Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Summary

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Abstract

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Introduction

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disability, sensory abnormalities, and autistic behaviors. In most cases, FXS is caused by a global loss of fragile X mental retardation protein (FMRP; encoded by Fmr1 gene) starting at early embryonic stages¹. FMRP is an RNA-binding protein that is normally expressed in most neurons and glial cells in the brain, as well as in sensory organs^2,3,4. In mammalian brains, FMRP is likely associated with hundreds of mRNAs that encode proteins that are important f....

Protocol

Eggs and chicken embryos were handled with care and respect in accordance with the animal protocols approved by the Jinan University Animal Care and Use Committee.

1. Egg and plasmid preparation

1. Egg preparation
   1. Obtain fresh fertilized chicken eggs (Gallus gallus) from the South China Agricultural University and store at 16 °C before incubation. For optimal viability, set all eggs for incubation within a week of arrival.
   2. Place eggs horizontally and incubate at 38 °C for 46-48 h until Hamburger and Hamilton (HH) stage 12²⁵ for neural tube transfecti....

Results

By performing in ovo electroporation at different sites and at different developmental stages, we achieved selective FMRP knockdown either in the auditory periphery or in the auditory brainstem.

FMRP knockdown in NM
Small hairpin RNA (shRNA) against the chicken Fmr1 was designed and cloned into the Tet-On vector system as described previously²⁰. The setup for in ovo electroporation is shown in Figure 1.......

Discussion

To determine the cell-autonomous function of FMRP, manipulating its expression in individual cell groups or cell types is necessary. Since one of the major functions of FMRP is to regulate synaptic formation and plasticity, selectively manipulating each synaptic component of a certain circuit is prerequisite for a full understanding of FMRP mechanism in synaptic communication. Using in ovo electroporation of chicken embryos, we demonstrated a method to target FMRP expression in t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Egg incubation
16 °C refrigerator	MAGAT		Used for fertilized egg storage.
Egg incubator	SHANGHAI BOXUN	GZX-9240MBE
Fertilized eggs	Farm of South China Agricultural University		Eggs must be used in one week for optimal viability.
Plasmid preparation
Centrifuge	Sigma	10016
Fast green	Solarbio	G1661	Make 0.1% working solution in distilled water and autoclave.
Plasmid Maxi-prep kit	QIAGEN	12162	Dissolve plasmid DNA in Tris-EDTA (TE) buffer; endotoxin-free preparation kit
Sodium Acetate	Sigma-Aldrich	S2889	Make 7.5M working solution in nuclase-free water.
Electroporation and Doxycycline Administration
Electroporator	BTX	ECM399
1 mL / 5 mL Syringe	GUANGZHOU KANGFULAI
Dissecting microscope	CNOPTEC	SZM-42
Doxcycline	Sigma-Aldrich	D9891	Use fresh aliquots for each dose and store at -20 °C.
Glass capillary	BEIBOBOMEI	RD0910	0.9-1.1 mm*100 mm
Laboratory parafilm	PARAFILM	PM996	transparent film
Pipette puller	CHENGDU INSTRUMENT FACTORY	WD-2	Pulling condition: 500 °C for 15 s
Platinum elctrodes	Home made		0.5 mm diameter, 1.5 mm interval.
Rubber tube	Sigma-Aldrich	A5177
Tissue Dissection and Fixation
Forceps	RWD	F11020-11	Tip size: 0.05*0.01 mm
Other surgery tools	RWD
Paraformaldehyde	Sigma-Aldrich	158127	Freshly made 4% PFA solution in phosphate-buffered saline can be stored in 4 °C for up to 1 week.
SYLGARD 184 Silicone Elastomer Kit	DOW	01673921	For black background plates, food-grade carbon powder is applied.
Sectioning
Cryostat	LEICA	CM1850
Gelatin	Sigma-Aldrich	G9391	From bovine skin.
Sliding microtome	LEICA	SM2010
Immunostaining
Alexa Fluor 488 goat anti-Mouse	Abcam	ab150113	1:500 dilution, RRID: AB_2576208
Alexa Fluor 555 goat anti-rabbit	Abcam	ab150078	1:500 dilution, RRID: AB_2722519
DAPI	Abcam	ab285390	1: 1000 dilution
Fluoromount-G mounting medium	Southern Biotech	Sb-0100-01
FMRP antibody	Y. Wang, Florida State University	#8263	1:1000 dilution, RRID: AB_2861242
Islet-1 antibody	DSHB	39.3F7	1:100 dilution, RRID: AB_1157901
Netwell plate	Corning	3478
Neurofilament antibody	Sigma-Aldrich	N4142	1:1000 dilution, RRID: AB_477272
Parvalbumin antibody	Sigma-Aldrich	P3088	1:10000 dilution, RRID: AB_477329
SNAP25 antibody	Abcam	ab66066	1:1000 dilution, RRID: AB_2192052
Imaging
Adobe photoshop	ADOBE		image editing software
Confocal microscope	LEICA	SP8
Fluorescent stereomicroscope	OLYMPUS	MVX10
Olympus Image-Pro Plus 7.0	OlYMPUS		commercial image processing software package