The cells of a 25 year old are not youthful. The chromosome has been degraded, leading the person rapidly toward a life dominated by wrinkles, sedentariness, and AARP. Telomeres are short repetitious chromosomal sequences. They prevent the degradation of the chromosome during DNA replication that is terminated early. When the telomere sequence becomes too short, the cell is unable to safely replicate its DNA and thus halts replication. As the cell becomes old, though, it becomes inefficient and will eventually die (this is a causative agent of aging). To restore the telomeres, most cells use an enzyme called telomerase. This enzyme attaches new strands of telomere to the 5’ end of the chromosome. Telomeres, which are a vital part of the chromosome, are vital to regulation of cell cycles, aging, and tumor development.

Telomeres are 5’ repetitious 8-10 base-pair sequences. Since DNA Polymerase terminates DNA replication before reaching the end of the template, the telomeres sacrifice themselves so that the rest of the DNA is fully replicated. Once telomeres become too short, however, newly replicated strands have no protection from premature termination. As a result, cells with too-short telomeres enter senescence, a phase during which they do not replicate. Extended senescence, though, results in reduced efficiency of the cell. It gradually becomes unhealthy and will eventually die without replacing itself. Fortunately, a polymerase named Telomerase extends the telomeres. This, in turn, prevents senescence. Telomerase carries its own template strand in the form of RNA. In this manner, it can replicate the telomere sequence beyond the chromosomal template.

Loss of telomere length might be a causative agent of aging. Guo-Liang Yu, of Blackburn laboratory, demonstrated in 1990 that immortality is dependent upon telomerase in Tetrahymena. Telomerase mutants lose cell mass and die. Similarly, other members of the Blackburn laboratory have observed similar degradation in other species of Telomerase mutants. Similarly, if Telomerase activity is low or absent in human cells then after a period of time the human body will undergo a similar process (shrinkage followed by death). Human newborn somatic cells divide 80 to 90 times in culture, while somatic cells from a 70 year old divide only 20 to 30 times. After division ceases, senescence occurs. During senescence, cells under morphological transformations and begin operating less efficiently. In addition, Greider, Harley and their colleagues observed that most normal somatic cells lost telomere segments during incubation. This means that Telomerase was not active. Similarly, Greider, Harly, and Hastie (of Medical Research Council in Edinburgh) found that telomeres in some human tissues shorten with age.

A treatment could be generated to exploit the connection between aging and telomere length. Since Telomerase is known to regenerate telomerase, then CMV-Telomerase could be transduced via a non-specific adenoviral vector into most eukaryotic cells. This would give the host cells the ability to encode Telomerase, which would then regenerate telomeres. In conjunction with a hormonal treatment to encourage cell proliferation, the cells would be able to exit senescence. Daughter cells would operate more efficiently than their ancestors, thus giving the patient a more youthful look. Such a medication would have tremendous benefit for the elderly, as it could have tremendous benefit physically. Mentally, though, the effect would be negligible since neuron proliferation is rare. For example, an ideal candidate might be a 25-year-old patient suffering from wrinkling, indigestion, and age-related discomfort. After being infected, the patient might notice that youth has been reinstated within just a few months. This treatment would have many risks, though, for it might induce immortality and lead to the development of tumors. A way to prevent Telomerase-related cancer development might be to encode a CMV-Telomerase promoter activator that is cleaved by Hfl protease (present during high cellular metabolic activity, as in cancer cells).

In conclusion, telomere loss leads to aging, and this process can be reversed by telomerase. Telomerase treatment might have great therapeutic and cosmetic benefits, but it also provides an environment hospitable to cancer. As a result, genetically engineering the human body to look young is currently a bad way to reverse the effects of aging. A good way is a face-lift.

References

Greider, E. & Blackburn, H. (1996). Telomeres, Telomerase and Cancer. Scientific American, February 1996. Retrieved February 1st from http://www.genethik.de/telomerase.htm

Children have 1,000 trillion synapses. By the time somebody is 30 years old, though, only 100-500 trillion of their synapses remain. This means that in just 7,000 days, as much as 90% of a person’s synapses disappear. Fortunately, though, this decline is not completely disastrous; many studies indicate that 30 year olds are not catatonic. Synapses are specialized neuronal junctions in which neurons, muscles, or glands can communicate with each other. Over time, synapses strengthen, die, and move based on the activities of the individual. Psychoactive drugs affect synapses either directly or indirectly, and can give the patient artificial happiness or emptiness. Synapses are constantly changing to accommodate activities, interact with chemicals, form memories, and encourage development.

Neurons communicate with each other, muscles, and glands via the synapses, which change with age. A synapse is a junction where the presynaptic neuron releases a neurotransmitter, and the postsynaptic cell undergoes transformations in response. As a result, synapses are crucial to thought, perception, mobility, and homeostasis. Humans initially have 1,000 trillion synapses. However, this number declines dramatically in babies as they eliminate excess synapses. This is done by programmed cell death, which makes sure that the growing brain does not get too big. Adolescents undergo further synaptic pruning; very little programmed cell death occurs, however. This makes sure that synapses are made more efficient; more neurotransmitter is available for the synapses, which are necessary. Unnecessary synapses, which impede efficiency, are eliminated. After adolescence, the body remains at a peak for several years. After this peak, though, the body quickly deteriorates in a process called aging. As people continue to get old, the human brain declines in function despite its changes in gene expression. It tries desperately to down-regulate synaptic plasticity genes and synaptic vesicle release. It focuses more on neuronal maintenance by up regulating genes associated with stress, DNA damage, and antioxidant attack. As the synapses become increasingly static and the neurons lose their youth, the effects of senescence take effect and the brain eventually becomes necrotized. This process begins around the age of 25.

To avoid this depressing reality, many people become addicted to drugs which function at the synapse. Agonists, such as nicotine, mimic neurotransmitters to activate receptors. Antagonists, such as atropine, bind to receptors and block their activation. Overexposure to a neurotransmitter results in the disappearance of its receptors. This is a response mechanism to too much neurotransmitter, resulting in drug tolerance. To avoid reality, then, druggies will need to take increasingly high drug doses to try to get their first high. Therefore, overexposure results in decreased synaptic efficacy. There are three primary mechanisms by which these psychoactive drugs take effect. The first is that psychoactive drugs can prevent an action potential from starting, thereby preventing any related synaptic activity. Examples include lidocaine, which binds to voltage-gated sodium channels. The second mechanism is that a drug can alter neurotransmitter synthesis, resulting in no, reduced, or overproduction of a neurotransmitter. A third mechanism is interference with neurotransmitter release. Black Widow Spider toxin increases release of a neurotransmitter, thereby heightening synaptic efficacy momentarily. Botulism and tetanus decrease neurotransmitter release.

When new memories are formed, synaptic changes occur. An extremely basic example of this is shown by Hebb’s Rule. According to Hebb’s Rule, a synapse that is repeatedly activated in conjunction with the firing of a postsynaptic neuron will be strengthened by structural and chemical changes. For example, many people associate UCLA with bears. The synapses between the UCLA neuron and the bear neuron will therefore strengthen. This strengthening occurs by release of more neurotransmitter, removal of excess synapses, and the presence of more receptors. As people age, though, the genes related to synaptic plasticity are down regulated. As a result, old people have a harder time forming new memories.

In conclusion, the brain is constantly changing. As people approach their third decade on this planet, they are losing hundreds of trillions of synapses. Fortunately, though, this is necessary to remove excess synapses (excess synapses reduce neuronal efficiency). As people get old and lose synapses, their minds also undergo tremendous morphological transformations.

There are two kinds of people: bunny huggers (Mukerjee, 1997) and cruel scientists (Barnard & Kaufman, 1997; Mukerjee, 1997). The overzealous bunny huggers (BH) argue that animal research is wasteful because animals are bad model organisms and deserve ethical treatment. The cruel scientists (BS) argue that animal research is necessary to scientific advancement because animals are good model organisms, and may be used after an in-depth cost-benefit analysis. Both of these arguments are seriously flawed because they are biased, emotional, and incomplete. This paper will offer a balanced approach to animal experimentation.

There are three persuasive arguments against animal research. First, the lab environment and methodology are very stressful for animals. Many lab animals experience isolation and enclosure. This unnatural lifestyle causes pain and suffering for subjects. The official web-site for PETA, a prominent BH organization, cites many cases where lab animals are placed under excessive pain, distress, and isolation. A second BH argument explains that animals are not good model organisms. For example, advancement in polio research was delayed because in vivo results misled researchers. A second example is that for many years, cigarettes were considered harmless because non-human primates react less to cigarette smoke. In both of these cases, many human lives were lost because the model organisms used did not accurately represent humans. The third BH argument is that there are many in vitro alternatives to in vivo. In the past, chemicals were tested for corrosiveness in vivo; synthetic skins, though, nearly eliminated the need for in vivo tests.

On the other hand, there are compelling proponents of animal research. The most persuasive CS argument is that animal research has led to tremendous human benefit. Research into dendritic cells has generated many novel new cancer medications with no adverse effects and complete tumor elimination (Antoni et al, 200). Animal research was necessary before Phase I clinical trials to test for autoimmune development. A second CS argument ascertains that certain animals are model organisms. For example, rats are considered “genetic stunt doubles” to humans, and results from rat studies have been successfully extrapolated onto humans. (Pearce, 1999) Rats and humans share 85% of the same gone, and rats are anatomically anthropomorphic. The third CS argument is that animal research is carefully regulated by both scientists and non-scientists. This ensures that animal research is conducted only if there is a low cost/benefit ratio. In addition, in places where animal research is conducted there is a department devoted to supervision. This third argument states that although there is although lab animals will always undergo some form of suffering, the benefit that arises will outweigh their sacrifice.

A compromise for BH’s and CS’s would discriminate between primates, rodents, invertebrates, plants, and microbes, and require rodent testing before primate testing. Primates, which undergo the most humanlike emotional transformations, require the most supervision and regulation. In-depth analysis and meta-analysis of animal research could give insight into which scenarios had low cost/benefit ratios. In addition, more literature review might occasionally eliminate the need for primates. By examining outliers (ie, rodents) then solid results with less variables can be generated. Rodent experiments, which are most common, are necessary for examining systemic diseases. For example, tumors cannot be fully studied in vitro because countless variables influence results. While rodent experiments require as much supervision as primate experiments, regulation should be more lenient. The third group of subjects, invertebrates, requires very little ethical intervention. The primary consideration with invertebrates is maintenance and disposal. It is important that invertebrates are maintained well for hygienic and ethical reasons, and they must be disposed of respectfully and cleanly (ie, not piled up in an alley behind CHS). However, since invertebrates lack the sophisticated vertebrate view of reality, many factors influencing vertebrate research—such as discomfort, isolation, and pain—are not as important. Fourthly, the only regulation necessary for plant research involves disposal. The cost of plant maintenance motivates research to treat the plants optimally. In addition, wasteful plant research will not get funding and will cease. However, plants still viable after research should be donated for domestic enjoyment. Lastly, microbes should be treated the same as plants. Since some microbes are pathogenic, though, a culture should be Clorox-washed before being tossed into a toxic waste. Essentially, financial factors will ensure that plants and microbes are not wasted.

In conclusion, there are both legitimate and illegitimate components of the animal research debate. Compromise is essential if the benefits of animal research are to dominate the costs to the creatures involved.

References

Anti, R. et al. (2000). Generation of T-Cell Immunity to a Murine Melanoma Using MART-1-Engineered Dendritic Cells. Retrieved January 13, 2006. http://www.immunotherapy-journal.com/pt/re/jimmuno/abstract.00002371-200001000-00008.htm;jsessionid=DR5nv2xvNXj0GCNth2416JcOlV3417gEJVYrviM5WYQQB5wqgMoi!1871300765!-949856144!9001!-1

Barnard, N. and Kaufman, S. (1997). Animal Research is Wasteful and Misleading. Retreived January 18, 2006. http://mipwww.life.uiuc.edu/404%20Docs/SciAm% 20articles/AnmResrchProCon.pdf

Mukerjee, M. (1997). Trends in Animal Research. Retreived January 18, 2006. http://mip www.life.uiuc.edu/404%20Docs/SciAm%20articles/AnmResrchProCon.pdf

Pearce, J. (1999). Of Mice and Men. Retrieved October 17, 2004,

http://www.ornl.gov/info/ornlreview/rev27-12/text/mnmmain.html

Rowan, A. (1997).. The Benefits and Ethics of Animal Research. Retreieved January 18, 2006. http://mipwww.life.uiuc.edu/404%20Docs/SciAm%20articles/AnmResrchProCo n.pdf

Introduction

Pseudomonas spp. are common soil-dwelling aerotactic Gram-negative proteobacteria with the unique ability to utilize exotic carbon sources for energy. While the majority of species can only perform respiratory metabolism, certain Pseudomonad species can anaerobically also use nitrate, nitrite or nitrous oxide as a terminal electron acceptor. Given a culture of Pseudomonad (Pseudomonas 80), I performed physiological, biochemical and molecular (Experiments 3C & 3D) analysis to determine its species.

In Experiment 3A, brightfield and phase-contrast microscopy were used to determine the size, motility, aerotactic behavior and Gram state of P. 80. Later in Experiment 3A, an oxidase test and oxidation/fermentation test gave me basic knowledge of P. 80’s metabolic pathway and ability to anaerobically ferment and deaminate (use amino acids for energy). Experiment 3B illuminated further physiological characteristics of P. 80, such as its optimal temperature, as well as tremendous biochemical information. Various growth media — both replica- and streak-plated — were invaluable instruments to confirm that I had a Pseudomonad and also narrow down the species. Lastly, Experiments 3C and 3D further described P. 80 via PCR, electrophoresis and sequence analysis.

Methods & Results

An important part of identifying my P. 80 was confirming it was even a Pseudomonad. To do this, I used Gram-stains, brightfield microscopy, wet mounts, and phase-contrast microscopy to identify a Gram-negative and motile 3-5µm bacillus with aerotactic behavior. I used standard tap water, ethanol, iodine, purple and pink stain on a heat-fixed glass slide for Gram-staining, and 10x, 40x and 100x magnification for microscopy. With my findings so far consistent with known data on Pseudomonads, I performed an oxidase test by smearing P. 80, S. epidimidis (negative control) and P. aeruginosa (positive control) to verify that cytochrome c was present in the cellular respiratory pathway. With a positive oxidase result, I confirmed that cytochrome c was present. Lastly, I confirmed that my sample could not anaerobically ferment glucose (a unique inability of Pseudomonads). I used the oxidation/fermentation (o/f) test, whereby I stab inoculated a glucose-rich media, and then either poured oil over the media (anaerobic conditions) or did not (aerobic conditions) before incubation. Sugar catabolism alters the pH, yielding a yellow color from a pH indicator; deamination of the added peptone amino acids yields a blue color. My sample could not catabolize sugar anaerobically (fermentation) but was able to deaminate anaerobically, as expected.

After examination to confirm I had a Pseudomonad, I went about determining what species was P. 80. I probed deeper into the nature of my P. 80 by streak- and replica-plating onto a myriad of agars.

To determine P. 80’s optimal temperature, I inoculated three tubes of YTB (similar to YTA, described above) and incubated at 4°C (no growth), 30°C (significant growth) and 40°C (moderate growth). Also, I expanded on the oxidation/fermentation test by streaking (aerobic conditions) and stabbing (anerobic conditions) King’s medium B agar supplemented with 0.2% KNO3. Examining the incubated tubes under UV light revealed that P. unkown did not fluoresce (no glow), reduce nitrate to N2 gas (which would form bubbles) nor reduce nitrate to nitrite (which causes a red color upon addition of a nitrite reagent). Now clear that P. 80 did not reduce nitrate at all nor express fluorescent pigments, I confirmed that my positive and negative controls (P. aeruginosa and P. fluorescens) did and did not, respectively, reduce nitrate fully to N2 gas and secrete a fluorescent pigment.

The next step was to analyze P. 80 at a molecular level. To do so, a polymerase chain reaction (PCR) was performed using universal- and Pseudomonad-specific primers. Universal primers amplify regions supposedly present in both my experimental organism (P. 80) and negative control (Bacillus subtilis); Pseudomad-specific primers amplify regions unique to Pseudomonads. To visualize the PCR results, I performed gel electrophoresis (shown below) with each column corresponding to: ladder; P. 80 + Pseudomonad-specific primers; Bacillus subtilis + Pseudomonad-specific primers; P. 80 + universal primers; Bacillus subtilis + universal primers. I expected to see bands in all four experimental columns except Bacillus subtilis + Pseudomonad-specific primers, but instead only saw a band on the column corresponding to Bacillus subtilis + universal primers. Since P. 80 did not resolve any bands, either the PCR itself was aberrant or the strain is mutated or contaminated.

At this point, I was ready to analyze P. 80 directly at the nucleotide level. I submitted amplified universal regions of the P. 80 genome to South Korea for sequencing. A few weeks later, I received the sequence (shown below) and used NCBI BLAST to see if the sequence is homologous to other species. After a few false starts with vague vaginal epithelial cultures, I found that it corresponded to Comamonas spp (Score = 878/878), Comomonas testosteroni (Score = 878/878) and P. testosteroni (Score = 878/878).

Discussion

Due to morphological, physiological, biochemical, metabolic and nucleotide consistencies, I identified P. 80 as P. testosteroni. Experiment 3A helped me confirm it was a Pseudomonad, and Experiment 3B explored which energy sources it could use as well as pigment production, nitrate reduction and hydryolase activities. Positive or negative results on the procedures of Experiment 3B were highly informative, narrowing down the strongest possibilities to P. putida, P. aeuruginosa and P. testosteroni. Each possibility conflicted on at least one criterion: gelatin hydrolysis and 42°C growth for P. putida; fluorescent pigment production for P. aeruginosa; and fructose catabolism and gelatin hydrolysis for P. testosteroni.

Sequence analysis Experiment 3D strongly indicated that I had P. testosteroni, which was surprising considering that until that point P. aeruginosa was the strongest candidate. If my culture had been P. aeruginosa, then the only inconsistency (no fluorescent pigment production) was possibly due to environmental conditions; after all, my P. aeruginosa control was not able to secrete fluorescent pigment either. A loss-of-function mutation is much more common than a novel or gain-of-functon mutation, as would be the case if my culture was P. testosteroni (as it was able to catabolize fructose and hydrolyze gelatin, two characteristics foreign to P. testosteroni. Alternatively, it is possible that there was an error in the PCR amplification, since I could not even resolve any bands upon electrophoresis of both universal- and Pseudomonad-specifc-primed P. 80 gene segments. Despite these inconsistencies between Experiment 3B and 3D, I chose to follow Experiment 3D’s results since it turned up multiple Pseudomonads, P. testosteroni-like species and even P. testosteroni itself.

What is the question asked or the overall problem?

The overall problem is that a complex of four maternal proteins is necessary for an embryo to progress beyond the first few cell divisions. Though the accompanying mRNAs are degraded during maturation of the oocyte, the translational products persist and are: FLOPED, MATER and TLE6; and Filia which binds MATER. This paper examines the distribution and role of FLOPED.

What is the experimental system/techniques?

After examining the embryonic distribution of FLOPED protein using antibody staining, survival and fertility of mice and their offspring carrying homozygous and heterozygous null mutations for FLOPED. Next FLOPED’s binding partners were identified via comparing motility of various proteins in wild-type and FLOPED-less oocyte extract. Then FLOPED was used for coimmunoprecipitation – if FLOPED does indeed bind the proteins whose motility shifted in the absence of FLOPED then they ought also coimmunoprecipitate.

What are the results and conclusions?

Mothers heterozygous for the FLOPED null mutation were able to bear offspring, though their homozygous null mutation FLOPED offspring were infertile. FLOPED was found to bind MATER, TLE6 and Filia — and without FLOPED the complex of these four does not form. This is evidenced by the shift in the motility of their bands in the absence of FLOPED, their coimmunoprecipitation with FLOPED antibodies and also their lack of coimmunoprecipitation with their individual antibodies in the absence of FLOPED. Thus FLOPED is necessary for the complex to form, explaining why heterozygotes both survive and are fertile. Homozygotes survive since their SCMC phenotype is maternal (not their own) but are infertile because their own oocytes cannot form the SCMC.

What is the question asked or the overall problem?

Vertebrate Crossveinless-2 (CV2) is a BMP4-dependent secreted protein that can potentiate or antagonize BMP4 signaling and is required for formation of crossveins in the Drosophila wing. What effects does CV2 depletion have in the cell? Since it is dependent on a robust BMP4 gradient, what is CV2′s function with respect to BMP4? Does it complex with BMP4? CV2 is indeed cleaved – but is it cleaved by tolloid proteases, as is Chordin? Does CV2 bind to purified Chordin with high affinity, and do CV2 and Chordin have similar in vivo activities?

What is the experimental system/techniques?

A series of experiments were performed to determine that CV2 is a BMP4 feedback inhibitor in Xenopus. Morpholinos were used: a morpholino oligomer against CV2 (CV2 MO); a BMP4 MO was coinjected with CV2 MO; and Smad1 phosphorylation was measured.
To determine if CV2 is cleaved by tolloids (as is Chordin) a sample of CV2 was incubated for 18hrs and then observed; the positive control was a sample of Chordin incubated under the same conditions. To determine if CV2 cleavage is required for wild-type phenotypes, various CV2 fragments were introduced as DNA constructs into 8-cell Xenopus embryos.

Tsg can have either an anti-BMP4 or a pro-BMP4 activity depending on the presence of Chordin. Xenopus development was analyzed in CV2- and Chordin-depleted embryos treated and untreated by Tsg MO, each of which were subsequently injected with CV2. The degree of CV2 activity was then determined morphologically and by measuring markers.

Prior researchers determined that BMP4 is bound and inhibited by CV2. Tsg/CV2 interactions were tested by immunoprecipitating CV2 and Tsg with an anti-CV2 antibody bound to protein G beads. In a pulldown assay, BMP4 was preincubated with CV2 in the presence of Tsg. Various quantities of BMP4 were used and Tsg levels were kept constant. Coincubation of CV2, BMP4 and Tsg followed by immunoprecipitation was performed to see if they will also form a complex. To test whether CV2 prevents BMP4 binding its receptor (recall that CV2 is an antagonist) BMP4 and CV2 (and later Tsg as well) were incubated prior to incubation of BMP4 with its receptor.

A chip much like a cDNA microarray but for protein, and also a pulldown assay, was used to measure binding of CV2 and Chordin in presence and absence of BMP4. Next the authors queries whether CV2 binds to Chordin overall or just to particular fragments – Chordin was thus cleaved enzymatically by tolloids (a task predominantly performed by Xlr in vivo) and then allowed to bind to CV2 beads. These beads were then eluted by anti-Chordin antibodies. This experiment was repeated with preincubation of Chordin with BMP4.

What are the results and conclusions?

CV2 MO caused an increase in high-BMP4 markers and a decrease in low-BMP4 markers. Depletion of BMP4 abolished the ensuing increase in CV2 transcripts caused by CV2 depletion. This is concretely in line with the notion that CV2 is a negative feedback inhibitor of BMP4. CV2 appeared to be entirely resistant to digestion by tolloid while Chordin was cleaved heartily. However, CV2 cleavage is required for wild-type phenotypes – perhaps it self-cleaves in a nonenzymatic pH-dependent pathway. The activity of CV2 is dependent upon Tsg – in the absence of Tsg, CV2 has very little effect. CV2 and Tsg coimmunoprecipitate, indicating that they bind each other. Furthermore, BMP4 binding by CV2 is greatly enhanced by the presence of Tsg, as confirmed by the pulldown assay. Conicubated samples of CV2, BMP4 and Tsg coimmunoprecipitated. Chordin and BMP4 are involved in a ternary complex, suggesting that CV2, BMP4 and Tsg also form a ternary complex. BMP4 binding to BMPR-1b was greatly reduced by preincubation with CV2 — and more so when preincubation was in the presence fo Tsg. CV2 and Chordin bind very capable, particularly in the presence of BMP4 – and with even greater affinity when Chordin is cleaved by tolloid proteases. Furthermore, BMP4 greatly increases CV2 affinity for Chordin fragments.

What is the question asked or the overall problem?

Hox genes express themselves in the same time order as their physical order in their cluster. The mechanism for this is unclear. Early studies involving examination of the genomes of various cell lines during Hox gene expression revealed specific genomic profiles during different phases. The first genes to be expressed are on the telomeric end of the cluster and the last genes to be expressed are at the centromeric end. This paper determines whether the genes need to be in a single cluster on the genome, as well as a mechanism critical for transcription that also can independently be responsible for transcription.

What is the experimental system/techniques?

The last Hox genes are expressed in mouse tail buds during late somitogenesis. Mouse tail buds were isolated at E8.5, E9 and E9.5 and gene expression of Hox and nearby genes was determined using tiling arrays; this procedure was repeated using embryonic stem cells, which ought have no expression of Hox genes. Chromatin immunoprecipitation combined with the tiling array hybridization was used to map sites occupied by RNAP II.

To determine whether there is a mechanism whereby the HoxD cluster chomatin decondensation propagates from the telomeric end to the centromeric end, mice were engineered with a HoxD cluster containing a 3Mb inversion that separated Hoxd1-10 from Hoxd11-13. Studying Hox gene expression with this construct reveals whether an internal gene cluster is required for progressive gene expression. The same techniques were used as in the wild-type.

What are the results and conclusions?

Chromatin decondensation correlated directly to areas of RNAP II binding and active transcription. This collinear chromatin dynamic suggests a mechanism whereby modifications spread from the telomeric extremity of the cluster to the centromeric extremity. In the mutant construct, Hoxd1-9 were expressed normally but Hoxd10 was expressed early. This suggests a telomeric activator of expression of these genes. Hoxd 13 was expressed early, likely due to activity of a spurious enhancer. Hoxd 11 and Hoxd12 had very low levels of expression. However, Hoxd11-13 were decondensed. This indicates the requirement of a telomeric enhancer element to activate Hoxd11 and Hoxd12 expression, and the change in Hoxd13′s location resulting in susceptibility to some third-party enhancer element. The fact that decondensation still occurred revealed that an internal gene cluster may be necessary for proper expression but is not necessary for decondensation.

Note: Received a 27/30.

What is the question asked or the overall problem?

Complex gene regulation propels vertebrate neurogenesis — and cell polarity also plays a critical role. This paper explores the relevance of the polarity protein PAR-1 and its regulatory kinase aPKC to neurogenesis, as well as the importance of the ubiquitin ligase Mind bomb (Mib). Mib is imperative for Notch ligand activity, and interactions between Mib and Par-1 are characterized.

What is the experimental system/techniques?

To verify that PAR-1 and aKPC are vital to neurogenesis, they were over- and under-expressed (via mRNA and morpholino injection, respectively). Direct examination and in situ hybridization for neuronal markers were two techniques used to quantify primary neuron populations. Markers included N-tubulin and Hox11L2 (for primary neurons), Sox2 and Sox3 (for neuronal progenitors). Phospho-histone-3 staining was used to measure mitosis.

To test whether differences between wild-type, abundant or ablated PAR-1 and apKC levels affect neuronal differentiation directly or via cytoskeletal disruption, the C17.2 strain of cells was used. This strain lacks epithelial morphology yet can differentiate into neurons — thus mutant differentiation in these cells must be independent of their cytoskeletal (in)activity. If PAR-1 and aPKC are unable to disrupt or enhance neuronal differentiation in these cells, then their own activity must involve cytoskeletal intervention.

The Notch pathway is a major regulator of neurogenesis. Perhaps PAR-1 and aPKC are mediated by the Notch pathway. To test this, a luciferase reporter with multimerized CSL-binding sites was used in C17.2 cells cotransfected with various aPKC and PAR-1 construct. Immunoprecipitation, spectrometry, over- and under-expression and other techniques were all used to probe the interaction of PAR-1 with Mib. Dil1 signaling activity depends on the ubiquitination of its intracellular domain by Mib — thus, fusing Dil1 to an ubiquitin would rescue it from the effects of PAR-1.

With earlier experiments revealing that PAR-1 phosphorylates Mib, researchers set out to identify Mib’s phosphorylation sites relevant to this pathway. Various Mib proteins were engineered with substitutions in putative serine/threonine phosphorylation motifs. Their levels were then assayed in presence or absence of PAR-1 in Xenopus embryo lysates.

What are the results and conclusions?

Over- and under-expression of PAR-1 and aKPC reveals that they influence neurogenesis in Xenopus embryos. Increased PAR-1 caused a surge in the number of primary neurons. Depletion of PAR-1 significantly reduced the number of primary neurons. Wnt signaling was unaffected, indicating that PAR-1 is not mediated by the Wnt pathway. Supportive of these findings, that aPKC increases decreased primary neuron quantities; aPKC decreases increased primary neuron quantities. Interestingly, populations of neuronal progenitors were not remarkably affected despite the presence or absence of PAR-1 activity causing wide rises and falls in primary neuron populations. This finding was supported by a corresponding consistence of mitotic activity despite activation or deactivation of PAR-1 activity. However N-tubuln activity was found to be remarkably increased within the bounds of its normal expression (as opposed to being expressed ectopically). Thus PAR-1 must promote neuronal differentiation yet not progenitor pool expansion. Increasing and decreasing PAR-1 and aPKC activity in C17.2 cells yielded parallel results – thus, their activity is intrinsic and does not rest upon a cytoskeletal pathway.

The luciferase reporter with multimerized CSL-binding sites was activated by aPKC and inhibited by PAR-1. Specifically, PAR-1 modulated Dil1 but not Notch ICD — PAR-1 repressed its activity while aPKC modestly enhanced it. Upstream of Dil1 is Mib. Mass spectrometry identified a mammalian PAR-1 homolog among proteins associated with Mib. Their physical interaction was verified by coprecipitation from cell lysates. An immune complex kinase assay revealed that PAR-1 phosphorylated Mib and a similar E3 ubiquitin ligase, identifying Mib as an in vitro substrate for PAR-1. In vivo, PAR-1 and homologs downregulated Mib levels in lysates from embryos and cultures. Mib mutated in its Eg ligase capability was unaffected by PAR-1, even when PAR-1 was overexpressed. Ablation of PAR-1 led to an increase in Mib protein levels. Treating with proteasome inhibitors cancelled the inhibitory effect of PAR-1 on Mib, suggesting that inhibition occurs via a proteasome pathway. Thus, PAR-1 likely phosphorylates Mib causing Mib’s proteasome-dependent degradation. Furthermore, Mib ubiquitinates Dil1; by fusing Dil1 to an ubiquitin and assaying neuronal development, it was shown that Dil1’s over-ubiquitination led to reduced neuronal growth and was impervious to overexpression of its inhibitor PAR-1. This shows that PAR-1 regulates Dil1 by phosphorylating E3-ubiquitin ligases such as Mib which in turn ubiquitinate Dil1.

Most Mlb mutants remained sensitive to PAR-1-mediated degradation, with the sites M2 and M8 each critical for this process; indeed, M2 and M8 mutations rendered the Mib mutants impervious to the effects of PAR-1. Thus, specific phosphorylation sites in Mib are required for PAR-1-dependent changes in Dil1 ubiquitination.

What is the question asked or the overall problem?

Vertebrate Crossveinless-2 (CV2) is a BMP4-dependent secreted protein that can potentiate or antagonize BMP4 signaling and is required for formation of crossveins in the Drosophila wing. What effects does CV2 depletion have in the cell? Since it is dependent on a robust BMP4 gradient, what is CV2′s function with respect to BMP4? Does it complex with BMP4? CV2 is indeed cleaved – but is it cleaved by tolloid proteases, as is Chordin? Does CV2 bind to purified Chordin with high affinity, and do CV2 and Chordin have similar in vivo activities?

What is the experimental system/techniques?

Cdx2 null mice die before gastrulation. Researchers thus engineered a conditional Cdx2-ablated mutant to study Cdx2′s role in the gut endoderm. Mice heterozygous for the conditional allele were crossed with wild-type mice; the heterozygotes were bed to form homozygous mutant F2 progeny. Cdx2 was equally ablated throughout the intestine.

Mutant duodenal epithelium proliferation was assayed by BrdU incorporation. Apoptosis was assayed via cleaved caspase-3 and also TUNEL staining. Mutant Cdx2 epithelial cells identity was directly observed by transmission electron microscopy of ultrastructural features. In addition, identity of these cells was probed by an immunohistochemical assay for keratin 13 and p63 (markers of keratinocytes, which TEM indicates is the identity of these mutant Cdx2 epithelial cells). For further study, markers of anterior foregut endoderm were assayed.

Gene expression profiling (via microarray) was performed using RNA samples extracted from total E18.5 control and mutant ileum as well as normal esophagus. Additional profiling was performed with PCR to examine the cell fate switch in the Cdx2-deficient posterior intestine, focusing on transcriptional regulators known to be crucial in regulating intestinal differentiation.
Cdx factors regulate Hox genes, which are expressed both within and outside the endoderm and affect gastrointestinal development. Analysis was performed on various Hox genes, including those whose early gut expression domains are well characterized. Further study was done, particularly on Wnt proteins and their targets.

What are the results and conclusions?

Cdx2 deficiency prevents colon formation and caused complete intestinal obstruction in Cdx2 mutants — the poor wretches. Despite live births, mutant pups died in postnatal day one (P1). Their posterior gut region was grossly abnormal. Wild-type controls had a colon and rectum at the terminus of their intestinal tract; mutants all lacked colons, with various other defects arising. Mutant duodenums were distended and translucent due to fluid retention caused by distal obstructed.

BrdU incorporation revealed an expanded proliferative compartment in the mutant duodenal epithelium. However, assays of apoptosis revealed a wild-type apoptotic rate. Expanded proliferation and a lack of enhanced cell death suggest that Cdx2 mutants have an epithelial proliferative pattern reminiscent of early embryonic stages prior to intestinal differentiation. Transmission electron microscopy revealed abundant tonofilaments in mutant posterior epithelial cells. Tonofilaments are typical of squamous epithelial cells — they are often seen in the desmosomal junctions of keratinocytes. Keratinocytes are typical of stratified esophageal epithelia but are extremely rare in the normal intestine. Immunohistochemical assays of keratin 13 and p63 — common in keratinocytes but rare in wild-type midgut and hindgut endoderm — revealed that the mutant epithelium was positive for these markers. Furthermore, the fact that an anterior foregut endoderm marker was detected in Cdx2-deficient ileum indicates it was indeed anteriorized and did not just undergo a significant developmental delay.

Mutant ileum was more transcriptionally similar to esophageal tissue than to normal ileum. Nearly all intestine-specific genes were downregulated in the mutant ileum. There were significant expression changes in 40% each of: genes signficantly expressed in E18.5 intestinal epithelium; and genes expressed more in the intestine than stomach. Also, genes expressed specifically in differentiated intestinal epithelium were all heavily downregulated. Genes involved in keratinocyte formation were significantly upregulated — including nine of those involved in esophageal formation. This strengthens the notion of the identity of the mutant epithelium.

Cdx2 mRNA obviously decreased — and so did several intestine-enriched transcription factors. Reverse-transcription and PCR revealed that this transcriptional modification occurred early in development. In fact, the expression of Math1 was reduced from E12.5 onward — this is significant as it is crucial for differentiation of intestinal secretory cell types. Conversely, foregut-enriched genes were ectopically activated in the mutant posterior intestine as early as E12.5. Additional genes were dramatically activated ectopically, and various more genes were repressed in their wild-type expression domains.

Hox9, expressed in the posterior hindgut, was underexpressed in Cdx2 mutants. In the early gut, Hoxc8, Hoxb9, Hoxc9, Hoxa13 and Hoxd13 mRNA levels were significantly lower in the mutant posterior intestine. However various Parahox genes maintained wild-type expression, indicating that not all factors along the AP axis of the gut were impacted by Cdx2 deficiency. Wnt proteins was upregulated due to anteriorization of the gut, causing subsequent upregulation of Wnt targets.

In conclusion, this paper finds that Cdx2 is necessary for posterior patterning of the gut along the AP axis. It controls many factors – prior studies postulated that it functions via of Hox factors. However, there are several Hox factors which are independent of Cdx2 yet still fall along the AP axis. Thus Cdx2 must function by targeting a litany of transcription factors, many of which are Hox factors. Cdx2 mutants are anteriorized, and can be rescued by Cdx1.