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In summary: - Dag1 glycosylation = formation + function - Dag1 ICD = function We think that extracellular interactions likely with a Nrxn family member is supporting synapse structure whereas indirect intracellular interactions are supporting synapse function.

What about intracellular interactions? I generated a PC specific Dystroglycan ICD truncation to get at this question. Interestingly, these mice had impaired synapse function but totally normal synapse formation.

First: Extracellular interactions. By conditionally deleting the glycosyltranferase Pomt2 I was able to abolish matriglycan expression - the extracellular business end of Dystroglycan. For all intents and purposes these mice had the EXACT same phenotype as Dystroglycan cKOs.

Now that we’ve established Dystroglycan’s role at these synapses: HOW is it accomplishing these roles? To do this I generated two mouse models: one in which extracellular interactions are disrupted and one where intracellular interactions are disrupted.

What was surprising was the increase in SIZE of GABAAa1 (but not VGAT) puncta. Reduced mIPSC amplitude + increased GABAAR puncta size? We think that Dystroglycan is regulating synaptic structure and we presume that there is a misalignment of synaptic microdomains in our mutants.

Perhaps to be expected, deleting Dystroglycan prior to synaptogenesis resulted in inhibitory synapse deficits. I found a reduced mIPSC frequency and amplitude by electrophysiology as well as reduced VGAT and GABAAa1 density by immuno.

This work really took off when I identified the Calb1-Cre line as a viable tool for deleting Purkinje cell genes as early as ~E14. This meant that for the first time we could study the role of Dystroglycan in synapse formation. (See my work characterizing Calb1-Cre in PCs below.)

The second half of my dissertation work is now up on bioRxiv! Here we examine the contribution of the functional domains of Dystroglycan to inhibitory MLI:PC synapses in cerebellar cortex 🧵

A couple important lessons here. (1) Characterize your Cre line! It might not do what you think it does. (2) Reporter expression ≠ target gene deletion.

For kicks, I also characterized the timecourse of reporter expression and Dystroglycan deletion using virally expressed Cre (AAV8-CMV-Cre at P0) and found a similar timecourse to that of Pcp2-Cre.

F i n a l l y, I found Calb1-Cre. This was the answer for me. Calb1-Cre turns on embryonically and has stable expression in Purkinje cells throughout life. My Dystroglycan conditional knockouts showed no synaptic Dystroglycan signal in Purkinje cells at any time point.

We think that Ptf1a expression is *too* transient. So transient that it recombines the tdTomato reporter allele in every GABAergic cell, but Dystroglycan in only a subset of them.

So I tried Ptf1a-Cre. Ptf1a is transiently expressed in cerebellar GABAergic precursors (Purkinje cells + MLIs in cerebellar cortex). I was encouraged to see tdTomato reporter expression in all Purkinje cells early postnatally, however Dystroglycan deletion appeared stochastic.

Surely, Nestin-Cre would be early enough, despite not giving me cellular specificity. Nope! Turns out the Nestin line we have does not drive Cre recombination in Purkinje cells at all!!

Two things were happening: (1) Cre was turning on in Purkinje cells gradually from P7-P14 and (2) Dystroglycan was taking forever to turn over. I needed a Cre line that turned on earlier.

I began with a simple experiment: use the popular Pcp2-Cre mouse line to conditionally delete Dystroglycan from Purkinje cells. To my surprise, Dystroglycan wasn't deleted from every Purkinje cell when I looked at P16 despite tdTomato reporter expression in all cells.

This paper was born out of a personal struggle to identify a Cre line specific to Purkinje cells in the cerebellum, *prior* to synaptogenesis. This is the paper that I wish existed in my first year of grad school.

Do you study cerebellar Purkinje cells? Do you use conditional genetics to manipulate said Purkinje cells? Friend, do I have a paper for you: Tools for Cre-Mediated Conditional Deletion of Floxed Alleles from Developing Cerebellar Purkinje Cells

It's hard to believe, but the first of the year's Advanced Research Training Courses is already over! Students in the Analytical and Quantitative Light Microscopy (@AQLMMBL) course arrived on campus on April 24 and head home today.