Cerebroventricular Microinjection (CVMI) into Adult Zebrafish Brain Is an Efficient Misexpression Method for Forebrain Ventricular Cells

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain – in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish

sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons

Sox transcription factors are crucial for vertebrate nervous system development. In zebrafish embryo, sox1 genes are expressed in neural progenitor cells and neurons of ventral spinal cord. Our recent study revealed that the loss of sox1a and sox1b function results in a significant decline of V2 subtype neurons (V2s). Using single-cell RNA sequencing, we analyzed the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four time points during embryonic development, employing the Tg(sox1a:eGFP) line. In addition to previously characterized sox1a-expressing neurons, we discovered the expression of sox1a in late-developing intraspinal serotonergic neurons (ISNs). Developmental trajectory analysis suggests that ISNs arise from lateral floor plate (LFP) progenitor cells. Pharmacological inhibition of the Notch signaling pathway revealed its role in negatively regulating LFP progenitor cell differentiation into ISNs. Our findings highlight the zebrafish LFP as a progenitor domain for ISNs, alongside known Kolmer-Agduhr (KA) and V3 interneurons

RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation.

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer’s disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.post-print10112 K

Morpholino-mediated gene knock-down using CVMI is dose-dependent.

<p>(A) The dose-response analyses were performed on dorsal regions of telencephalic sections. PCNA immunohistochemistry coupled to DAPI nuclear counterstaining on 500 µM control morpholino-injected (B), 500 µM PCNA morpholino-injected (C), 250 µM PCNA morpholino-injected (D), 100 µM PCNA morpholino-injected (E), 50 µM PCNA morpholino-injected (F) brains. (G) Graph depicts the relative amount of PCNA-positive cells in dorsal telencephalon after every dose in control and PCNA morpholino-injected brains. Scale bars 50 µm. N = 3 adult fish for every dose.</p

CVMI of FGF8 increases ventricular cell proliferation.

<p>(A) Pairwise sequence alignment between human FGF8 (NP_149355) and zebrafish Fgf8 (NP_571356) showing 86.4% similarity between two proteins. Red boxes indicate the residues required for ligand-receptor interaction and they are completely conserved between human and zebrafish. Asterisks indicate identical residues; semicolons indicate conservative substitutions. (B) PCNA immunohistochemistry (IHC) on the telencephalon of BSA-injected brains at 1 dpi. (C) DAPI counterstaining on A. (C') High magnification of dorsal telencephalon. (D) PCNA immunohistochemistry (IHC) on the telencephalon of FGF8-injected brains at 1 dpi. (E) DAPI counterstaining on C. (E') High magnification of dorsal telencephalon. (F) Graph depicts the average number of PCNA-positive cells per section. Scale bars 50 µm. N = 3 adult fish for each injection.</p

Morpholino-mediated gene knockdown using CVMI in <i>Tg(her4.1:mCherry)</i> reporter line.

<p>(A) Fluorescence reporter activity of <i>Tg(her4.1:mCherry)</i> transgenic line in the radial glial cells (red arrows along the ventricular surface) of the rostral telencephalon. (A') Higher magnification of the dorsomedial region of A. (B) Fluorescence reporter activity in rostral telencephalon injected with translation-blocking vivo morpholinos for mCherry transgene. (B') Higher magnification of the dorsomedial region of B, indicating the significant reduction of reporter activity. (C) Fluorescence reporter activity of <i>Tg(her4.1:mCherry)</i> transgenic line in the radial glial cells (red arrows along the ventricular surface) of the caudal telencephalon. (C') Higher magnification of the medial region of C. (D) Fluorescence reporter activity in caudal telencephalon injected with translation-blocking vivo morpholinos for mCherry transgene. (D') Higher magnification of the medial region of D, indicating the significant reduction of reporter activity. (E) Graph depicts the average mCherry fluorescence intensity in mCherry antisense morpholino-injected brains (green line) over a time course relative to control morpholino-injected brains (red line). Scale bars 50 µm. v: ventricle, tel: telencephalon, hpi: hours post injection, dpi: days post injection. N = 6 adult fish for each time point.</p

Knocking-down PCNA as an endogenous gene using CVMI.

<p>(A) PCNA immunohistochemistry (IHC) on rostral telencephalon of control morpholino-injected (ctrl-MO) brains. (B) DAPI counterstaining on A. (C) High magnification image of medial ventricular region of B. (D) PCNA IHC on rostral telencephalon of PCNA morpholino-injected (PCNA-MO) brains. (E) DAPI counterstaining on D. (F) High magnification image of medial ventricular region of E. (G) PCNA IHC on rostral telencephalon of ctrl-MO brains. (H) DAPI counterstaining on G. (I) High magnification image of medial ventricular region of H. (J) PCNA IHC on telencephalon of PCNA-MO brains on a more caudal level. (K) DAPI counterstaining on J. (L) High magnification image of medial ventricular region of K. (M) PCNA IHC on rostral optic tectum of ctrl-MO brains. (N) DAPI counterstaining on M. (O) High magnification image of dorsal region of N. (P) PCNA IHC on rostral optic tectum of PCNA-MO brains. (Q) DAPI counterstaining on P. (R) High magnification image of dorsal region of Q. (S) Graph depicts the average number of PCNA-positive cells in PCNA antisense morpholino-injected brains (green line) over a time course relative to control morpholino-injected brains (red line). Scale bars 50 µm. hpi: hours post injection, dpi: days post injection. N = 3 adult fish for each time point.</p

Knocking-down PCNA reduces neurogenesis as a functional consequence.

<p>(A) Schematic representation of the neurogenesis assay after PCNA knock-down. Following morpholino injection (control and PCNA-antisense), BrdU is given between 12 and 36 hours. Brains were analyzed at 7 days post injection (dpi) for BrdU (marker for proliferated cells), HuC (neuronal marker) and DAPI as a nuclear counterstain. (B) BrdU immunohistochemistry on the dorsal region of telencephalon from control morpholino-injected brain. (C) Co-staining for HuC and BrdU in control morpholino-injected dorsal telencephalon. (D) BrdU immunohistochemistry on the dorsal region of telencephalon from PCNA morpholino-injected brain. (E) Co-staining for HuC and BrdU in PCNA morpholino-injected dorsal telencephalon. (F) Graph depicts the average number of newborn neurons (HuC-BrdU double-positive cells) in control and PCNA morpholino-injected brains. Scale bars 50 µm. N = 4 adult fish.</p