) A reporter gene is fused to a weak promoter that cannot direct transcription on its own. This recombinant gene is injected into the nucleus of an egg and integrates randomly into the genome. If it integrates near an enhancer, the reporter gene will be expressed when that enhancer is activated, showing the normal expression pattern of a gene normally associated with that enhancer. (B) Reporter gene expression (dark region) in a Drosophila embryo injected with an enhancer trap. This expression pattern demonstrated the presence of an enhancer that is active in the development of the insect nervous system and which was unrecognized before the procedure.

 The c-myc gene synthesizes very short-lived mRNA and protein products when stimulated by a variety of growth factors, appearing suddenly as cells are induced from the G0 state into G1, and are degraded shortly thereafter. The c-myc proteins signal cell division, and if they are not degraded rapidly, the cell will continue proliferating, thereby increasing the risk of tumor formation. The translocation of the c-myc gene to an IgG enhancer in a single cell can cause a form of leukemia called Burkitt lymphoma.

2. FAMILIES OF TRANSCRIPTION FACTORS bind to the enhancer or promoter regions for specific gene expression a. Homeodomain factors- genes coding for proteins with a highly conserved homeobox of 61 amino acids (183 nucleotides) implicated in cranio-caudal segmentation of the body.  First discovered in Drosophila; mice and humans contain at least 38 Hox genes arranged in 13 paralogous groups in 4 different chromosomes.

 Hox gene mutations in mice have been shown to produce homeotic transformation of vertebral or spinal segments.  HOX13 mutations in man cause synpolydactyly (interphalangeal webbing and extra digits in hands & feet).  The fibroblast growth factor (FGF) selectively activates posterior homeobox genes  Transforming growth factor-F (TGF-F) selectively activates anterior Hox genes  Retinoic acid cause morphological abnormalities by posterization of Hox genes.

b.LIM domain- cysteinerich zinc-binding region responsible for protein-protein interactions.  Also shown to play roles in cytoskeletal organization, organ development and oncogenesis.  Absence of Lim proteins results in development of headless mammalian embryos (Lim-1 in the organizer and Islet-1 in motor neurons).

c. Paired box- consist of 9 known proteins (Pax1-9) consisting of paired domain of 128 amino acids that bind to DNA, and may also contain a homeodomain (Pax6 in the eye, Pax3 in the developing somite).

d. Zinc-finger proteins- DNAbinding region contains projections or fingers with Cys/His residues folding around a Zinc atom (WT-1 in the kidney, Krox20 in the rhombomeres of the hindbrain).

The family of zinc finger proteins are named after the amino acids that grasp the zinc.

Estrogen receptor

 The WT1 factor binds to the regulatory regions of several genes that are active during kidney development & inhibits the expression of certain growth factors in the developing kidney People with one mutant WT1 allele have urogenital malformations and develop Wilms tumor of the kidney

Functional domains of zinc finger transcription factors. Cysteine (C) and histidine (H) coodinate a zinc atom, causing the looping out of the "zinc fingers."

e. Nuclear receptor superfamily- On binding of the hormone (e.g. estrogen, progesterone, testosterone, cortisone, ecdysone, nonsteroid lipids such as retinoic acid, thyroxine, and vitamin D), the NR/hormone complex can form active dimers which can now bind to target genes. The DNA sequences capable of binding nuclear hormone receptors can be either be enhancers or promoters.

f. Basic helix-loop-helix (bHLH) proteins

 Active forms are heterodimers containing a basic DNA-binding region & a hydrophobic helixloop-helix region responsible for protein dimerization (e.g., E12, E47, myogenic factor MyoD).  Those with HLH but not the basic part form inactive dimers with other bHLH proteins & inhibit their activity (e.g., Id, a myogenesis inhibitor).

g. Winged helix proteins- have 100 amino acid winged helix domain which forms a type of DNA-binding region known as Fox proteins (Forkhead in Drosophila embryonic termini, HNF3F in the vertebrate main axis). h. T-box factors- DNA-binding domain is similar to the prototype gene product known as T in the mouse & Brachydury in other animals (endodermal determinant VegT, limb identity factors Tbx 4 and Tbx5). i. High mobility group (HMG) box factors- with no activation nor repression domains, but bend DNA to bring other regulatory sites into contact with the transcription complex (testis determining factor SRY, TCF factors activated by the Wnt pathway).

j. POU- contains a homeobox, plus a region coding for 75 amino acids which bind to DNA through a helixloop-helix structure (Pit-1 activates the genes encoding growth hormone, prolactin, & other pituitary proteins; Oct1 & Oct2, activate IgG genes; & UNC-involved in determining neuronal cell fates.

k. Leucine-zipper- bind DNA as dimers. A leucine zipper is formed by two a helices, held together by hydrophobic interactions between leucine residues, which are located on one side of each helix.  Examples include AP-1, CREB, and Gcn4.
The structure of the AP-1/DNA complex. AP-1 is a dimer formed by Jun and its homologous protein Fos. It contains a leucine zipper motif where two a helices look like a zipper with leucine residues (green color) lining on the inside of the zipper.

3. SELECTIVE INHIBITION OF MRNA TRANSLATION  During the growth of the oocyte, mRNAs are stored in mRNP (message ribonucleoprotein) complexes, localized within a specific region of the cytoplasm, or homogeneously dispersed within the cytoplasm of the entire oocyte. They are not translated until some condition is satisfied.  Oocyte maturation inhibitor (OMI) prevents translation of mRNAs unless cytoplasm alkalinization takes place after fertilization.  Similar mechanisms require specific signals on the mRNA, typically located in the 3 UTR.

4. CONTROL OF RNA EXPRESSION BY CYTOPLASMIC LOCALIZATION  Selective localization of messages is accomplished through their 3 UTRs.  Certain mRNAs in Xenopus embryos are selectively transported to the vegetal pole of the frog oocyte. After fertilization, these messages make proteins that are found only in the vegetal blastomeres
 In Drosophila, the bicoid & nanos messages are each localized to different ends of the oocyte. This localization allows the Bicoid protein to form a gradient wherein the highest amount of it is at the anterior pole, while the Nanos protein forms a gradient with its peak at the posterior.  The ratio of these two proteins will eventually determine the A-P axis of the Drosophila embryo & adult.

The bicoid and nanos mRNAs in Drosophila egg are localized near the anterior and posterior poles, respectively. The caudal, hunchback, and pumilio mRNAs are distributed throughout the egg cytoplasm. The gradients of Bicoid (Bcd) and Nanos proteins lead to accumulation of Hunchback protein in the anterior and caudal protein in the posterior of the egg. Because Pumilio protein requires Nanos protein for its activity as a translational repressor of hunchback, it functions only at the posterior end.