Of interest

Hello, It has been awhile Internet world!

After I finished my applications for admission to graduate school I jumped on a plane to Europe. I celebrated thanksgiving in Sweden. It was magical - Stockholm seemed like a beautiful snow globe and the people were just as amazing. I did stop in London and Paris, both cities are great as well, but for some reason I have a strong connection with Sweden.

Regardless, I'm back in lab with tons to do.
Today I received a new batch of Math1-Lacz spinal cords back-filled from the thalamus with Fluorogold and last week I prep a huge litter of E19.5 Tamox-induced (@E8.5) Math1-myrGFP spinal cords for examination.
I am really looking forward to examining these cords and getting an idea of what’s going on. Especially the Tamox line - I haven't looked at spinal cords older than E13.5 Sooo it will be interesting to see if I can detect any projections into the brain at this stage. (Or really detect anything at all - the literature and in situ's done suggest that Math1 expression in the spinal cord begins at E9.5 and continues till E13ish. Therefore, my tamoxifen treatment may not have induced any GFP expression OR dependent on how long the tamoxifen stays active in the system it may induce and label the first round of cells to differentiate into the D1 class.)







Anyways, here's your food for thought today.


Please press that link and read the letter.

MTL?!

Soon is the 33rd Annual W. Alden Spencer Award and Lecture is taking place here at Columbia. They are honoring Larry Zipursky and Marc Tessier-Lavigne.
Zipursky intitally discovered and explored the family of Dscam proteins – Oweing to much of our understandings of neuronal circuitry.

Tessier-Lavigne has been a name in my mind for six years now; he has uncovered many mechanisms relating to axon guidance. He has just been donned as Rockefeller University’s new president and has lead quite a wealthy career keeping up with basic research while also pursuing the biotech industry. He is definitely someone I look up to when I imagine life in terms of my own career. I welcome him to New York City in March 2011 (I’m hardly even an ant but still)!

Recently, He published in Journal of Neuroscience with Alexander Jaworski and Hua Long at Genentech on “Collaborative and Specialized Functions of Robo1 and Robo2 in Spinal Commissural Axon Guidance”.
Here are some main discoveries:
They analyzed the amount and ability of axons to cross the midline at the floor plate during project at E11.5 and E12.5 in various genetic backgrounds. Robo1 and Robo2 are the receptors which respond to the Slit1-3 repellants produced in the floor plate.
Robo3.1 is implicated in repressing Robo1’s function therefore diminishing axon repulsion and allowing commissural projecting cells to target their growth towards the floor plate. After midline crossing, Robo3.2 is then unregulated in axons and acts as a classic Slit repellent pushing axon growth towards its next target.
Robo3 mutant animals have a major deficit in commissural projecting neurons in the spinal cord. In previous studies it was found that in the mutant Robo3 background loss of Robo1 partially rescued the deficit, as well as, loss of both Slit1 and Slit2.
In this study they found that in the Robo3 mutant background a loss of both Robo1 and Robo2 restored the crossing deficiency significantly. Indicating the failures of Commissural axons to project through the midline in the Robo3 mutant are partially due to Robo1-depedent repulsion but also Robo1- and Robo2-repulsion.
Due to combination mutants of Robo1/2/3 they end up illustrating that Robo1 and Robo2 work together to prevent post-crossing axons from reentering the midline and they have individualized roles in the sorting of axons into specific positions within the ventrolateral funiculus.
Lastly, I find most interesting, they provide evidence that there is most likely a Robo3-dependent mechanism not involving Robo1 or Robo2 (or the combination of both) allowing midline crossing in a subset of contralateral neurons.


I will post soon on the talks given at the Spencer award & lecture!

Seminar: Embryonic stem cells as a tool to study Motor Neuron development and diseases

Today I attended a PACKED seminar by Columbia's own Hynek Wichterle in the department of Pathology and Cell Biology. The walls were lined with standing people and the floor covered with sitting students.
Hynek's lab studies stem cells in hopes to better understand motor neuron development and diseases.
He began his talk introducing Hans Driesch's experiments with Sea Urchins in 1892. He, studying the embryo, separated the cells resulting after the first round of cell division. He imagined that the cells would then develop into the respective but separated portion of the sea urchin corresponding to the location where the cells were harvested. Instead each of the cells developed into fully functional complete sea urchins.
The initial cells Driesch harvested where embryonic stem cells, totipotent in nature, able to differentiate into any cell (not to be confused with: Pluripotent able to become cells that compose any of three germ layers, multipotent able to become many cells of a related family, Oligopotent differentiate into very few set of cells, and unipotent can only become themselves). So awesome, right? Let's just play with embryonic stem cells. But, really - let's!

In any developed organism the amount of cellular diversity, the systems of cross talk and proximity between these highly diversified cells makes studying specific cells very difficult. In vivo animal mutant models can be tricky and lead to embryonic lethality before gaining any new insight. Therefore, learning more about the genetic pathways and environments needed to induce stem cells to become specific types of cells can allow researchers to study those very cells more concretely. Wichterle aim's to do just that with motor neurons.

Hynek continues on to point out that due to the diligence of labs previous research (please visualize the head nod that ensued in the direction of his previous mentor, Tom Jessell) we know ways to distinguish motor neurons based on molecular identity and functionally. The signals required to develop spinal cord motor neurons are also being elucidated: starting from the rostral or cervical end of the spinal cord (the neck) retinoic acid (RA) signals at its strongest then lessens as a gradient down the spine through the brachial region towards the middle of the cord meeting the least of the increasing gradients of GDF, FGF and WNT (signaling morphogenic molecules).
Wnt, GDF, and FDF increase along the thoracic level till reaching the end of the lumbar or caudal spinal cord. Lastly, across the entire ventral portion of the spinal cord, SHH signals as a gradient strongest from ventral lessening upwards into the dorsal (top half) area of the cord.

Using these tools and signals, the Wichterle lab has been successful in inducing pluripotent stem cells in vitro to differentiate into cervical and brachial motor neurons. They express the appropriate markers, when injected back into a developing spinal cord they migrate to the correct distinct positions, and can then function perfectly as the in vivo developed cells within the tissue. Now with the model cells to work with the lab moves on to test the role of repressive chromatin in the pattern of developing motor neurons in the rostral to caudal axis, microRNA's in the dorsal-ventral axis, and modeling neuronal diseases like ALS.


Hox gene products, homeobox proteins, are an important family of transcription factors during development; they aid in the creation of different motor neuron classes across the Rosto-Cadual axis (length from the neck to tail). Different sets of Hox genes are required to be expressed in different anatomical locales along this axis. For example, starting Rostal, the cervical region requires Hoxc genes 4-5, Brachial requires Hoxc6-8, and (moving caudual) Thoracic Hoxc9 and Lumbar Hoxc10-13.
The hoxc genes are interestingly assembled in the genome on chromosome 12 in a similar fashion to expression; hoxc gene 4-5 are organized in the 3' end in chromatin (DNA) and increase in order of number till the 5' end. Therefore, it is thought there must be repression of specific hox expression in these distinct anatomical locations to allow for the differential gene expression (i.e. repression of hoxc4 & 5 in the lumbar section but none of hoxc10-13 permitting their expression). It was postulated through correlations that during development repression is lifted from the primary hoxc genes, priming the Chromatin structurally, allowing transcriptional molecular machinery access for the later relaxation (de-repression) of the numerically higher hoxc genes. Therefore, it was postulated that there is a progressive clearance of repressors on the chromatin starting with Hoxc4 and continuing to Hox13 during the due time of need for induction of those cells.
Wichterle and Dansen labs collaborated (NYU, NOTE: post-doc from Jessell lab as well) to confirm the postulated nature of the Hoxc chromatin. They performed chromatin immunoprecipitations on stem cells and found that all hox genes had a considerable amount of repressive markers across them. They looked at the different cell types in the cord and found in development cervical motor neurons lack of repressive markers. Puzzling, they decided to do Quantitative PCR to profile the hoxc gene expression during time points in the developmental cascade of inducing stem cells to motor neurons. They found that there was no progressive clearance of repression, the profile of hox gene repression did not correlate with expression. They decided to do more chromatin immunoprecipitations but looked for the density of retinoic acid receptors. They found a correlation between the presence of RA receptors located on the hoxc 4-5 genes and the loss of repressive markers. Wichterle states that somehow the recruitment of RA receptors initiates the clearance of repression. Cool, How?
Its pretty convenient that the two most well know players in the patterning of motor neurons along this axis show tight correlation between their activities. Can you further confirm RA receptors role by stunting it's gradient or prolonging its gradient along the axis, then look at methylation (this is the marker they used for repression, methyl groups added on chromatin usually mark a repressed gene)? That is, at a time when the changing RA gradient won't completely mess up the fate of the cells...?
What are the molecular complexes that are responsible for providing the methylation on the chromatin? What is the gene expression profile for these chromatin remodelers - Micrroarray and look for enrichment of certain known chromatin modifiers?

Wichterle goes on to question how the caudal neurons develop. It seems throughout the development of the cells there is no lack of repressive markers. They looked at a microarray to see what genes were enriched in the lumbar cells versus the cervical. They found one interesting gene, Cdx2. Inducing stem cells with cdx2 seemed to have no role, but they found in cooperation with FGF they could induce expression of hoxc8 motor neurons. Through further experimenting they found they could also induce hoxc6-9 genes but repress hoxc1-4 genes. He concludes showing that hoxc9 likely cooperates in the repression of hoxc1-4. (For more information see paper)
The orchestra is getting meatier regarding the instruments to play out development of even one cell type: Signals, transcription factors and chromatin modifiers...

Sorry for the length of this one...

Hynek continues with part two, what I found to be the most interesting data, about the dorso-ventral patterning of the spinal cord. SHH signals as a gradient from the notocord and floorplate (Ventral/bottom of Spinal cord aka the side facing the front of your body) which is responsible for inducing different varieties of Interneurons and Motor neurons based on the receieved amount of SHH. In the ventral portion, closest to the floor plate, cells are induced to express NKx2.2 as the gradient lessens dorsally the next class of express Olig2 (Motor neurons) and then further lessening of the gradient the next class express Irx3 (picture: http://en.wikipedia.org/wiki/File:Sonichedgehog.jpg). These genes are markers of progenitor cells, the cells expressing them have matured from stem cells and based on gene expression decided to be a specific type of cell. (ex. nkx2.2-motor neurons, olig2-oligodendrocytes).
Hynek's lab investigated the role of microRNAs in cells that border an area with overlapping gradient signals.
Presumably these two gradients are providing the cells with signals that instruct competing progenitor gene expression to either become one type of cell or another. Somehow cells naturally overcome this confusion and the spinal cord results in a clear pattern of segregated neuron types.
The lab noticed previous research done that identified a gene that when knocked out would eliminate all microRNAs, Dicer. Dicer mutants do not live past gastrulation, therefore studying it's affects have been proven difficult. Wichterle's lab formulated a conditional tamoxifen-inducible line for dicer. Allowing them to knockout dicer's expression during specific time frames in development. They still had issues with survival of the animals but had enough success to illustrate an expansion of motor neurons and losses of Irx3 cells. Seemingly, in the natural background microRNA represses the expression of Olig2 allowing only a discrete segment of the spinal cord to develop into motor neurons. This indicates that the p2 set of cells (Irx3 expressing) must transiently express olig2 at some point. The further investigated the situation by looking at the microRNA profile of cells exposed to high vs. low levels of SHH.

To Be Continued...


This is very long. I do apologize. I think I just got really excited about the research...my head is exploding with information.

Seminar: Developing Therapies for motor neuron diseases.

I've been meaning to start this blog for quite some time now. The basic point is that I have too many thoughts; I can end up talking too much and making way too many tangent conversations.
I've created this blog for me to summarize certain seminars I attend, papers I read and just blab about my work/things that interest me. Not only will I be able to clear my mind but I am hoping some will read this and have questions/answer my questions/comment.

Disclaimer:: I do not think I have any literary abilities. Please do not focus on my writing capabilities or grammatical errors.

Here it goes:

Today I attended a talk hosted by the Motor Neuron Center at Columbia University Medical Center. The speaker was Brian Kaspar from Ohio State University, he presented a short talk on his lab's work on Gene therapeutics targeting SMA and ALS.
He began the talk introducing the general problems with targeting diseases in the CNS; the blood brain barrier, diversity of cell types, magnitude of the amount of cells from the Brain reaching down throughout the spinal cord, etc. Kaspar then introduced Adeno-associated viral vectors (AAV). AAV is a virus naturally found in humans and nonhuman primates, it has no known pathological responses and can integrate into post-mitotic cells. Once infected in cells it integrates it's genomic information stably for long term trans gene expression (on Chromosome 19). With these attributes it sounds like an awesome vehicle to use when searching for ways to deliver gene therapies to various live systems. Currently there are clinical trials ongoing for Cystic fibrosis and Parkinson's disease.
The trick is figuring out which family member of the virus to use, how to administer it, when to administer it and what are the effects of the trans gene expression. For example, some familial genetic disorders present themselves because of a lack of a certain gene's expression (therefore, lack of a certain protein). Some gene therapies have a long list of issues in clinical trials because of the body's natural immune response to a protein it has never 'seen' before - even if that protein can rescue the disease over time - one would still need to battle the immune system to allow it. In light of this issue, another problem occurs = lack of focus on efficacy. FDA clinical trials usually focus on the safety of the treatment but not the efficiency to alleviate the disease. Moving on...
The lab initially started testing AAV6-GFP (green fluorescent protein) and AAV8-GFP through tail vein injection but had no success in penetrating the blood brain barrier to the CNS. However, when they tested AAV9 they were very successful in finding GFP expression in a robust amount of astrocytes all over the nervous system and some neurons. Astrocytes are mainly born a few days postnatal. With this in mind they tried to target mostly neurons by injecting intravenously through a facial vein visible postnatal 1 and 2 (tails are too small then). The result was great GFP expression in the dorsal root ganglia and motor neurons throughout the spinal cord and various neurons in the brain (neocortex, hippocampus, cerebellum).
Both scenarios present possible good routes for therapies against either SMA (neonatal injection) and ALS (adult tail injection). SMA is luckily known to be attributed to a loss in the SMN1 gene expression. Therefore, animals affected by SMA have at some point 'seen' smn1 protein physiologically speaking. Using an SMA mouse model, they tested the ability of AVV9-SMN1 treatment to rescue the disease phenotype. In short, they were successful in finding that the treated SMA mice compared to untreated SMA controls were able to become stronger and eventually live normal mouse life spans (tested through muscle growth, behavior, electrophysiology, and life span). However, SMA semi-adult mice (they only live about 15 days) past neonatal do not response similarly to the treatment, as the mice age the AAV starts targeting mainly astrocytes and less neuronal populations which ends up not rescuing the illness.
The Kaspar lab is now testing this gene therapy on non-human primates and having great success. They plan to move forward with human clinical trials.
They are also doing a lot of work on ALS, but I'm tired of writing about it for now. Need to do some image analysis.

Very exciting stuff in terms of possible treatment options.

Here is a link to their recent paper about AAV9 injections: http://www.nature.com/nbt/journal/v27/n1/abs/nbt.1515.html

"Expression of major guidance receptors is differentially regulated in spinal commissural neurons transfated by mammalian Barh genes"

Expression of major guidance receptors is differentially regulated in spinal commissural neurons transfated by mammalian Barh genes.
Developmental Biology
Kawauchi, Muroyama, Sato, and Saito

The authors claim that the competence of spinal cord neurons to send axons ventrally across the midline to become commissural neurons is temporally dependent on embryonic stage. They focused their studies on the known guidance receptors and transcription molecules that mark and aid Math-1 derived D1 interneurons in their characteristic maturation. During said embryonic stage misexpression of mbh genes activates guidance receptor genes, Rig1 and Np2. Lhx2 has been shown in our lab to have a strong potential genetic relationship with Rig-1, furthermore, Lhx2 mutants do not extend their axons through the midline, instead their trajectory stops adjacent to the floor plate in the ventral spinal cord. Kawauchi claims to have revealed that mbh genes (mbh1 and mbh2) control pathways either lhx2-dependent or independent of guidance receptor expression.

I was under the impression this information was already known…

Kawauchi introduces the basics of commissural neuron phenotypes in the developing spinal cord:
The transient and simultaneous expression of chemoattractants and receptors, as well as, chemorepellents and their receptors is a very intriguing display of the amazing molecular circus. Expression and function of these groups must be temporally and distinctively expressed locations allows cells types to obtain their distinguishing properties for a functioning growing adult.
Ignoring the Roof plate for the moment, we know that attractants, like netrins and sonic hedgehog, and repellents, like slits and semaphorins, are produced from the floor plate acting upon commissural neurons aiding in their trajectories. Some of the key chemo-guidance molecule/receptor pairings attracting to and across the floor plate are: Netrin-Dcc, Sonic hedgehog-Boc, Slit–Robo1, and Semaphorin-Npr2.

So please imagine this for a moment: tons of neuronal cells are being born and oozing out of the ventricular zone in the spinal cord. They have been seemingly fated by their progenitor gene expression to begin a cascade of further gene expression requiring them pretty much instantly to migrate their cell bodies to a particular position and simultaneous send their axons to specific locals. Numerous cells are engaged in this activity at the same time but we end up with very diverse groups owing to evolutionarily distinct fully functioning organisms. While in this discussion we are concerning ourselves with the understanding of a specific cell type and genetic pathway, if you think of multiplying these ideas by the millions, the image of orchestration of development becomes a mystifying tale. (A tale that always leaves me hungry and smiling).

Focusing on Rig-1 (Robo3), it functions to repress the role of the Robo1 receptor allowing cells expressing Rig-1 to ignore Slit repulsion until after crossing the floor plate. Robo1 is then used to suppress Dcc to prevent re-crossing of the axon through the floor plate (Plus, it has also been shown with growth cone studies that Slit’s repulsion dominates Netrin’s attraction). Kawauchi et al, go on to investigate the transcriptional relationship between Lhx2, Rig-1, and the bar-class homeobox genes mbh1 (barhl2) and mbh2 in fating the commissural Math1derived dorsal spinal neurons.
After in vivo electroporation of mouse embryos at E11.5 & E12.5 with Mbh1, Mbh2, Lhx2, Lhx9, and Math1 they provided some evidence supporting the bar-class genes playing an important role between Math1 expression and the Lim HD transcription factors.
One first notices d1 neurons by the expression of Math1. Math1 is a neuronal progenitor solely responsible for birthing all commissural projecting Lhx2+/Lhx9+ cells in the dorsal most area of the spinal cord beginning at E9.5. Once cells have turned off Math1 they, based on embryonic day, express Lhx2 and Lhx9 (more specifically, in my studies they express them temporally not only based on their dorsal-ventral migration location but also based on their RostroCaudal location). But the genetic-molecular control of this transcriptional control is not well understood.
They found that ‘misexpression’ of mbh1 and mbh2, but not Lhx2, by transfection into right side of the spinal cord at E11.5 (then allowed expression for 2 days till analysis at E13.5) have almost redundant roles in providing an >10% increase in commissural projecting axons. (*Of note to me, there are seemingly some ipsilateral projecting cells labeled on the medial lateral edges). Transfection at E12.5 analysis at E14.5 does not provide the same effect and cells obtain a phenotype copying transfection of Fluorescent reporter DNA only.

(*I would like to note that at this point in the paper there is a shift of focus. In my opinion this shift is modestly expressed. If I were writing the paper I would have tried to highlight the importance of the information/analytical ideas expressed. I understand that the shift is based on theoretical ideas - that they only support these ideas they do not concretely provide evidence. But it lays down, to me, a good path for investigation).

This is exciting -maybe. As in, the cells are not as malleable to gene expression instructions that they may have been a day or few earlier. Have they past some threshold in development where new waves of genes have been expressed to allow a suppression of the old ones (similar to the Rig1/Robo1/Dcc story)?? The authors say the cells transfected at E12.5 are not ‘competent’ to become commissural neurons.
ORRRR (bear with me) have a certain amount and type of genes relating to specific functions of that neuronal cell already been expressed and sequestered to specific areas in the cell, so, when they are needed (as RNAs with the machinery available to express them as functioning proteins when signaled to be needed) to respond to the transient distinct spatial signals from the environment they have a stock to do so efficiently.??
Kawauchi again transfects cells at E11.5 with mbh1 and mbh2 then examined changes in gene expression for most of the known guidance receptors. Robo1, Robo2, Kit, Boc, and Dscam were not activated by misexpression of either Bar-class genes. However, Rig-1 and Nrp-2 were both elevated. Nrp-2 elevated expression does not change 2 days after transfection when the cells have reached the deep dorsal horn settling position (mirroring the endogenous expression). However, Rig-1 expression remarkably disappears by the time the cells reach the deep dorsal horn 2 days after transfection. The authors imply that this indicates that Rig-1’s activated misexpression by mbh is able to be saved/regulated by innate pathways naturally downregulating Rig-1 at the appropriate time/settling position. Mbh1 did not activate Mbh2 and mbh2 did not activate mbh1. Transfection with Lhx2 activated Rig1 but Lhx9 transfection did not (both Lhx gene tranfsections had no activating affects on Mbh1 or 2).
Interestingly, Mbh1 and 2 transfection has been shown to increase Dcc protein levels. However the misexpression does not result in an increase/activation of Dcc gene transcripts, suggesting an increase in Dcc protein is based on activating post-transcriptional expression as the authors state. AWESOME.
Math1 transfection into the spinal cord activated Mbh1, Mbh 2 , Lhx2, and Lhx9 gene expression. (Note: Lhx9 activation was weaker by Mbh genes than my Math1 suggesting there are more Mbh-independent factors involved or a timing issue). To decipher the order of gene expression, since Mbh1 and Mbh2 also activate Lhx2/9 genes, Kawauchi transfected Math1 and a construct that expresses dominant negative form of either Mbh1 or Mbh2. They found the activation effect on Lhx2 and Lhx9 genes by Math1 was repressed in the presence of the inactive form of Mbh1. But Mbh2 activation by Math1 was not repressed in the presence of the dominant negative Mbh1 proteins. (Note: repression of Lhx9 was not complete therefore more of an indication that there are mbh-independent pathways leading to lhx9 expression).
They found that it is the repressive abilities of Mbh1 and Mbh2 (through expression of chimeric Mbh proteins consisting only of the homeodomain and transcriptional repressor domain) that allow for activation of Lhx2 and Lhx9, therefore, most likely activation is an indirect effect.
Further strongly supporting the idea that there is another in-between factor from Mbh’s to Lhx2/9.
Well I guess we can somewhat confidently say that between Math1 and Lhx2/9 Mbh1 and Mbh2 repress expression/activity of another factor that represses Lhx2/9 therefore aiding in their expression.
I think animal models with single or combinational mutant knockouts might provide more evidence.
I wonder if there have been studies involving axon severing then analyzing the ability of the growth cones to express/perform different proteins/functions in response to different signals (or move…probably would definitely not be able to move…but maybe reorient)...
I'm thinking that if mRNA transcripts are produced and localized to specific areas in the axon or growth cone AND if the cell has produced transcripts but not the protein - The cell may be waiting for further signal to express the protein, therefore, if they are pre-produced and localized based on the cell type (mutant background, etc.) we can make some comments on how the severed growth cone acts in response to extracellular signals.
Just brainstorming...