The MGE produces INs (SST+ somatostatin, PV+ Parvalbumin) that will migrate into the cortex and striatum.6,7,8,9,10,11
The LGE generates medium spiny neurons (MSNs, BCL11B+) for the striatum and INs that will migrate into the olfactory bulb (CALB2+, PBX3+).12,13,14,15,16
Finally, the CGE produces INs (VIP+ vasoactive intestinal peptide-positive, LAMP5+) that will travel to the striatum, cortex, and amygdala.
Single-cell dissociation of brain organoids for single-cell RNA Sequencing (10x Genomics Chromium platform)
The following protocol is a modification of the Worthington Papain Dissociation kit manufacturer's protocol, as used in previous works from our lab [3,8].
1. Before starting the protocol, reconstitute the following reagents:
1a. Add 5 mL of Earle’s medium into Papain vial (5 ea/kit - use 1 vial for 2 organoids);
1b. Add 500 μL of Earle’s medium into DNase vial (5 ea/kit - use 1 vial for 2 organoids);
1c. Add 32 mL of Earle’s medium into Inhibitor vial (1 ea/kit - use 1 vial for up to 10 organoids).
2. Mix 500 μL of reconstituted DNase with 5 mL of reconstituted Papain.
· CRITICAL STEP: Mix gently as DNase is sensitive to shear denaturation.
3. Gently transfer each organoid to an individual 60 mm dish.
4. Carefully aspirate excess media and add 2.5 mL of Papain + DNase solution to the 60 mm dish.
5. Using a new, sterile razor blade, mince organoids into small pieces (< 1 mm).
6. Transfer the plate to an orbital shaker at 70 rpm inside a humidified tissue culture incubator at 37 °C and 5% CO2 for 30 minutes.
7. Use a 1 mL pipette to gently dissociate and break up minced pieces.
8. Return the plate to the orbital shaker using the same conditions as Step 6 for 10 more minutes.
9. During this time, add 5 mL of Earle’s medium plus 3 mL of reconstituted Inhibitor solution to a 15 mL conical tube (prepare 1 tube per organoid).
10. Using a 10 mL pipette, gently pipette the minced pieces up-and-down 10 times.
11. Transfer the entire volume to an empty 15 mL conical tube and wait for the debris to settle (1-3 minutes).
12. Transfer the cell suspension (avoiding debris) to the tube prepared in Step 9.
13. Invert the tube a few times to mix.
14. Centrifuge the cells at 300g for 7 minutes.
15. Aspirate the supernatant, and gently resuspend the cells in 500-1,000 μL of 1x PBS.
16. Filter the resuspended cells.
17. Dilute and count the cells.
18. Resuspend the cells at 1,000 cells/μL.
19. Add BSA to a final concentration of 0.04%.
The core circadian clock mechanism is composed of 2 interlocked transcriptional negative feedback loops14. In the primary loop, transcriptional activators, BMAL1 (ARNTL) and CLOCK (or its ortholog NPAS2), form a DNA-binding heterodimer and drive expression of the PER1-3 and CRY1/2 genes, which ultimately repress BMAL1-CLOCK activity in a feedback manner. This loop also drives rhythmic expression of the nuclear hormone receptors, NR1D1 (Rev-erbα) and NR1D2 (Rev-erbβ), which in turn rhythmically repress the expression of BMAL1 and CLOCK as the second loop15.
Formaldehyde (CH20) can crosslink of biomolecules (a well-known reaction called crosslinking reaction) between protein-protein or protein-DNA. The former of which is essential for mass-sepcturm to test protein interaction (by matching the measured mass to a theoretical total mass of the two peptides plus the mass of the cross-linker). The latter of which is a almost mandatory step for CHIP, for the reason that FA can enhance the linkage between protein and DNA at the interaction site, which could be otherwise missed or destructed during isolation process.
Above is the chemical interaction between FA and molecules.
Antigen retrival
Although fixation is essential for the preservation of tissue morphology, this process can also have a negative impact on IHC/ICC detection. Masking of the epitope can be caused by cross-linking of amino acids within the epitope. Antigen retrieval refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored.
When discussing antigen retrieval methods, techniques generally fall into two main categories, protease-induced epitope retrieval (PIER) and heat-induced epitope retrieval (HIER). Once optimized, the effects of antigen retrieval can be pronounced.
Protease-induced Epitope Retrieval (PIER)
In the PIER method, enzymes including Proteinase K, Trypsin, and Pepsin have been used successfully to restore the binding of an antibody to its epitope. The mechanism of action is thought to be the cleavage of peptides that may be masking the epitope. The disadvantages of PIER are the low success rate for restoring immunoreactivity and the potential for destroying both tissue morphology and the antigen of interest.
Heat-induced Epitope Retrieval (HIER)
HIER is believed to reverse some cross-links and allows for restoration of secondary or tertiary structure of the epitope. In general, HIER has a much higher success rate than PIER. For all HIER methods, slides must be cooled before commencing IHC/ICC incubations.
In addition, the technique is often too harsh for cryostat tissue sections and alcohol-fixed tissue. The possibility of artifactual staining should always be considered when using any antigen retrieval methodology. The use of controls to demonstrate specific antibody binding should be included since staining is influenced by multiple variables in any given experiment.
The figure above shows that the PH (either acidic, neutral or basic) can majorly influence the outcome of HIER. Choose the correct one for your own samples.
Supplementary - the difference between formaldehyde / formalin and PFA (paraformaldehyde)
Formaldehyde is CH2O, the simplest aldehyde.
Formalin is the name for saturated (37%) formaldehyde solution. Thus, a protocol calling for 10% formalin is roughly equivalent to 4% formaldehyde. Beware though, that some solutions have methanol in them to stop polymerization but this could have a negative effect on your sample.
Paraformaldehyde (PFA) is actually polymerized formaldehyde. "Pure", methanol-free formaldehyde can be made by heating the solid PFA. This might be called paraformaldehyde, but it actually isn't because it’s not the polymer form. You can buy EM grade formaldehyde or you can make your own . . .
The emerging drug inducible CRISPR/Cas9 systems can be arbitrarily divided into two categories depending on whether at transcription or protein level does chemical control occurs. The first category includes Tet-On/Off system and Cre-dependent system, in which the transcription of Cas9 or sgRNA are subject to chemical control. The second category includes chemically induced proximity (CIP) systems, intein splicing system, 4-Hydroxytamoxifen (4-OHT)-Estrogen Receptor (ER) based nuclear localization systems, allosterically regulated Cas9 (arC9) system, and destabilizing domain (DD) mediated protein degradation systems. Finally, we will discuss the advantages and limitations of each system.
1. Systems of Drug Induction at the Transcription Level
Tet ON/OFF
The Tet system is one of the most wildly used drug inducible transgene expression system. It is based on releasing the Escherichia coli Tet repressor protein (TetR) from its bound tet operator (TetO) sequence upon addition of tetracycline or its derivative dox (doxycycline). Fusion of the VP16 activation domain to TetR results in a transcriptional activator tTA. In the Tet-Off system, dox binds to tTA and triggers its release from the tetO-containing promoter (Ptet), thus switching off its driven transgene. The reverse-tTA (rtTA) is derived from tTA mutant with a reverse activity, which only binds to TetO in the presence of dox. Therefore, in the rtTA based Tet-On system, addition of dox triggers rtTA binding to the Ptet and switches on the target gene. M2rtTA2 is an alternative version of rtTA showing reduced basal expression. The Tet-On 3G protein (TRE3G), which contains 5 amino acid distinctions from M2rtTA2, further increases the sensitively to dox and decreases the background expression when used in combination with optimized tetracycline response element (TRE) repeats, which consists of 7 repeats of the 19 bp TetO sequence (TCCCTATCAGTGATAGAGA) separated by spacers to control Cas9 expression. Further, an optimized M2rtTA2-TRE system was adopted to generate an inducible Cas9 for less background activity in human pluripotent stem cells, named iCRISPR. Replacing Cas9 with dCas9 fused with transcription activation domains such as VP64-P65-Rta (VPR), results in inducible target gene activation.
sgRNA expression can also be regulated by Tet system. sgRNA is driven by the RNA promoter, such as H1. TetR, bound to TetO repeats in between H1 promoter and sgRNA, serves as a block for transcription, thus leading to suppression of sgRNA expression. Dox binding to TetR triggers its release from TetO, thus relieving the suppression of sgRNA expression.
Cre Dependent System
Cre recombinase can efficiently excise DNA fragments flanked by loxP sites in mammalian cells. A loxP-stop-loxP (LSL) cassette can be placed in between a promoter and Cas9 coding sequence. Cre mediated loxP recombination removes stop signal, thus activating Cas9 expression (Fig. 1B). A mouse line containing a LSL-Cas9 cassette knocked in the Rosa26 locus has been generated (ROSA26 is a locus used for constitutive, ubiquitous gene expression in mice. ROSA26 gene itself ubiquitously transcriped over body and over developmental stages but not translated into protein. Thus turn out to be a unessential protein and perfect for knock in experiment. The human version is called AAVS1, AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a well-validated “safe harbor” for hosting DNA transgenes with expected function. It has an open chromatin structure and is transcription-competent). Tissue specific expression of Cas9 can be achieved by crossing with mice harboring Cre recombinase driven by tissue specific promoters. In addition, Cre activity can be regulated by fusing with the ligand binding domain (LBD) of the ER (estrogen recptor, in the absence of estrogen, the ER is sequestered by heat shock protein 90 (Hsp90) in the cytoplasm. Upon ligand binding, it disassociates from Hsp90 and translocates to the nucleus, acting as a transcription factor. The LDB of ER has been widely used as a drug inducible tool) or its mutant (Distinct ER mutants with selective affinity to the synthetic 4-OHT over the endogenous β-estradiol were identified, which are critical for reducing undesired background activity), rendering a tamoxifen (has high affinity to modulate ER activity, and its active metabolite version 4-Hydroxytamoxifen, 4OHT, is more frequently used in practice)-dependent Cre action, or by fusing with the LBD of a mutated progesterone receptor which responds to the synthetic steroid RU486 but not endogenous progesterone (progesterone and estrogen is the two most frequent hormone in female).
2, Systems of Drug Induction at the Posttranslational Level
Inducible system at posttranslational level is theoriotically way faster than the previous one in transcriptional level. Some of the following system can reach the response within several hours.
2.1 CIP system
The most widely used CIP system is rapamycin and its derivatives, expanded to the recent developed systems including S-(+)-abscisic acid (ABA)-inducible ABI-PYL1 and gibberellin (GA)-inducible GAI-GID1, both of which were derived from plant hormone signaling pathways.
Rapamycin, an inhibitor of mammalian target of rapamycin, induces heterodimerization of FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB). To generate an inducible CRISPR/Cas9 system, Bernd Zetche et al. splitted Streptococcus pyogenes Cas9 (SpCas9) into two parts, Cas9(N) and Cas9(C) based on information from its crystal structure, and fused them with FRB and FKBP, respectively. The two split parts can be reconstituted to the full and functional Cas9 in the presence of rapamycin (Fig. 2A, upper panel). However, the Cas9 background activity was high due to auto-assembly in the absence of rapamycin. To damp the background activity, the authors compartmentalized the two split partners by fusing a nuclear export sequence (NES) to Cas9(N)-FRB fragment and two nuclear localization sequences (NLS) to Cas9(C)-FKBP. In addition, Bernd Zetche et al. applied this strategy to dCas9 and fused VP64 activation domain to one split fragment to achieve rapamycin inducible transcription activation.
Similarly, ABA induces heterodimerization of ABI1 and PYL1, which are fused with dCas9 and activation domains (ADs) respectively. GA, on the other hand, introduces dimerizaiton of GAI and GID1. Importantly, ABA- and GA-inducible systems were demonstrated to be reversible and dose responsive.
2.2 Intein splicing
Inteins are similar to self-splicing introns, however, they are transcribed and translated together with their host proteins. After translation, Inteins catalyze auto-excitation of themselves and concomitantly join the flanking peptides without perturbing their biological function.
The 4-OHT-responsive intein were created by insertion of the human LBD of ER into the M. tuberculosis RecA intein, and an evolved clone, 37R3–2 with higher splicing efficiency were identified. To generate an optimally recovered Cas9 upon splicing, 37R3–2 were inserted in Cas9 at specific sites (S219 or C574) after testing 15 candidate sites. The authors further demonstrated that this conditionally active Cas9 induced a comparable genome editing efficiency as wild-type Cas9 at several endogenous genomic loci. However, a major limitation of the intein-based system is that it is irreversible.
2.3 Systems based on 4-OHT driven ER nuclear translocation
As the figure suggested, at the absence of 4OHT, ERT2 is captured by HSP90 at cytoplasm. Since Cas9 is designed to binded to ERT2, Cas9 can not be transported to nuclus and inactivated. When 4OHT is applied, HSP90 is replaced by 4OHT and CAS9-ERT2 complex can be transported into nuclus thus be activated.
2.4 Allosterically regulated Cas9
In sum, a confirmation changed is designed to make sure, when 4OHT is applied, ER-LBD change its confirmation and thus change the confirmation of Cas9 to make it be activated.
2.5 Destabilized domains (DDs) controlled CAS9 systems
DD is an engineered unstable LBD that degraded in the absence of ligand. When DD fusion to a protein of interest, the stability of the fusion protein will be under small molecule control. In the absence of small molecule ligand, DD directs the fusion protein to proteosome dependant degradation. Ligand binding to DD shields the fusion protein from degradation. Several ligand-DD pairs have been generated through mutant library screen, including Shield1-FKBP12 mutant [[75], [76]], trimethoprim (TMP)-Escherichia coli dihydrofolate reductase (DHFR) mutant [[77], [78], [79], [80], [81]] and CMP8/4-OHT-Estrogen receptor destabilized domain (ER50 DD).
These info are extracted from this paper followed.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709367/
The morphology of the growth cone can be easily described by using the hand as an analogy. The filopodia (shows in green labeled actin) are like the "fingers" of the growth cone; they contain bundles of actin filaments (F-actin, from Cristein Holt presentation she emphesize the beta-actin but anyway) that give them shape and support. Filopodia are the dominant structures in growth cones, and they appear as narrow cylindrical extensions which can extend several micrometres beyond the edge of the growth cone (believed to search for further direction like animal tentacle). The filopodia are bound by a membrane which contains receptors, and cell adhesion molecules that are important for axon growth and guidance (e.g., Netrin receptor DCC).
In between filopodia—much like the webbing of the hands—are the "lamellipodia (also shows in green, but the meshwork shape)". These are flat regions of dense actin meshwork (not bundled) instead of bundled F-actin as in filopodia. They often appear adjacent to the leading edge of the growth cone and are positioned between two filopodia, giving them a "veil-like" appearance. In growth cones, new filopodia usually emerge from these inter-filopodial veils.
The growth cone is described in terms of three regions: the peripheral (P) domain (enriched with actin, shows in green), the transitional (T) domain (between peripheral and central), and the central (C) domain (bascially the microtubules).
Growth cones are molecularly specialized, with transcriptomes and proteomes that are distinct from those of their parent cell bodies, i.e., local translation occurs majorly in the axon grow cone (a great number of mRNA are carried into the distal grow cone and accumulated there. Once netrin or other secreted singals been captured by the membrane receptors, the mRNA beneath the receptor are immediately, like with in 5 minutes, translated into protein). A very simple model is, netrin bind to the membrane recptor DCC to activate beta-actin local translation and make a new branch of fliopodia nearby the singal direction, whereas repulsive signal like sema activate cytoskeletal disassembly on near signal side and push the growth cone to the opposite direction.
There are two most common models to study axon guidance: commissures and topographic maps.
1, Commissures are sites where axons cross the midline from one side of the nervous system to the other.
As described above, axonal guidance cues are often categorized as "attractive" or "repulsive.", but this is too simple to model the same axonal growth cone can alter its responses to a given cue based on timing. These issues are exemplified during the development of commissures. The bilateral symmetry of the nervous system means that axons will encounter the same cues on either side of the midline. Before crossing (ipsilaterally), the growth cone must navigate toward and be attracted to the midline. However, after crossing (contralaterally), the same growth cone must become repelled or lose attraction to the midline and reinterpret the environment to locate the correct target tissue.
Two experimental systems have had particularly strong impacts on understanding how midline axon guidance is regulated:
1.1 The ventral nerve cord of Drosophila
The use of powerful genetic tools in Drosophila led to the identification of a key class of axon guidance cues, the Slits, and their receptors, the Robos (short for Roundabout). The ventral nerve cord looks like a ladder. There are two commissures, anterior and posterior, within each segment of the embryo.
The currently accepted model is that Slit, produced by midline cells, repels axons from the midline via Robo receptors. Ipsilaterally projecting (non-crossing) axons always have Robo receptors on their surface, while commissural axons have very little or no Robo on their surface, allowing them to be attracted to the midline by Netrins and, probably, other as-yet unidentified cues. After crossing, however, Robo receptors are strongly upregulated on the axon, which allows Robo-mediated repulsion to overcome attraction to the midline. This dynamic regulation of Robo is at least in part accomplished by a molecule called Comm (short for Commissureless), which prevents Robo from reaching the cell surface and targeting it for destruction (before reaching to the middle line).
1.2 The spinal cord of mice and chickens
In the spinal cord of vertebrates, commissural neurons from the dorsal regions project downward toward the ventral floor plate (neuron reside at the dorsal part, detial seen in the figure below). Ipsilateral axons turn before reaching the floor plate to grow longitudinally, while commissural axons cross the midline and make their longitudinal turn on the contralateral side. Strikingly, Netrins, Slits, and Robos all play similar functional roles in this system as well. One outstanding mystery was the apparent lack of any comm gene in vertebrates. It now seems that at least some of Comm's functions are performed by a modified form of Robo called Robo3 (or Rig1).
2, Topographic maps
Topographic maps are systems in which groups of neurons in one tissue project their axons to another tissue in an organized arrangement such that spatial relationships are maintained.
In the simplest type of mapping model (retina neuron project to optic tectum), we could imagine a gradient of Eph receptor expression level in a field of retina neurons with the anterior cells expressing very low levels and cells in the posterior expressing the highest levels of the receptor. Meanwhile, in the target of the retinal cells (the optic tectum), Ephrin ligands are organized in a similar gradient: high posterior to low anterior. Retinal axons enter the anterior tectum and proceed posteriorly. Because, in general, Eph-bearing axons are repelled by Ephrins, axons will become more and more reluctant to proceed the further they advance toward the posterior tectum. However, the degree to which they are repelled is set by their own particular level of Eph expression, which is set by the position of the neuronal cell body in the retina. Thus, axons from the anterior retina, expressing the lowest level of Ephs, can project to the posterior tectum, even though this is where Ephrins are highly expressed. Posterior retinal cells express high Eph level, and their axons will stop more anteriorly in the tectum.
