130 BICD -- Embryos, Genes, and Development Winter 2006
(Four parts, 200 points total)
Final Exam
______________________________________________________ Please PRINT your name here
ANSWER KEY
Please write your answers in ink. Requests for regrades will not be considered for exams done in pencil.
Part I (74 pts.)________________________ Part II (28 pts.)________________________ Part III (68 pts.)_______________________ Part IV (30 pts.)_______________________
I. (72 pts.) 1. (8 pts.) Signaling via two pathways is responsible for dorsal-ventral patterning of the vertebrate neural tube. What are the two pathways? Which one acts primarily on the dorsal neural tube and which on the ventral neural tube? What tissue is the original source of each signal? What tissue becomes the secondary signaling center in each case? BMP/TGF-B pathway 1 -acts dorsally 1 Shh pathway 1 - acts ventrally 1 BMP signal originates in ectoderm 1 / epidermis 1 Secondary BMP signalling center in roofplate 1 / Dorsal neural tube or neural tube 0.5 Shh Signal originates in notochord 1 / mesoderm 0.5 2nd signal center in floorplate 1 / ventral neural tube or neural tube 0.5 Diagrammatic answers must be fully labeled and clear for points to be awarded. D-V orientation should be labeled.
2. (8 pts.) Initially both the Mllerian and Wolffian ducts are formed in both males and females. Explain specifically how the hormones produced by the ovaries or testes determine which duct(s) will be maintained in each sex. Also name the structures eventually derived from each duct. Anti-Mullerian hormone AMH 1 -secreted by Sertoli cells 1 -degrades mullerian duct in males 1 Testosterone in males 1 -secreted by Leydig cells 1 -sustain Wolffian ducts in males, Wolffian ducts become epidydimis, vas deferens, seminal vesicles 1 (name at least one) / sperm ducts 0.5 -no Wolffian ducts in females due to absence of testosterone 1 In females Mullerian duct becomes Fallopian tubes, uterus, cervix, upper vagina 1 (minimum of one structure must be named) Incorrect answers: Wolffian ducts give rise to scrotum, penis, prostate, ovary, testes -0.5
3. (8 pts.) Referring to the development of the chick forelimb, what is the expected outcome of the following experimental manipulations? (a) Removal of the AER No limb development occurs. (2) (b) Replacement of forelimb mesoderm with leg mesoderm Proximal wing structure and distal leg structures forms (2) (c) Replacement of the AER with an FGF-soaked bead Normal Limb development (2) (d) Implantation of a Sonic hedgehog-expressing cell pellet into the anterior portion of the limb bud Mirror image duplication of digits (2) 4. (6 pts.) Treatment of fertilized Xenopus eggs with UV light leads to the development of "ventralized" embryos by preventing the cortical rotation. Treatment with high concentrations of lithium chloride (LiCl) has the opposite effect--development of excess dorsal tissue. LiCl treatment can even partially rescue UV-irradiated embryos, in effect bypassing the need for the cortical rotation. With the information that LiCl is an inhibitor of GSK-3, a key component of the Wnt signaling pathway (see figure), propose a specific molecular model for how LiCl treatment brings about the effects described above. Your answer must include a description of the normal function of the cortical rotation.
Cortical rotation serves to relocate Dsh at the vegetal pole. (1) Dsh inhibits GSK-3, (1) that prevents beta-catenin degradation(1), allowing its nuclear accumulation(1) and activation of downstream targets involved in organizer formation(1). LiCl serves as an inhibitor of GSK-3, (1) thereby allowing beta-catenin stabilization and activation of dorsal program, independent for the requirement of cortical rotation and possibly causing excess formation of dorsal tissue because LiCl is acting in a diffuse and widespread manner.
5. (6 pts.) The Sry gene was identified by careful molecular genetic analysis of phenotypically female XY individuals and phenotypically male XX individuals. Describe two pieces of evidence that Sry encodes the major testis-determining factor in mammals. Any one of the examples cited worth 3 pts Inaccuracies in detail and/or incomplete descriptions of evidence-deduct 0.5-3 pts XX mice with the SRY transgene form testes(3) Sry DNA injected into XX mouse zygote cause testes formation (3) XY human females have a mutant form of Sry AND XX human males have an extra copy of Sry on one of the X chromosomes where Sry should normally be absent. (3) Mesonephric migration experiment: Culturing XX or XY mesonephrons with XY gonads or with gonads from XX mice containing Sry transgene causes mesonephric cells to enter the gonads (3) 6. (8 pts.) Describe two specific examples of a change in Hox (homeotic) gene expression that results in a change in animal body plan. For each example, include the organisms and Hox gene(s) involved, the specific nature of the change in Hox gene expression, and the specific difference in body plan that this change makes. Inaccurate gene names 1, wrong gene names no points Mouse Hoxc8 KO: lumbar vertebra becomes a thoracic vertebra with ribs Hoxa11 and Hoxd11: lose radius and ulna Human HOXD13, digits fuse Drosophila Ubx When Ubx is deleted, 3rd thoracic segment becomes a 2nd thoracic segment and so fly has additional pair of wings Antp When Antp expressed in head, legs instead of antennae grow out of head sockets When Antp not expressed in second thoracic segment, antenna sprout in leg positions. Snake/python vs chick Hoxc-6 and Hoxc-8 expressed in most of the length of snake, causing ribs to be formed throughout most of body, limbs not formed. Caterpillar vs maggot Maggot:Dll excluded in abdomen by abdA and Ubx therefore no limbs form in abdomen region. Caterpillar: later in gastrulation Dll is expressed in 3rd through 6th abdominal segments in so it forms abdominal prolegs. Shrimp/lobster Brine Shrimp:uniform exprewssion of Ubx and abdA in thoracic segments-lack maxillipeds Lobster:lack ubx and abdA in 1st and second thoracic segments and these segments have paired maxillipeds.
7. (10 pts.) Expression of gene F in the snowy lemur is controlled combinatorially by DNA-binding transcription factors encoded by genes A-E. You know from genetic tests that mutation of gene A results in complete loss of expression of gene F. Mutation of either gene B or gene C results in ectopic expression of gene F. Mutation of gene D and gene E results in loss of expression of gene F in the brain and tail bud ectoderm respectively (these are gene F's normal expression domains). The regulators of gene F are expressed in the following patterns: A: Ubiquitous B: CNS except for brain C: Epidermal ectoderm and mesoderm from just anterior of the forelimb buds to just anterior of the tail bud. D: All CNS E: Epidermal ectoderm from just anterior of the hindlimb buds to the posterior tip of the tail bud. Consider transcriptional enhancer modules that contain the following combinations of binding sites. If you were to test the in vivo activity of these enhancers using GFP reporter genes in transgenic lemurs, where would you expect to see GFP expression in each case? (a) AADAD All CNS (1 pt) (b) ADBBDAD BRAIN (1pt) (c) AAEBAEB Epidermal ectoderm from just anterior of the hindlimb buds to the posterior tip of the tail bud (1 pt) (d) AEACECA TAIL BUD ECTODERM (1 pt, 0.5 for TAIL BUD) (e) ADCACAD All CNS (1 pt) (f) Enhancer b with all B binding sites mutated All CNS (1 pt) (g) Enhancer c with all B binding sites mutated Epidermal ectoderm from just anterior of the hindlimb buds to the posterior tip of the tail bud (1 pt)
(h) Enhancer d with all C binding sites mutated Epidermal ectoderm from just anterior of the hindlimb buds to the posterior tip of the tail bud (1 pt)
Which of the above modules are likely to correspond to normal enhancers associated with gene F? b and d (2 pts)
8. (10 pts.) A mutation in the wimpy gene prevents muscle precursor cells from differentiating into either smooth or striated muscle. A genomic DNA clone is isolated for this gene, and antibodies are produced that react with the protein. In situ hybridization experiments using the cloned DNA as a probe show that, in wild-type animals, mRNA encoded by wimpy is present at equal levels in all muscle tissue throughout development. RNA gel blot experiments reveal, however, that the wimpy mRNA in striated muscle is of a different size from the mRNA in smooth muscle. Antibody staining of wild-type animals shows that protein encoded by the gene is present in both types of muscle, but that it appears much later in the striated muscle. Propose an explanation for these results in terms of the molecular mechanisms that are involved in controlling the differential activity of wimpy. Size: state any one-5points Differential/alternative splicing Usage of different promoter Timing: state any one-5points, Masking Sequestration MicroRNA Nuclear retention Post-translational modification does not count in temporal difference unless antibody recognition depends on it Presence of different protein factors in each type of muscle- half credit only If fail to mention whether the mechanism accounts for size or timing, half credit.
9. (10 pts.) During the development of the vulva (egg-laying apparatus) in the nematode C. elegans, vulval precursor cells (VPCs) can adopt one of three fates. Cell-cell interaction plays a crucial role in determining VPC cell fates. (a) Which VPC cell fate(s) require(s) the inductive signal from the anchor cell, and which fate(s) do(es) not? Primary lineage: central vulval cells required high levels of signal (1) Secondary Lineage: lateral vulval cells require low levels of signal (1) Tertiary lineage: hypodermal cells do not required signal (1) Notch signals from central to lateral vulval cells no points Not indicating low level of signaling for secondary lineage: -0.5 points (b) Ablation of the anchor cell with a laser results in a remarkable phenotype. What is this phenotype (be specific; i.e., what fate do the VPCs express, and what is the consequence for the worm)? No Central Vulval cell forms. (1) VPCs become hypodermis. (1) Worm can't lay eggs but eggs do get fertilized. Bag of worms phenotype in which progeny eat their way out of adult worm (2)
(c) The anchor cell signal is received on the surface of the VPCs by the product of the let23 gene, a receptor tyrosine kinase. Deletion of the extracellular domain of a transmembrane receptor protein generally makes the receptor constitutively active and ligand-independent. What do you think would be the effect of expressing such a mutant Let-23 receptor in all the VPCs? RTK pathway in VPCs are constitutively activated and so all VPCs take on the central vulval cell fate (3).
II. (30 pts.) 1. (4 pts.) In the nematode C. elegans, the lin-14 gene, which encodes a transcription factor, is transcribed both early in development and later. However, Lin-14 protein is present only early in development. The lin-4 gene encodes a small RNA that is never translated into protein. When lin-4 is mutated, Lin-14 protein is found to be present in later stages of development as well as in early stages. Explain how the wild-type lin-4 gene functions to regulate lin-14. Lin4 encodes a microRNA gene, which is only transcribed in later stages of development--1pt Lin4 miRNAs recognize and bind to seven sites within 3'UTR of lin14 mRNA, forming imperfect dsRNA regions--2pt Binding of lin4 to lin14 mRNA targets the mRNA for degradation--1pt Loss of function mutation of lin4 leads to protein expression of lin14 in later stages.
2. (4 pts.) You have identified a mouse mutant that affects the behavior of neural crest cells. Specifically, you observe that these cells never migrate away from the neural tube. When you examine the expression of N-cadherin in the mutant animals, you find that it is expressed strongly throughout the neural tube and the neural crest cells. Propose a specific hypothesis to explain the abnormal behavior of the neural crest cells in this mutant. Cadherins are cell adhesion molecules. Their extracellular domain can recognize and bind to each other--1pt In wild type mice, N-cadherin is initially expressed in the neural plate (prospective neural tube and neural crest cells). Later on, as neural tube forms, N-cadherin expression is down regulated in neural crest cells, allowing them to migrate away from neural tube (N-cadherin) and epidermis (E-cadherin)--2pt In this mutant mouse, N-cadherin is not subject to down regulation any more. Its persistent expression in neural crest cells holding them from migration--1pt
3. (6 pts.) Define the terms diploblast and triploblast. Give one example of an organism of each type. Diploblast: organisms with only two true germ layers (ecto and endoderm)--2pt Example: Cnidaria sea (jellyfish, anemones, corals, hydra, et al)--1pt Triploblast: organisms with three germ layers (ecto, meso and endoderm)--2pt Example: Vertebrates ( humans, mice), echinoderms, insects, nemotodes et al.--1pt
4. (6 pts.) Familial adenomatous polyposis (FAP) is a dominantly inherited predisposition to colorectal cancer. Individuals with this disease are found to carry mutations in the APC gene (which encodes a component of the Wnt signaling pathway) and to display elevated levels of c-myc oncogene expression in the cancerous tissue. Explain the links between these facts. In the absence of Wnt, wild type APC recruits -catenin, together with GSK-3, directs -catenin for degradation, keeping -catenin at a low level and in the cytoplasm--2pt FAP patients carry a loss-of-function mutation of APC, which leads to -catenin accumulation and translocation into the nucleus. In the nucleus, -catenin binds to and turns LEF/TCF factor from transcriptional repressor to transcriptional activator--2pt Oncogene c-myc is one of the target genes of -catenin/LEF/TCF. Its transcription is elevated in FAP tissue. Elevated c-myc promotes cell proliferation, and contributes to the development of FAP--2pt
5. (4 pts.) For each of the following organisms, name the primary determinant of sex and describe the mechanism of dosage compensation: (a) mammals Primary sex determinant: Y chromosome determines male--1pt Dosage compensation: inactivation of 1X in XX individuals--1pt (b) Drosophila Primary sex determinant: X/A ratio, 1.0 female, 0.5 male--1pt Dosage compensation: hypertranscription of X in XY individuals--1pt Note: 1pt is taken if individual sex chromosome composition is not mentioned. 6. (4 pts.) Consider the role of Notch signaling in germ-cell development in the gonad of the nematode C. elegans. What cell(s) express the ligand for the Notch receptor? What cell(s) express the Notch receptor itself? Specifically what role does the Notch pathway signaling play in the germ-cell development process in this organism? What is the expected germ-cell phenotype of worms that are mutant for the gene encoding the Notch ligand? The distal tip cell expresses the Notch ligand (1 point) The germ line cells express the Notch receptor (1 point) Germ line cells in which Notch signaling is active due to contact with the ligand in the distal tip cell undergo mitosis (1 point) Notch signaling is not active in germ line cells not in contact with the distal tip cell. These germ cells undergo meiosis (1 point)
III. (68 pts.) 1. (12 pts.) You are investigating the role of Hox genes in the development of a recently discovered organism, which has a head, a trunk region containing forelimbs and hindlimbs, and a tail. You find that this organism has a Hox gene cluster with four genes in it, arranged as follows: HoxA HoxB HoxC HoxD
You find that HoxB is expressed in the front half of the trunk region of the body (which includes the forelimbs), while HoxD is expressed in the tail. (a) Where would you expect to see HoxC expression? Why? HoxC is likely to be expressed in the 2nd (rear) half of the trunk region, the region containing the hindlimbs--1pt As it's well conserved through evolution, Hox genes' arrangement along the chromosome correlates well with their expression along AP axis of the body--2pts Since HoxB is expressed in the front trunk and HoxD is expressed in the tail, it's reasonable to expect HoxC expression in the rear trunk--1pt
(b) You would like to find a loss-of-function mutation in HoxC. What mutant phenotype would you expect? Explain. Loss-of-function of HoxC might lead to disappearance of rear trunk characters (hindlimbs), and very likely a conversion into a forelimb segment--2pts Hox genes have been found to function partially by repressing the genes expressed more anterior to them. In this case, HoxC represses forelimbs and promotes the development of hindlimbs--2pts
(c) HoxB is not expressed in the head. What might be the effect of mis-expressing it there, and why? You might find forelimbs coming out of the head, or a complete conversion of the head into the forelimb segment--2pts This is similar to the Drosophila antennapedia gain of function mutant. HoxB specifies the forelimb segment, and likely overrules the HoxA function in the head more or less--2pts
2. (12 pts.) Spemann and Mangold found that if the organizer from a frog embryo is transplanted into the ventral side of a recipient embryo, the transplanted organizer establishes a completely new axis, resulting in the formation of twinned tadpoles joined at the ventral side. Describe in molecular detail what happens to the normal ventralizing signals when the organizer is transplanted into the ventral side of the recipient embryo. In your answer name two specific ventralizing signals and two dorsalizing factors, and describe how they interact. Two ventralizing signals (from ventral mesoderm): X-wnt8 and BMP-4--4pts Dorsalizing factors (from Spemann organizer)--4pts X-wnt8 inhibitors: Cerberus, Frzb, Dickkopt BMP-4 inhibitors: Noggin, Chordin, Nodal-related3 (Xnr3), Follistatin When a second organizer is transplanted to the ventral side of the embryo, the organizer forms a new source of X-wnt8 and BMP4 inhibitors, antagonizing the ventralizing signals in a ventral to dorsal reducing gradient. Combining the dorsalizing signals from the original organizer of the recipient embryo, only the region in the middle has low concentration of inhibitors, still responds to the ventralizing signals, and becomes the adjoined ventral sides of the twinned tadpoles--4pts
3. (16 pts.) Describe how the anterior-posterior axis is specified in the Drosophila embryo. In your description include the distributions and specific functions of bicoid, nanos, hunchback, and caudal mRNAs and/or proteins. The AP axis is first specified by the maternal genes in the embryo, namely bicoid, nanos, hunchback and caudal. Bicoid and hunchback specify the anterior. Nanos and caudal specify the posterior--2pts Bicoid mRNA is localized to the very anterior of the oocyte. After fertilization, Bicoid protein diffuses from the anterior the middle line--2pts Nanos mRNA is localized to the very posterior of the oocyte, and Nanos protein diffuses from the posterior to the midline--2pts Hunchback and caudal mRNA distribute evenly along the AP axis--2pts Bicoid protein activates zygotic hunchback expression. Bicoid protein also inhibits caudal mRNA translation, leading to the posterior half expression of caudal protein--4pts Nanos protein inhibits hunchback mRNA translation by binding to hb translational activator Pumilio, leading to the anterior half expression of hunchback protein--4pts
4. (16 pts.) The seven-stripe pattern of even-skipped expression is one of the first indications of segmentation in the Drosophila embryo. (a) How are these specific bands of expression generated? The expression of each stripe is controlled by a specific enhancer--2pts Each enhancer is activated by a specific combination of transcription factors, which are either maternal effect genes or gap genes--2pts
(b) Explain the roles of the Hunchback, Giant, Bicoid, and Krppel proteins in ensuring the precise expression of even-skipped in stripe 2. Bicoid and Hunchback are transcriptional activators, required for activation of evenskipped expression--4pts Giant and Kruppel are transcriptional repressors. Their presence confines the borders of the even-skipped stripe2, with Giant on the anterior side and Kruppel on the posterior side--4pts
(c) If the giant gene is mutated, what would you expect to happen to even-skipped stripe 2? Explain your reasoning. Giant mutation likely results in an anterior expansion of the even-skipped stripe2-- 2pts The anterior side repressor Giant is no longer functioning in an area where the activators Bicoid and Hunchback are present--2pts
5. (12 pts.) There has been a high level of interest recently in therapeutic cloning (or "somatic nuclear transplantation") as a means of obtaining embryonic stem (ES) cells for medical purposes. This approach seems particularly promising for treating diseases of the hematopoietic (blood cell-forming) system. Describe how therapeutic cloning was combined with gene therapy to cure a mouse homozygous for a mutation in the Rag2 gene (Rag2-deficient mice cannot recombine the DNA in their white blood cells to make antibodies). Culture tail tip cells, transfer the nucleus into the enucleated oocyte--2pts Activate and culture to the blastula stage--2pts Isolate and culture Rag2-/-ES cells--2pts Require Rag2 gene in ES cells by injecting Rag2 DNA vector--2pts Culture and induce hemotopoietic differentiation--2pts Thansfer the fixed hemotopoietic stem cells back the mouse, generating functional white blood cells--2pts
IV. (30 pts.) Distinguish between... the epithelial and mesenchymal cell states. Epithelial cell state: Layer of tightly associated or organized cells 1.5 Mesenchymal cell state: Loosely associated cells or migratory cells 1.5 segmentation and homeotic genes in Drosophila. Segmentation genes: Mediate transition from specification to determination 1.5/ specify the AP axis 1.5/ mediate formation of segments in embryo 1.5/ mediate 14 stripe pattern in embryo 1.5/ 2 or more examples of segmentation genes gap, pair-rule etc. 1.5 Homeotic genes: Specify identity of each segment 1.5/ mediate formation of limbs and other appendages 1.5/ mediate specific character of segments1.5 / 2 or more specific examples of homeotic genes 1.5 homeotic genes are expressed broadly 0.5 invagination and ingression. Invagination: Infolding of a sheet of cells or of epithelium 1.5 / Infolding of cells 1 Ingression: Migration of individual cells or of mesenchyme 1.5 / Migration or movement of cells 1 Detailed, clear diagrams illustrating the key difference may be awarded variable points splanchnic and somatic mesoderm. Splanchnic Mesoderm: Ventral part of lateral plate mesoderm 1.5/ Gives rise to circulatory system or heart or cardiogenic epithelium or endocardium 1.5 Somatic Mesoderm: Dorsal part of lateral plate mesoderm 1.5 / Gives rise to limbs or limb bud 1.5 / Forms part of body cavity lining 1.5/ Limb connective tissue 1.5 / limb skeletal tissue 1.5 / Lateral plate mesoderm 1 Unacceptable: muscle, somites, paraxial mesoderm cell fate specification and differentiation. Cell fate specification: First stage of commitment 1.5/ Restriction of cell fates in development 1.5 / Reduction of totipotency to several limited fates 1.5/ Progress in cell development, but not final stage 1.5/ " Telling the cell what to become" 1 Differentiation: Development of specialized cell types 1.5/ Final cell fate 1.5/ Irreversible, end point in cell development the yellow crescent and the gray crescent. Yellow Crescent: Mesodermal cells in tunicates that give rise to muscle cells 1.5/ Give rise to muscle cells 1.5/ Forms in chick embryo 0.5
Gray Crescent: Certain cytoplasmic determinants that induce gastrulation in Xenopus embryo development 1.5/ Induce dorsal-ventral axis patterning in embryo 1.5/ Site of dorsal axis in embryo/ Form after cortical rotation that occurs after sperm entry or fertilization 1.5/ Forms in frog or Xenopus embryo 0.5 homologous and analogous genes or body structures. Homologous genes or structures: Similar in structure or function, resulting from a progenitor in a common ancestor 1.5/ Common evolutionary pathways 1.5 / Same structure 0.5 Analogous genes or structures: Similar in structure or function, but arise from independent evolutionary origins 1.5/ Convergent evolutionary paths, distinct ancestors 1.5/ Same function 0.5 Good examples of each category may be awarded full points primary and secondary sex determination in mammals. Primary sex determination:Determination of the gonad to develop as ovaries or testes 1.5 / Ovaries in females, testes in males 1.5 / Genotypic sex 1.5 Secondary sex determination: Specification of the body phenotype for sexually dimorphic structures homophilic and heterophilic interactions between cell adhesion molecules. Homophilic interactions: Interactions between CAMs of identical type 1.5 / Interactions between CAMs of similar cells 0.5 Heterophilic interactions:Interactions between CAMs of different types 1.5 /Interactions between CAMs of different cells 0.5 post-transcriptional and post-translational gene regulation. Post transcriptional gene regulation: Regulation of the RNA for production of protein 1.5 / Examples - differential splicing, nuclear selection, differential mRNA stability, selective mRNA translation (minimum one example) 1.5 Post translational gene regulation: Regulation of protein activity after its formation/ Examples - differential protein modification, phosphorylation, differential protein stability (minimum one example) 1.5