Two color MARCM differentiates sister cells

Twin-spot MARCM
Twin-spot MARCM to reveal the developmental origin and identity of neurons [Nature Neuro]

We mentioned the innovative MARCM technique in a previous post. Here, Lee and colleagues extend MARCM (Mosaic Analysis with a Repressible Cell Marker, pronounced mark-em) to twin-spot MARCM, where both cells from a mitotic event are labeled with different colors fluorescent proteins. In regular MARCM, only one cell is labeled and the other daughter cell remained unlabeled. Like the original MARCM, this technique lets you distinguish between what would otherwise be identical pairs/clonal populations of cells during development and gain insight into the (lack of) stereotypy in development. Under the hood, twin-spot MARCM is a bit different: Instead of relying on GAL80 suppression of GAL4-driven transcription (regular MARCM), twin-spot MARCM uses RNAi directed against the protein-coding transcripts.

Since MARCM can be difficult to understand, here’s an excellent, detailed yet easy-to-understand description written for a bio lab class from Richard Vogt at the University of South Carolina:

  1. A fly is constructed with the following genotype: (promotor)Gal4; UAS-GFP. In this fly, the promoter drives the expression of a transcription factor called Gal4, and Gal4 binds to and activates a regulatory site referred to as “UAS” (upstream activating sequence). Activation of the UAS site drives expression of GFP (green fluorescent protein) which fluoresces green when stimulated by blue light.
  2. This fly also contains a gene encoding and expressing a protein called “Gal80”; Gal80 suppresses the action of Gal4. If Gal80 is expressed, no GFP is made and no green fluorescence can occur.
  3. This fly also contains a complex of genes referred to as the FLP/FRT system; FLP is a transcription factor that activates the FRT site, which is situated adjacent to the Gal80 site. Further more, at least in our case, the FLP is driven by a “heat shock” promoter (hs). All this means is that when you raise the temperature of the animal to 37oC, this activates the hs promoter which activates the expression of FLP which activates the FRT site.

            Something I’ve not mentioned yet… there is also an FRT site adjacent to the UAS-GFP site. Something else I’ve not mentioned yet, the FRT-UAS-GFP site and the FRT-Gal80 site are on the same chromosome, but importantly on different chromatids.

  4. So we make a bunch of fly embryos that have all this stuff in them. Procedurally this is really easy, since the genes have already been put in the flies, and all we have to do is take virgin females of one stain (FRT-Gal80) and mate them to males of another strain (FRT-UAS-GFP) and… POW… we have fly embryos that have all this stuff in them.
  5. All the cells in the embryos we now have are capable of expressing GFP except for the one problem… all the cells are expressing Gal80 which is blocking the expression of GFP. We need to turn off Gal80 expression. We do this by activating the FLP/FRT system.
  6. Normally, a cell has two copies of each chromosome called chromatids. In our case, the chromatids are different, one containing by FRT-Gal80 and the other containing FRT-UAS-GFP. This cell can not express GFP because Gal80 is present. During mitosis, the chromatids are duplicated and sort to produce two identical chromatid pairs, both pairs consisting of a FRT-Gal80 chromatid and a FRT-UAS-GFP chromatid. Like their mother, neither daughter cell would be able to express GFP, again because Gal80 is present.

            HOWEVER, AND HERE IS THE TRICK… if the FRT is activated during mitosis, it induces a recombination event (recombination normally only occurs during meiosis), creating one chromatid pair that contains only UAS-GFP and another chromatid pair that contains only Gal80. One of the resulting daughter cells now contains no Gal80, and suddenly is able to express GFP and fluoresce green light. And any additional cells produced by this daughter will also express GFP.

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Transcriptomics of the fetal human brain

A cutting-edge application of the Affy total human exome GeneChip (4X coverage per exon, 40X coverage per gene): Functional and Evolutionary Insights into Human Brain Development through Global Transcriptome Analysis.

From the News and Views, I was intrigued to learn that previous transcriptome analyses of adult human brains found very little difference in gene expression between brain areas:

[…] this suggests that it is the gene expression during development that largely determines higher brain functions by specifying the complexity of neural connections. Numerically, the most important genes relating to cognitive differences between species may be genes that specify how the machinery is put together. In support of this hypothesis, many of the identified differentially expressed genes in this study are related to processes involved in connection formation, such as axonal guidance and cell adhesion.

An impressive 76% of all human genes are expressed in the developing fetal brain. Of those, 33% are differentially expressed over brain regions (13 regions were examined) and 28% are alternatively spliced. The differentially expressed genes are also ones that seem to have evolved the most recently. Even in these early (midgestation) stages, left-right asymmetry was seen, such as the localization of the language-associated FOXP2 genes to Broca’s area.

Of interest to computational folks, they find that gene expression follows power-law scaling (as many other naturally occurring “small-worlds” networks do) with certain hub genes connected to many others and certain spoke genes with relatively few connections. Unsupervised hierarchical clustering is used in this analysis.

Visualizing synaptic tagging and capture

A set of two articles recently came out in Science that directly visualize two different (and likely complementary) approaches to synapse specific delivery of gene products. Plasticity at specific synapses (input specificity — we’re restricting the discussion to the dendrites of the post-synaptic neuron) requires proteins (eg. new AMPA receptors) to get to those post-synaptic compartments and membranes. But the intructions for these new proteins are contained in the nucleus with the rest of the genome. Clearly, new proteins synthesized in the soma can’t just be sent everywhere, since only specific inputs (eg. particular dendritic spines) need these new proteins. How does this happen? Hence, the postulated synaptic tag.

Two approaches

Broadly, there are two approaches to synaptic tagging: 1) mRNA is distributed widely and translated locally at tagged locations; 2) protein products are distributed widely in the bodies of dendrites but only contact/off-load at tagged synaptic specializations. This News & Views gives a nice overview of the two papers, which find approach 1) in Aplysia cultures with sensorin mRNA and approach 2) in rat hippocampal neurons with Vesl-1S/Homer-1a protein. It amazes me that both were found pretty much simultaneously, but that might have more to do with the use of the photoconvertible Dendra2 protein than anything else.

With both approaches, we still don’t know why mRNA/protein is directed to a certain location. That is, we can visualize synaptic tagging but we don’t know what is the tag, its ontogeny, or the mechanism of tagging. But that might not be so important to understanding more about neural function. These new tools might allow us to image plasticity at many synapses at once, perhaps even in vivo. But before that, more work is needed… does the optical signal (from the Dendra fusion protein) correlate with degree of potentiation? Can we detect plasticity in the opposite direction, ie. synaptic depression, through another tag?  (As a sidenote to approach 1), the use of 5′ and 3′ UTRs as a sort of molecular zipcode is also intriguing.)

Sunday afternoon reading: Genetic tools "primer"

I came across this fantastic review of tools for the Genetic Dissection of Neural Circuits in Neuron a few days ago. It’s by Liqun Luo, Ed Callaway, and Karel Svoboda. I highly recommend it, as it spans the gamut from genetic targeting (recombination, binary logic, viral delivery) to circuit reconstruction (super resolution LM, EM, brainbow) to activity modulation and functional mapping (uncaging, artificial GPCRs, light-gated channels, MIST). I don’t think I’ve ever seen quite a review of so many cutting edge neurotechnologies in one place. I can’t recommend this piece enough really. For me, with my lack of molecular expertise, the first sections on combinatorial gene targeting/expression techniques were great, pulling together Gal4, Cre/Flp, and Tet systems into a unified framework, along with more general concepts like site-directed integration, enhancer-trap, and repressor trap (eg. Thy1 mice).

Neuroengineering memory: Something old, something new

Over the last week, it seems like everyone has sent me this NYT piece on PKM-zeta (about work in Todd Sacktor’s lab). I’m not sure why this work is being featured in the Times right now, since it’s a few years old. But it was news to me and I think it is of interest to anyone trying to understand structure-function relationships in the brain. In the original Science paper (from 2007), a pseudosubstrate inhibitor of PKM-zeta caused irreversible loss of a conditioned taste aversion memory (news and views here). I was unfamiliar with PKM-zeta, which appears to be a constitutively active form of PKC-zeta (a kinase that some might be more familiar with) and that lacks the autoinhibitory regulatory domain of PKC. The amazing phenomena is that, after treatment with ZIP (the pseudosubstrate that ties up PKM-zeta), the memory is permanently erased and doesn’t seem to return.

What’s going on? One tantalizing possibility is that the enzyme itself is directly related to the memory trace. This contradicts the (unproven) assumption of modern neuroscience that memories are stored solely in the synaptic strengths (ie. membrane-bound receptors) of a neuron. The other suggestion is that PKM-zeta is actively maintaining synapses and that enzymatic inhibition disrupts the precise maintenance of receptors or synaptic machinery. The effects happen quite fast (within 2 hours after drug injection), which seems short for receptor recycling but perhaps long enough for structural change to occur. I’m no expert on receptor movement: Is 2 hours long enough to add/remove a significant number of receptors?

Fascinating work but the method is blunt, wiping all experimentally-induced memories (and probably others too). Last month, another group reported (also in Science) selective erasure of a fear-conditioned memory using an interesting new genetic tool. Here, neurons in the amgydala that overexpressed CREB were found to be preferentially recruited into a fear memory trace (as shown in a previous Science paper). Incorporation into the memory trace was assayed by expression of the immediate-early gene (ie. activity-dependent) Arc. In the present study, they combine overexpression of CREB in a subset of neurons with cell death (via Diphtheria toxin in a transgenic mouse vulnerable to diphtheria). Apparently, normal mice lack the receptor (here a simian version is used) that confers pathogenicity for diphtheria. Thus, the viral construct both overexpresses CREB in a subset of neurons and selectively makes the same subset vulnerable to diphtheria. Ablation of just these neurons causes a permanent loss of the memory. Subsequent similar learning proceeds just fine (using the remaining neurons).

Can we say that the race is officially on to ablate just the synapses involved in the memory? I think so. Extra points if the ablation is reversible too!

Deisseroth on new step ChR2

Nature methods has a small piece interviewing Karl Deisseroth on properties of the new step ChR2.

Some shortcomings of step ChR2 and future research directions:

Deisseroth expects ongoing efforts to improve key features of these channels. “One disadvantage is that some of the mutants have reduced current compared to wild type, so multiple mutations may help to bring those current levels back up to wild-type levels,” he says. Projects designed to improve membrane targeting and to apply a composite of opsins, including the red light–responsive channelrhodopsin from Volvox carteri, are also in the works in his laboratory.