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transcript of second neuroscience journal club

David Mckalip dmmckali at gibbs.oit.unc.edu
Thu May 26 14:33:44 EST 1994


Following is a transcript from the Second Neuroscience Journal Club.  the 
meeting was attended by about 15-20 people, 12 of which signed up to speak.

In attendance were: TJ (moderator), Eric, Norm, Gustavo, Irit, KaiG, 
Anders, Guests (beetle, Eukaryotic, Avian), Martin and Nev. 

The meeting lasted about an hour and was performed entirely at the BioMOO 
(telnet bioinformatics.weizmann.ac.il 8888) in real time.  The slides 
were prepared ahead of time using the BioMOO slide projector. 

BioMOO was recently featured in the May 13, 1994 issue of Science 
(264:900-961).

***************************************
Second BioMOO Journal Club, 1600 GMT, Wednesday, May 11
 
  Authors
    Galileo DS.  Majors J.  Horwitz AF.  Sanes JR.
  Institution
    Department of Anatomy and Neurobiology, Washington University School of
    Medicine, St. Louis, Missouri 63110.
  Title
    Retrovirally introduced antisense integrin RNA inhibits neuroblast
    migration in vivo.
  Source
    Neuron.  9(6):1117-31, 1992 Dec.
  Abstract
    We used retrovirus-mediated gene transfer to ask whether integrins are
    involved in the development of neuroblasts in the chicken optic tectum.
    Vectors were constructed with the E. coli lacZ gene in the sense
    orientation and beta 1 integrin sequences in the antisense orientation.
    Tests in culture showed that the progeny of cells infected by these vectors
    were identifiable by expression of LacZ and had reduced levels of beta 1
    integrins on their surfaces. We then injected these vectors into optic
    tecta on E3, at the height of neuronal production. Clones of LacZ-positive
    cells were analyzed 3-9 days later, as they migrated along radial glia to
    form the tectal plate. Antisense sequences had little effect on the
    proliferation of progenitors, or on the radial stacking of their progeny in
    the ventricular zone (E6). However, many antisense-bearing cells
    accumulated in the ventricular zone and failed to migrate into the tectal
    plate (E7.5 and E9). At later stages (E12), few antisense-bearing cells
    could be found. Thus, integrin appears to be required in the migratory
    process, and cells that fail to engage in integrin-mediated interactions
    may die.

Eric has arrived.
Norm waves to Eric
Eric grins at Norm, and waves to everyone
Norm says  Anyone, who  would like to be on the list to speak, type
 request.
Norm says, "TJ, have you put yourself down as the moderator?"
Norm smiles
TJ [to Norm]: Yes
TJ is nervously fidgeting
Norm chuckles
Anders says, "Think he need an extra cup of coffee?""
Norm hands TJ a bottle of Guinness
TJ laughs.
 the side.
TJ guzzles Guinness and wipes his face with a satisfied grin
KaiG finds his way in.
The beetle guest materializes out of thin air.
Irit materializes out of thin air.
The beetle guest says, "What is the purpose of the seminar"
TJ [to the beetle guest]: We are discussing the following paper:

TJ shows slide #1.

                      * * * * * * * * * * * * * * * * * * * *
 
Retrovirally Induced Antisense Integrin RNA Inhibits Neuroblast Migration
_In Vivo_

Deni S. Galileo, John Majors, Alan F. Horwitz and Joshua R. Sanes

Neuron 9:1117-1131, 1992.








                      * * * * * * * * * * * * * * * * * * * *

TJ says, "I have a lot of slides, I will show each one and then leave some
 time for discussion between each.  Also, there will be a more general
 discussion at the end of each major section.  Everyone ready to start?"
Eric says, "Wow TJ! Four projector? Is this going to be like those music
 videos?"
Norm nods and laughs
Gustavo materializes out of thin air.
Norm nods to Gustavo
TJ nods to Gustavo, too
Gustavo excuses himself for being a bit late and sinks into a comfy chair.
TJ says, "I know a joke from a scientific meeting: 'In my first carousel...'"
Anders says, "Please start, TJ"

TJ shows slide #2.

                      * * * * * * * * * * * * * * * * * * * *
 
              Key Events in the Development of Brainstem Structures

--> Neurons are "born" in the center of the central nervous system, at the 
    ventricular surface

    In the tectum the cerebral aqueduct (of Sylvius) is the ventricle

--> "Birth" involves a final cell division (which always occurs at the     
    ventricular surface) and differentiation away from the stem cell type

    In tectum, this pattern is "outside-out": cells which differentiate    
    earliest end up in the most lateral part of the tectum


                      * * * * * * * * * * * * * * * * * * * *

Eric [to TJ]: Ouch!
TJ [to Eric:]: Discussion?

TJ shows slide #3.

                      * * * * * * * * * * * * * * * * * * * *
               Key Events (cont'd)

--> Neurons migrate along radial glial guides to their final locations

    Radial glia in the tectum extend from the aqueduct to the pial surface

--> While the general pattern is to migrate from ventricle to final        
    location radially, some tangential movement may occur

    Two questions then arise:
    1) How do neurons "know" when to get off?
    2) Why and how do they "jump" from one radial glial guide to another?



                      * * * * * * * * * * * * * * * * * * * *

Norm says, "pretty basic stuff....yet always a mystery."
Eric says, "Just a comment/question: The tectum then develops in the opposite
 pattern from the cortex?"
Eric says, "Outside out vs. Inside out?"
TJ [to Eric]: Yes.  So does thalamus, brainstem, spinal cord.  Retina and
 cerebellum are another matter entirely, different from cerebral cortex
 *and* from brainstem.  That's a story for another day.
Eric nods
Norm nods and watches slide show
TJ wonders if anyone else has seen confocal pictures of migrating neurons
 moving radially and tangentially
Norm nods, "Fascinating indeed"
TJ says, "It's very striking.  There seems to be *a lot* of tangential
 migration, which may influence the interpretation of today's paper."
Norm says, "How so?"

TJ shows slide #4.

                      * * * * * * * * * * * * * * * * * * * *
  It is of obvious value to perturb these developmental processes.


  In the past, three strategies have been used:

    1) Ablation by injection of antibodies

    2) Antisense transgenics

    3) Homologous recombination (Ann Rev Neurosci 15:115, 1992)





                      * * * * * * * * * * * * * * * * * * * *

TJ [to Norm]: Remind me of that later, we can cover it after methods.
Norm says, "Antibodies don't always work too well do they?"
Eric says, "My understanding is the work well in culture or isolated cell
 system."
TJ says, "Right.  We don't really know what happens to them, whether they are
 stable, or subject to proteolysis; whether they remain localized, are taken
 up by cells, or diffuse large distances."

TJ shows slide #5.

                      * * * * * * * * * * * * * * * * * * * *
Sanes' group has previously used retroviral vectors to 
derive 'fate maps' of central nervous system structures.


This method labels a single precursor cell, and since the
virus is replicated but not shed, labels all 'daughter cells'
without a decrease in signal due to cell division.


Because the virus is injected at low concentration,
each columnar group of cells is assumed to be clonally related.


*** These 'clones' are the basic unit being studied here ***

                      * * * * * * * * * * * * * * * * * * * *

Norm nods at slide. "great technique."
TJ says, "This last point relates to Norm's question.  If there is substantial
 tangential migration, then Galileo et al cannot tell which are clonal
 populations."
Norm eyes widen a bit. "Aha..."

TJ shows slide #6.

                      * * * * * * * * * * * * * * * * * * * *
Why study chicken optic tectum?


--chick visual pathway is from retina -> tectum

--compare mammals, where retina -> LGN -> visual cortex is the
  main pathway

--there is a need to set up a retinotopic map in addition to the
  layering of cells found in other locations





                      * * * * * * * * * * * * * * * * * * * *

TJ says, "Plus, they had worked in this system for the past few years."
TJ smiles wryly
Norm says, "no visual cortex in the chick?"
TJ says, "Not to speak of.  Some argument about that, but certainly nothing
 comparable to mammalian visual cortex."
Norm nods
TJ says, "I know in pigeon there is a visual cortical area, called the _wulst_
 I think.  Anyone know of this?"
Norm shakes his head slowly
Eric [to TJ]: I believe that is a telencephalic structure, no?
TJ says, "Well, all I know is a name."
TJ [to Eric]: Yes, some people argue it's the visual cortical equivalent

TJ shows slide #7.

                      * * * * * * * * * * * * * * * * * * * *

Precursor cells labeled at the ventricle give rise to 


    * several types of neurons

    * two kinds of astrocytes

    * radial glial cell(s)






                      * * * * * * * * * * * * * * * * * * * *

Eric says, "It is one of the easiest to find cortical structures in birds at
 early ages, so I've run into it as a common area for study. For bird folks."
TJ says, "Is this point clear to everyone?  If not, I can suggest a good
 review article."
Norm says, "It is quite clear...."
Eric nods
TJ says, "The origin and fate of the 'neuroblast', 'glioblast', or
 'neuroglioblast' has been *very* controversial for the better part of the
 last century."
Gustavo [to TJ]: what's the review article?
TJ says, "But these new fate mapping techniques, and some fine _in vitro_
 work, has established (to my satisfaction) the identities and fate(s) of the
 precursor cells"
TJ [to Gustavo]: Cameron and Rakic, Glia 1991, I can mail you the 'real'
 citation after :-)
Eric says, "ISn't it the case that the initial restriction process may be
 different among different structures?"
The cereal guest has arrived.
Gustavo [to TJ]: Maybe you can give it for the record..
Norm waves to cereal guest. "you can type: "request" to speak here.
TJ says, "Cameron RS and Rakic P (1991)  Glial cell lineage in the cerebral
 cortex: a review and synthesis.  Glia 4:124-137"
TJ lied, earlier :-)

TJ shows slide #8.

                      * * * * * * * * * * * * * * * * * * * *

Events at the ventricular zone:


   1) Cells undergo their last division at the ventricle (aqueduct)

   2) They begin migrating along radial glial guides

   3) Note that the process is 'outside-first' as compared to 
      'inside-out' process in cortex


  INTEGRINS are important for neuronal migration _in vivo_ and
    _in vitro_.

                      * * * * * * * * * * * * * * * * * * * *

Norm says, "What sort of work has proven integrins to be important?"
TJ says, "Off the top of my head, I know that *overexpression* of integrins 
 (or was it extracellular matrix?) is inhibitory to neural crest cell
 migration."
Norm thumbs through article's reference section
Norm says, "Sanes 1989, Annual Review of Neuroscience: ECM molecules that
 influence neural development"
TJ says, "I also recommend Kreis T and Vale R (eds) _Guidebook to the
 Extracellular Matrix and Adhesion Proteins_, Oxford 1993.  An excellent entry
 in an excellent series, I have the one on cytoskeleton too."
TJ says, "From which the next slide is taken:"
Norm nods.."Thanks"

TJ shows slide #9.

                      * * * * * * * * * * * * * * * * * * * *
             Integrin Subunit Combinations and Ligands
                    (from Kreis and Vale, 1993)
Alphas1-8 and alphaV, alphaIIb, alphaL, alphaM, alphaX and betas1-8 exist;
   Combinations of alpha and beta subunits form distinct
   extracellular matrix proteins, each with different ligands.
   Known ligands for combinations with beta1 are:

alpha1      collagen, laminin
alpha2      collagen, laminin
alpha3      fibronectin, laminin, collagen
alpha4      fibronectin, vascular cell adhesion molecule-1
alpha5      fibronectin
alpha6      laminin
alpha7      laminin
alpha8      ?
                      * * * * * * * * * * * * * * * * * * * *

Eric says, "A whole lot of work in vitro shows their important for axon
 extension on variously coated surfaces, but less work has been done on
 migration."
Norm abosrbs the glory of the last slide. "amazing summation of years of work."
TJ says, "So you can see one potential problem with this study.  Even if the
 antisense experiment works, and interferes with beta1 expression by neurons
 or glia, we still don't know if they would bind normally to"
TJ says, "collagen, laminin, fibronection, VCAM-1, or all of the above."
Norm nods 
TJ says, "It's unclear whether they wanted to hit the system with a
 sledgehammer first, then try more finesse, or whether they tried finesse and
 it didn't work."
Norm chuckles. "Well, it is still an important piece of work."
TJ says, "Finesse would be defined as expression of say alpha 2 antisense, or
 one of the other, more specific, beta's"
TJ says, "Time for some methods.  Are we all clear on the intro?"
Norm nods

TJ shows slide #1.

                      * * * * * * * * * * * * * * * * * * * *






                             METHODS








                      * * * * * * * * * * * * * * * * * * * *

TJ says, "Discussion on this slide?"
Norm laughs
TJ grins crookedly

TJ shows slide #2.

                      * * * * * * * * * * * * * * * * * * * *
A number of different vector constructs (next slide) were injected
into E3 chick embryo tectum, as the cells of the ventricular zone
are still dividing.

pLZ10 and its viral homolog, LZ10, have been used previously for 'fate
mapping' as discussed above.
           ____ _ __ _________ _________ _________ ____ _ __
virus -//-|_U3_|R|U5|___gag___|___pol___|___env___|_U3_|R|U5|-//-

           _________   _________ _______________   _________
pLZ10 -//-|_RSV_ltr_|-|___gag___|____lacZ_->____|-|_RSV_ltr_|-//-
                             Bgl^II          Xba^I
                             BamHI

U3|R|U5 = long terminal repeat (ltr) ;  ->  = direction of transcription
                      * * * * * * * * * * * * * * * * * * * *

TJ says, "Note that the long terminal repeat (ltr) is the same as the U3, R,
 U5 sequence.  U3 and U5 are promoters, R is called a 'true repeat'."
TJ says, "Is this slide clear to everyone?  If not, it gets worse."
Norm looks around room

TJ shows slide #3.

                      * * * * * * * * * * * * * * * * * * * *
pLZ14/LZ14 are the same, but the _gag_ sequence has been reduced from 
1260 to 254 bases to accommodate additional genes.

           _________   _____ ________________   _________
pLZ14 -//-|_RSV_ltr_|-|_gag_|_____lacZ_->____|-|_RSV_ltr_|-//-
                         Xho^I            Xba^I









                      * * * * * * * * * * * * * * * * * * * *


TJ shows slide #4.

                      * * * * * * * * * * * * * * * * * * * *
pLZ15/LZ15 have lacZ (beta-galactosidase) and beta1-integrin genes, with 
beta1-integrin in the 'antisense' orientation, and the U3 promoter between.
     *** Concern: promoter interference.
pLZ16/LZ16 are the same, but with no U3 promoter.
     *** Concern: transcript contains a long stretch of beta-galactosidase 
        'sense' transcript; this may interfere with hybridization to 
        'sense' mRNA

pLZ15_________   _____ ________________ ____ ______________   _________
-//-|_RSV_ltr_|-|_gag_|_____lacZ_->____|_U3_|_beta1_int_<-_|-|_RSV_ltr_|-//-
                   Xho^I            Xba^I   ^BglII

           _________   _____ ________________ ______________   _________
pLZ16 -//-|_RSV_ltr_|-|_gag_|_____lacZ_->____|_beta1_int_<-_|-|_RSV_ltr_|-//-
                         Xho^I            Xba^I            Xho^I
                      * * * * * * * * * * * * * * * * * * * *

TJ says, "These are also shown in figure 4, if you have the paper in front of
 you."
Norm asks a question
TJ says, "Shoot"
Norm says, "How does the "p"LZ-- differ from a viral associated gene?"
The cereal guest has disconnected.
The housekeeper arrives to remove the cereal guest.
###
TJ says, "p is for the plasmid form.  I'm not clear on this, and our
 virologist did not come today, so bear with me."
Norm nods appreciatively. "oh, there it is in the start of the methods section"
TJ says, "They coinfect host cells with the pLZ**, a wild-type virus
 construct, and a neo gene for neomycin resistance"
TJ thinks the last of these is kinda superfluous
Norm says, "*p* denotes the plasmid encoding the viral genome"
The eukaryotic guest has arrived.
Norm says, "LZxx alone is the virus itself"
Norm waves to eukaryotic geust. "you can type : "request" to speak"
TJ says, "Then the host cells shed DNA from the construct, that can be used to
 inject into the ventricle (in this case, the cerebral aqueduct)"
Norm nods
TJ is unclear on this point, spent several happy hours yesterday trying to
 figure it out.
TJ says, "I'm not sure whether the material injected is 1) an RNA copy of
 pLZ** and the virus; 2) a DNA copy; or 3) something else I've not considered"
Norm says, "I think it is the virus itself"
Anders asks if the precursor cells of interest are in direct contact with the
 ventricle
TJ says, "It could be 1), because I found out yesterday that retroviruses
 (like RSV used here) carry a little reverse transcriptase *protein* inside
 the viral capsid."
Eric says, "Has to be the virus, or the transfer into the cells in vivo would
 be vanishingly rare."
TJ [to Anders]: Yes, they maintain physical contact with the ventricle at all
 times.
Norm says, "top of page 1129..."And virus was concentrated from the
 medium"...."
TJ [to Anders]: The primitive neuroepithelium is a *pseudostratified columnar
 epithelium*
Eric says, "The 'host' cells are a producer cell line designed to produce high
 titers of virus."
TJ is just showing off, don't mind him
TJ says, "Ok, so we agree that it is the RNA form, enclosed in a viral capsid"
KaiG says, "What method did the use transformation?"
Norm nods eagerly
TJ shows slide #1.

                      * * * * * * * * * * * * * * * * * * * *







                                RESULTS







                      * * * * * * * * * * * * * * * * * * * *

Irit says, "don't they say in the description of the 3T3 control that in that
 case they had to use a different method of transfection, because mouse cells
 can't be infected by their virus?"
TJ nods to Irit

TJ shows slide #2.

                      * * * * * * * * * * * * * * * * * * * *
1) Immunohistochemistry of tectum at embryonic days 5, 7 or 9 (E5, E7, E9)

   see staining of extracellular matrix and blood vessels

   can't make out individual cells

2) Immunohistochemistry of isolated cells

   [Integrin staining on arbitrary scale by TJ]

   50% neurofilament+       integrin 0/+ to ++     presumably neurons
   25% vimentin+            integrin + to +++      radial glia/dividing cells
   25% flat cells nf- vim-  integrin ++++          fibroblasts?


                      * * * * * * * * * * * * * * * * * * * *

Norm [to KaiG]: They injected the isolated viral particels directly into the
 ventricle
TJ says, "Point #1 is shown in figure 2, point #2 is paraphrased from their
 description in the results section"
TJ says, "A few photos of point #2 are shown in figure 3"
The eukaryotic guest has disconnected.
The housekeeper arrives to remove the eukaryotic guest.
TJ says, "So I would expect naively that antisense beta1 integrin would have a
 greater effect on radial glia and other glia than neurons."
TJ says, "Any discussion on this?"
Norm nods
Eric says, "Depends on what 'effect' you're talking about."
Eric says, "Radial glia don't migrate, so it doesn't effect them so overtly."
Norm says, "That makes sense...except, I had thought that integrins would be
 located on the Neuronal cell surfaces and then bind to ECM secreted by the
 glia"
TJ thinks to answer Eric's question, we will have to wait for two more slides
Eric [to Norm]: And what they're doing on the glia is a lot less clear.
TJ says, "And in response to Norm's point, my memory of neurons and radial
 glia at EM demonstrates no extracellular matrix-type material between them"
Norm nods to Eric
Norm [to TJ]: "really?"

TJ shows slide #3.

                      * * * * * * * * * * * * * * * * * * * *
3) Infection of QT6 quail fibroblasts with LZ10, LZ15, LZ16
                             FIGURE 5A

   Double-label immunofluorescence with antibodies to beta-galactosidase,
   chick beta1-integrin

                Results of Beta1-Integrin Immunohistochemistry
_________________________________________________________________________
   LZ10-infected
        or                                            control levels
   LZ16-infected, B-gal negative
_________________________________________________________________________
   LZ15-infected, B-gal positive
        or                                     60% of control levels
   LZ16-infected, B-gal positive
                      * * * * * * * * * * * * * * * * * * * *

TJ [to Norm]: I'll have to check after, if I'm wrong I'll edit it out of the
 log :-)
Norm [to TJ]: Just like David Letterman
TJ says, "So you can see, there is not a *total* block of beta1 integrin
 expression, just a reduction."
Norm nods
Norm says, "Maybe a reduction will be enough to shift the balance toward
 adhesion and away from migration."
TJ says, "But there is no effect on mammalian beta1 integrin, showing that
 their antisense agent is sequence-specific"
Norm nods

TJ shows slide #4.

                      * * * * * * * * * * * * * * * * * * * *
4) 3T3 mouse fibroblast transfection with LZ14, LZ15 or LZ16

                             FIGURES 5B, 5C


% control values                       Anti-Chick         Anti-Mouse
                                        integrin           integrin
                                       __________         __________

LZ14 (no beta1-integrin antisense)       control              n.d. 
LZ15 (U3 promoter, beta1 antisense)        40%                n.d.
LZ16 (lacZ/beta1 antisense transcript)     40%                100%
LZ16 (uninfected, lacZ-negative cells)     n.d.               100%


                      * * * * * * * * * * * * * * * * * * * *

TJ says, "Everyone with me?"
TJ looks around the room
Norm says, "Yup!"
KaiG nods
Eric nods

TJ shows slide #5.

                      * * * * * * * * * * * * * * * * * * * *
5) The pivotal experiment: inject LZ10 (control), LZ15, LZ16 into 
   E3 chick cerebral aqueduct

   *** This is at the beginning of neurogenesis in the chick tectum
   *** Compare LZ10 (lacZ only) to LZ15 and LZ16 (lacZ/beta1 integrin
       antisense constructs)

In the following set of three slides:

Data have been taken from Tables 1 and 2 (p. 1123), grouped by AGE
   (original data were grouped by treatment)
Significance levels indicated by:     *   p < 0.05
                                      **  p < 0.005
                                      *** p < 0.001
Note that N= number of clones, NOT number of tecta
                      * * * * * * * * * * * * * * * * * * * *

TJ says, "I changed the tables around to suit me.  I thought grouping by age
 and treatment made more sense."
Norm agrees
TJ says, "The data are the same :-), I just changed the bins"

TJ shows slide #6.

                      * * * * * * * * * * * * * * * * * * * *
                              Embryonic Day 6
                       Labeled cells / infected clone

Virus        Ventricular Zone      Intermediate Zone          Tectal Plate
-----        ----------------      -----------------        ----------------
LZ10         15.4 +/- 1.2               n/a                       n/a

LZ15         12.6 +/- 0.9  n.s.         n/a                       n/a

LZ16         13.3 +/- 0.7  ?            n/a                       n/a


Neither intermediate zone nor tectal plate is present at this age.

    n.s.  p > 0.05                       ? significance not determinable
                      * * * * * * * * * * * * * * * * * * * *

TJ says, "Remember that the injection was on E3 in each case."
Norm nods and eagerly awiats next slide
TJ says, "There is not much of an effect three days post-injection, before the
 tectum has developed (much) layering"

TJ shows slide #7.

                      * * * * * * * * * * * * * * * * * * * *
                              Embryonic Day 7.5

Virus        Ventricular Zone      Intermediate Zone          Tectal Plate
-----        ----------------      -----------------        ----------------
LZ10         10.0 +/- 1.6           3.5 +/- 0.4              7.7 +/- 0.7
             12.3 +/- 1.4           3.3 +/- 0.5              7.9 +/- 0.9

LZ15         16.7 +/- 1.4  *        1.7 +/- 0.3 ***          1.0 +/- 0.4 ***

LZ16         13.8 +/- 1.6  n.s.     2.0 +/- 0.3  *           1.7 +/- 0.3 ***




*  p < 0.05                  *** p < 0.001               n.s.  p > 0.05
                      * * * * * * * * * * * * * * * * * * * *

TJ says, "Now we start to see an effect.  Cells seem to be 'stuck' at the
 ventricle if they are infected with virus, which contains the beta1 antisense
 construct."
TJ says, "Look at how few cells have reached the tectal plate, the layer
 furthest from the ventricle (aqueduct)"
Norm says, "marked decrease in migration at this age"
TJ [to Eric]: See the outside-out pattern?  A lot more cells in the tectal
 plate than intermediate zone at this age.

TJ shows slide #8.

                      * * * * * * * * * * * * * * * * * * * *
                              Embryonic Day 9  

Virus        Ventricular Zone      Intermediate Zone          Tectal Plate
-----        ----------------      -----------------        ----------------
LZ10          5.0 +/- 0.6           2.2 +/- 0.3             15.3 +/- 1.2
              6.1 +/- 0.7           2.5 +/- 0.6             19.0 +/- 2.7

LZ15          8.6 +/- 1.0  **       1.0 +/- 0.1 ***          3.7 +/- 0.7 ***

LZ16          9.4 +/- 0.9  **       1.8 +/- 0.3 n.s.         7.2 +/- 1.1 ***




** p < 0.005                 *** p < 0.001               n.s.  p > 0.05
                      * * * * * * * * * * * * * * * * * * * *

Eric nods
Martin materializes out of thin air.
Norm says, "Trend continues to E9"
TJ says, "We are starting to see a statistically-significant drop in the total
 number of cells in treated vs. control tecta, a number I did not put in the
 tables for the sake of simplicity."
Norm waves to Martin

TJ shows slide #9.

                      * * * * * * * * * * * * * * * * * * * *


                              Embryonic Day 12

          Almost no clones seen in LZ15- or LZ16-injected chicks










                      * * * * * * * * * * * * * * * * * * * *

The amoeban guest materializes out of thin air.
TJ says, "The infected cells have apparently died."
Martin waves to all (sorry I'm late- cell culturing)
The amoeban guest has disconnected.
The housekeeper arrives to remove the amoeban guest.
TJ says, "It seems as though they failed to migrate, and went belly-up"
Norm says, "Well, that makes sense....lack of permissive environment at
 ventricular zone"


TJ shows slide #1.

                      * * * * * * * * * * * * * * * * * * * *
                             CONCLUSIONS
======================================================================
=======
   A drop in beta1-integrin expression is the only effect on infected cells.
               There is no effect on mouse integrin expression.
-----------------------------------------------------------------------------
    There is no difference in morphology, size, or doubling time of cells.
            [Where and how was doubling time determined? --- TJ]
-----------------------------------------------------------------------------
             There is no effect on skin, pial, or vascular cells.
-----------------------------------------------------------------------------
The degree of beta1-integrin inhibition is independent of the promoter used.
                LacZ alone has no deleterious effect on cells.
-----------------------------------------------------------------------------
  RADIAL MIGRATION OF TECTAL CELLS IS INHIBITED BY ANTISENSE
BETA1-INTEGRIN
          There is likely to be an effect on cell survival as well.
                      * * * * * * * * * * * * * * * * * * * *

Norm says, "The controls showed that the transfection was not toxic, therefore
 it was likely due to lack of a permissive environment for the cells that didi
 not migrate."
TJ nods

TJ shows slide #2.

                      * * * * * * * * * * * * * * * * * * * *
                          QUESTIONS FOR THE FUTURE

======================================================================
=======

                  What step in migration requires integrins?

-----------------------------------------------------------------------------

          What ligands in the tectum are bound by beta1 integrin?


======================================================================
=======



                      * * * * * * * * * * * * * * * * * * * *

Eric says, "Cells may require different conditions in vivo vs in vitro. It's
 the easiest test, but it's not very strict."
Eric says, "It may also be the ONLY reasonable test they could have done."
Norm says, "Eric, I am not sure what you mean.,.."
TJ says, "That was the last slide, folks.  Time for any general discussion."
Anders Thanks TJ for a great lecture
Norm turns on the lights
TJ is also unclear, and looks at Eric
Gustavo blinks.
Eric [to Norm]: The infected cells may reveal non-specific toxicity of
 infection in vivo that the wouldn't show in vitro.
Eric says, "In one case you are feeding cells serum and other concentrated
 growth factors, and in the other you have real life."
Norm says, "Yeah, but you would think that it would show up in the controls"
Eric says, "The paper hinges on the anti-sense activity being specific, but
 the only good test is in vivo and that's the experiment itself."
Eric says, "They have done all the reasonable controls they can, but this
 can't constitute definitive proof becasue of this flaw."
Eric says, "Not that I would have minded having my name on the paper, heh."
Norm says, "The controls of LacZ + controls in vivo still migrated and didn't
 die"
TJ wonders, would a sense transcript, possibly leading to overexpression, get
 around Eric's criticism?
Eric says, "But the control didn't expess the anti-sense (that was the
 control). If the anti-sense is non-specific, then you get a mess."
Norm nods. "I see"
Gustavo wouldn't mind seeing a knock-out - much more specific..
Eric [to TJ]: No, becasue a sense transcript would of course show different
 binding specificity from the antisense.
Norm learns something.
Eric nods to Gustavo. Bingo. But maybe a dead animal and no data to collect.
TJ agrees with Gustavo, but shares Eric's reservations
Gustavo says, "But maybe a brain-dead animal, and lots of data."
Gustavo grins.
Eric says, "BUT, a targeted knockout, targeted to a subpopulation of cells,
 THAT would work."
Martin says, "how about an inducible promoter hooked up to the antisense "
Norm says, "To make it clinically applicable...knockouts are impratical"
Martin says, "then could knock it out when wanted and not disturb normal
 development"
Eric [to Martin,]: no good ones around work yet. But you CAN do cell type
 specific knockouts.
TJ [to Eric]: Can you suggest an alternative experiment that *would* be
 responsive to your concerns?
Martin agrees
Norm [to Eric]: Cell specific knockouts in vivo?
Eric [to Norm]: True, but you don't want knowledge there; you want clinical
 activity, and worry about side-effects like non-specificity later.
Eric [to TJ]: Sure, but I'll have to explain my own work a bit.
Norm says, "Yikes, tell that to the IRB"
TJ scans the room for the volunteer for next journal club
Eric [to Norm]: Don't tell me doctors flinch at side effects, as long as their
 not too pronounced.
The avian guest has arrived.
Eric [to TJ]: You can express a site specific recombinase in a limited cell
 population, to specifically knock out a gene in that population.
Norm [to Eric]: Of course not...but if they are too profound..and it doesn't
 work...no one would support followup work
TJ points at Martin!
Martin says, "maybe"
Eric [to Norm]: But it'd be worth finding out.
Martin says, "tis rude to point"
Eric [to Norm]: BUt NOT with retrovirus -> adenovirus.
Martin says, "give me a couple of days to figure out a good topic"
Norm says, "I think the line of research trying to attack specific molecules
 is the best..not cells"
Martin says, "ok...... will do it - not sure on what though - neuro-something"
Norm [to Eric]: ADENOVIRUS>>>YES YES YES
Eric [to Norm]: But if the molecules are too widely expressed, the data is
 easier to interpret if you only manipulate a limited cell population.
Norm says, "But you must of course find a way to
 target the cell."
Martin says, "what promoter was used for the antisense?? (please humor my
 ignorance)"
Eric [to TJ]: I spend my days developing the technology for engineering
 cell-type specific genomic rearrangements. Knockouts included.
Norm [to TJ]: What ligand do YOU think is responsible for the migration
The avian guest has disconnected.
The housekeeper arrives to remove the avian guest.

TJ shows slide #2.

                      * * * * * * * * * * * * * * * * * * * *
A number of different vector constructs (next slide) were injected
into E3 chick embryo tectum, as the cells of the ventricular zone
are still dividing.

pLZ10 and its viral homolog, LZ10, have been used previously for 'fate
mapping' as discussed above.
           ____ _ __ _________ _________ _________ ____ _ __
virus -//-|_U3_|R|U5|___gag___|___pol___|___env___|_U3_|R|U5|-//-

           _________   _________ _______________   _________
pLZ10 -//-|_RSV_ltr_|-|___gag___|____lacZ_->____|-|_RSV_ltr_|-//-
                             Bgl^II          Xba^I
                             BamHI

U3|R|U5 = long terminal repeat (ltr) ;  ->  = direction of transcription
                      * * * * * * * * * * * * * * * * * * * *

-----------------------------------Eric------------------------------------
DUAL-TARGET INHIBITION OF HIV-1 INVITRO BY MEANS OF AN
ADENOASSOCIATED VIRUS ANTISENSE VECTOR
   by CHATTERJEE-SR JOHNSON-PR WONG-KK
   SCIENCE  VOL:258 (5087):1485-1488(1992)
-----------------------------------Eric------------------------------------
Eric [to Norm]: Not adenovirus, but almost as good.
Norm says, "Well maybe I am just ignorant on what a knockout is...I percieve
 it as a genomic modification of the entire organism before birth"
Eric says, "This technology of Galileo has also been ued on developing heart
 cells, and cancer cells in an in vivo model."
Norm [to Eric]: did you see my question?
Eric says, "Apologizes for the general paste. It was for NORM."
Eric [to Norm]: About the ligand?
Norm [to Eric]: about what a knockout really represents.
Martin says, "how about NGF, BDNF, all those things....."
Norm [to TJ]: so what ligand DO you think is responsible?
Eric [to Norm]: Oops, I lost it. Please ask again?
KaiG says, "I have to go guys, it's getting late here in Europe. I will read
 the remaining part "
TJ says, "g'night, kai!"
KaiG waves
Eric waves
TJ says, "I would vote for laminin, but just because neurons adhere to it so
 well _in vitro_"
Eric says, "Has laminin been knocked out yet?"
Martin says, "we use collagen and poly-lys .........that works fine....."
Norm says, "Not yet that I know of..."
Martin says, "why laminin"
KaiG has disconnected.
TJ says, "Plus, there is a lot of work on laminin in neuron-glia interactions
 in my favorite system, mammalian lateral geniculate nucleus"
Martin says, "i know poly-lys is artificial"
Martin says, "ahh"
Martin nods
TJ says, "Everyone happy?  Ready to go home?"
Eric says, "And David Anderson's lab uses laminin/poly-lysine for
 promoting neuronal diff'n of neural crest cells."
You say, "In real patients....thanks TJ"
Norm appluads TJ enthusiastically
Eric applauds also.
Martin jumps up to the podium and hugs tj
TJ smiles contentedly
Anders stands up and applauds
TJ blushes at the hug from Martin
You say, "Excellent Job...great slides and discussion!"
Martin claps.....
Norm cheers wildly
Martin says, "sorry i missed the most of it.....slides looked
great"
Norm nudges Martin..."YOU are NEXT"...he cackles evilly.
Martin says, "nev is the BSE guy"
Nev finds its way in.
Martin says, "arrghhh. unless Eric wants to do the next one"
Eric whispers, "I'm working on an adenovirus now, so it's
something I'm interested in"
Martin says, "Hi Nev, want to do a BSE related seminar...."
Martin says, "You just missed this seminar"
Nev nods.
You say, "Whoever. Eric, you want to do it?"
Eric says, "Bad timing for me. Maybe the next one after."
Martin says, "I will help Nev do a BSE related one - whenever
(who is next)"





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