VOKES EXAM 1 STUDY GUIDE
VOKES EXAM 1 STUDY GUIDE BIO 349
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This 7 page Study Guide was uploaded by Clayton Payne on Sunday February 21, 2016. The Study Guide belongs to BIO 349 at University of Texas at Austin taught by Dr. Steven Vokes in Spring 2016. Since its upload, it has received 98 views. For similar materials see Developmental Biology in Biology at University of Texas at Austin.
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Date Created: 02/21/16
EXAM 1 STUDY GUIDE C. elegans, Drosophila, Xenopus, Hox Genes, and Gene Regulatory Networks Xenopus Gastrulation ● Induction of the dorsal hemisphere ○ Sperm centrosome is transported into the oocyte at the sperm binding site in the animal hemisphere. ○ Centrosome organizes microtubules that extend down to the vegetal cortical cytoplasm, binding the cytoskeleton of the cortical cytoplasm. ○ Wnt11 mRNAs and other Wnt pathway proteins are transported down the microtubules by kinesin and concentrated in the vegetal hemisphere. ○ The entire cortical cytoplasm is rotated approximately 30 degrees, forming the socalled grey crescent where the cortical and internal cytoplasm overlap. This is the future site of the dorsal blastopore lip and organizer region. ○ Wnt11 prevents betacatenin degradation in the future dorsal side of the embryo, which induces dorsal cell fate. ● Induction of mesoderm ○ VegT and other Veg proteins exist in vegetal hemisphere prior to midblastula transition as maternal mRNAs. ○ At MBT, VegT drives transcription of endoderm genes, inducing endodermal formation. ○ VegT also drive transcription of Nodal ligands, which are secreted onto the cells above the endodermal cells, inducing mesodermal cell fate. Vg1 is also secreted, inducing nodal signalling. ○ Thus, on their own animal and vegetal tissue will differentiate into ectoderm and endoderm, respectively. When stacked on top of each other, the vegetal tissue will induce the animal tissue to form mesoderm. This doesn’t happen in a normal embryo because the animal and vegetal caps are separated by a blastocoel cavity, and thus mesoderm will only form along the equator. ● Formation of the organizer ○ The cells of the dorsal blastopore lip, head mesoderm, (above the dorsal mesoderm), chordamesoderm, and pharyngeal endoderm, are collectively referred to as the “organizer”, because they 1) induce formation of the neural tube from ectoderm, 2) form the notochord (from dorsal mesoderm), 3)induce ventral mesoderm to become somites, 4) initiates gastrulation movements. ○ The organizer is induced by convergence oodaland eta catenindorsal) signaling. ○ VegT and betacatenin work synergistically to drive transcription of Xnr genes, the highest concentration of which is seen in the Nieuwkoop center, a region of dorsal tissue directly below the future organizer. ○ Xnr is a Nodal ligand and is secreted onto the future mesodermal cells. The dorsalmost mesoderm, where Xnr concentrations are the highest, becomes the organizer. ○ Transcription factor produced by Xnr signalling and transcription factors produced by beta catenin together drive transcription of bone morphogenetic protein (BMP) inhibitors such as Noggin and Chordin. ● Migration of the organizer and gastrulation ○ Gastrulation begins with the involution of the organizer. ○ ○ The pharyngeal endoderm and head mesoderm are the leading edge of the involution and induce fore and midbrain, the chorda mesoderm induces hindbrain. ○ ○ Most of the dorsal mesoderm will form the notochord, an anteriorposterior structure lying below the neural tube. ● Induction of the epidermis and the neural tube ○ The DEFAULT differentiation state of ectoderm cells is neural tissue. ○ BMPs, expressed throughout the ectoderm, inhibit neural differentiation and induce epidermal fate. ○ Noggin and Chordin, expressed in the notochord and secreted onto ectoderm above it, inhibit BMPs, and thus allow neural tissue to develop. ○ A gradient of Wnt8 along the anteriorposterior axis determines antpost patterning, with the highest concentration posteriorly. ○ Wnt8 inhibitors, like Cerberus allow formation of head structures, injection of Cerberus mRNAs will produce a multiheaded embryo. Hox transcription factors ● Hox gene loci and expression patterns ○ Hox genes are arranged in linear clusters on chromosomes. ○ They are an ancient gene family. Drosophila have a split cluster on 1 chromosome. Humans have 4 clusters across 4 chromosomes which arose from gene duplication events, leading to high genetic redundancy. In knockout studies, 1 particular hox gene you want to investigate = 4 knockouts you have to do. ○ If you assess Hox gene expression from anterior to posterior body segments, you find that they are expressed in the same linear order they are found on the chromosome. ○ Expression pattern persists in adulthood(!) ● Defining what body part will form in a particular body segment. ○ Generally, when you knock out a hox gene, a posterior structure will be transformed into an anterior structure, i.e., you will turn lumbar vertebrae, into thoracic vertebrae. ○ The stereotypic nature of these homeotic transformations is due to the colinear nature of their expression. ● Translational regulation of hox genes ○ Mice who have the Ts gene knocked out develop with multiple homeotic transformations. ○ Ts is not a hox gene, but encodes ribosomal protein L38, which is part of the ribosomal machinery. This was discovered by injecting Ts knockout mice with Rpl38 mRNA. ○ Rpl38 is necessary for translation of some, but not all hox genes. Since multiple genes are affected, multiple homeotic transformations occur. Drosophila DorsalVentral Patterning ● Germ cells vs. Somatic cells ○ Pole cells develop at the posterior end of a Drosophila embryo. These pole cells eventually become germ cells. ○ A female’s germ cell (oogonium) divides to form 1 oocyte (egg) and several nurse cells during oogenesis. Nurse cells provide maternal mRNAs to the oocyte prior to fertilization. ○ The nurse cell/oocyte complex is surrounded by a layer of follice cells, which are somatic. The oocyte and follicle cells are separated by a thin layer of extracellular space called the perivitelline space. Germ cells vs. somatic cells are an important distinction when considering genetic crosses and tissue transplant experiments ● After fertilization, the embryo’s nucleus goes through 10 cycles of replication and division, and localize to the outer layer of the embryonic cell. In the 11th cycle, zygotic transcription begins, as well as cellularization of the syncitial nuclei. ● Prefertilization events 1. Nurse cells secrete Gurken mRNAs into the oocyte, where they are localized between the nucleus and posterior side of the cell. Nurse cells also secrete Dorsal mRNAs, which remain translationally repressed throughout the oocyte until cellularization. Note that Dorsal RNA is evenly distributed throughout the oocyte. 2. Gurken mRNAs are translated and secreted into the posterior perivitelline space, where they bind Torpedo, a membrane receptor protein expressed in follicle cells. Posterior follicle cells then send a signal back to the oocyte, which recruits PAR1 proteins to the posterior end. PAR1 organizes and polarizes the microtubules, with the capped () ends being anterior and the dynamic (+) ends being posterior. 3. Motor proteins transport the nucleus along microtubules to a more dorsal position. Gurken mRNAs are translated again and secreted into the dorsal perivitelline space, where the again bind and activate Torpedo. 4. Torpedo activates a cascade which represses Pipe expression in dorsal follicle cells. ● Postfertilization events 1. In ventral follicle cells, where Pipe is synthesized, Pipe adds sulfate groups to membrane proteins facing the perivitelline space. These sulfated proteins recruit a GD complex, which recruits Snake protein. The GD/snake complex cleaves and activates Easter, which in turn cleaves and activates Spatzle, generating a Spatzle gradient along the ventral axis. Active Spatzle binds Toll, a receptor in the membrane of embryo cells facing the perivitelline space. Note that Toll is evenly distributed throughout the egg but only activated ventrally. 2. Dorsal is synthesized from mRNA about 90 minutes after fertilization, just before cellularization begins, and forms an inactive complex with Cactus protein. 3. The activated Toll receptor activates Pelle kinase, which phosphorylates Cactus. Phosphorylated Cactus is degraded. 4. Unbound Dorsal is able to be translocated to the nucleus, and does so in the ventral embryonic cells where Cactus has been degraded. Once translocated, Dorsal acts as a transcription factor for genes which drive a ventral cell fate. Gene Regulatory Networks ● Temporally and spatially precise transcription of genes is essential for proper development and cellular function. Organisms have evolved mechanisms to ensure genes are kept under tight transcriptional control. ● Doublenegative linear pathways: let’s say you want to Gene C to be transcribed after Gene A is transcribed, and ONLY after Gene A is transcribed. The easiest way for this to happen would be fore gene product A to act as a transcription factor for gene C. However this poses a problem: in the absence of gene product A, Gene C may undergo “leaky” expression. Thus, a more secure way of regulating is to have Gene C be repressed by gene product B, and Gene B repressed by gene product A. Thus, Gene C is actively repressed until Gene A is transcribed, at which point the repression can be lifted. ● Positive feedback loops: L et’s say you want Gene B transcribed after Gene A, but you want lots and lots of product B. Have product B be a trxn factor for Gene A, and B be a trxn factor for Gene B. This is good for amplifying signal but the loop poses the problem of becoming unstable. ● Negative feedback loops: L et’s say you want to limit the signal to a very brief period of development. Have product B repress Gene A, which can no longer drive transcription of B. ● Feedforward regulation: I n this case, B is a trxn factor for C, and A is a trxn factor for B and C. As a result, C is transcribed twice as much as it normally would be (signal amplification). A Gene Regulatory Network Summarizing Cell Specification in C. elegans. ● Note that the schematic is slightly inaccurate in that it shows the E and MS cells already divided. MOM1 (Wnt ligand) would in fact be signaling to POP1 (TCFLEF homolog) at the EMS cell stage, before cell division. ● Par proteins, as well as SKN1, are maternal effects genes. Par1/2 are sequestered into the Pcell via the sperm centrosome and microtubules, in a mechanism similar to the Wnt mRNA sequestration in Xenopus, and lead to PIE1 expression. ● SKN1 is present throughout the entire Pcell prior to division into P and EMS cells. SKN1 drives the expression of genes specifying an EMS cell fate, and also plays a role in driving an E cell fate. In the Pcell, it is unable to drive E fate due to the presence of PIE1, which inhibits transcription by inhibiting RNA polymerase. In MS cells, POP1 actively inhibits the expression of Egenes.
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