Spatial High-resolution Analysis of Gene Expression Levels in Tendons

Summary

This article describes how to perform an optimized in situ protocol for tendons. This method discusses tissue preparation, section permeabilization, probe design, and signal amplification methods.

Abstract

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich tissues, and specifically, tendons were left behind due to their low cell number, low RNA amount per cell, and high matrix content, which made them complicated to analyze. One of the recent and most important single-cell tools is the spatial analysis of gene expression levels in tendons. These RNA spatial tools have specifically high importance in tendons to locate specific cells of new and unknown populations, validate single-cell RNA-seq results, and add histological context to the single-cell RNA-seq data. These new methods will enable the analysis of RNA in cells with exceptional sensitivity and the detection of single-molecule RNA targets at the single-cell level, which will help to molecularly characterize tendons and promote tendon research.

In this method paper, we will focus on the available methods to analyze spatial gene expression levels on histological sections by using novel in situ hybridization assays to detect target RNA within intact cells at single-cell levels. First, we will focus on how to prepare the tendon tissue for the different available assays and how to amplify target-specific signals without background noise but with high sensitivity and high specificity. Then, the paper will describe specific permeabilization methods, the different probe designs, and the signal amplification strategies currently available. These unique methods of analyzing transcription levels of different genes in single-cell resolution will enable the identification and characterization of the tendon tissue cells in young and aged populations of various animal models and human tendon tissues. This method will also help analyze gene expression levels in other matrix-rich tissues such as bones, cartilage, and ligaments.

Introduction

Tendons are connective tissues that enable the transmission of force between muscle and bone¹. Developmentally, axial tenocytes are derived from mesenchymal cells within the sclerotome of the somites²; limb tendons derive from the lateral plate mesoderm; and cranial tendons arise from the cranial neural crest lineage^3,4. Tendon can be characterized by the expression of the scleraxis transcription factor⁵, although several markers also play a key role in tendon development, including tenomodulin, mohawk, and early growth response 1/2^6,7,8,9.

Despite the few known markers of the tendon, in general, a more in-depth characterization remains challenging because the tendon contains cells that span across a gradient of biomechanical properties. From the myotendinous junction, tendon mid-body, and the more calcified enthesis, the tendon cells reside in extracellular matrices that range in tensile properties. Since the tendon must withstand tensile stress imposed by the difference in mechanical strength between soft and hard tissue, the spatial organization of the cells in the tendon is particularly important for its function. However, little is known about these tendon subpopulations.

Many high-resolution spatial transcriptomic tools can be used to begin to elucidate cell subpopulations, including but not limited to, single-cell RNA Seq or in situ hybridization. However, while these spatial profiling assays help uncover RNA expression across tissue after microdissection or sectioning, these methods can be challenging when performed on tendon tissue. Tendons are matrix-rich tissues composed of nearly 86% of collagen by dry mass¹⁰, making it challenging to extract the cells for sequencing. Due to both the complications in isolating cells from the matrix, the hypocellular nature of the tendon¹¹, and the relatively low RNA count, the tendon is a difficult tissue to analyze.

In this paper, we present a method to optimize novel in situ hybridization assays to leverage them for tendons by providing tissue preparation, permeabilization, and probe design methods. Coupled with existing sequencing technologies, this may help researchers spatially characterize tendon subpopulations across developing, adult, or injured tendons with increased assay sensitivity and specificity.

Protocol

All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) and AAALAC guidelines. Experiments were performed under approved protocol #2013N000062 at Massachusetts General Hospital. In this study, C57BL/J6 mice (5 weeks of age and P0) were used. See the Table of Materials for details related to all materials, reagents, and instruments used in this protocol. 1. Sample preparation and fixation Euth…

Representative Results

Figure 1: Poly A RNA expression in adult mouse Achilles tendon using RNAScope. Representative image of successful Poly A labeling in mouse Achilles tendon (left panel) using the commercialized ISH assay. Colocalization with DAPI confirms the specificity of the probe (middle and right panels), allowing control for background noise. Images were t…

Discussion

In this paper, we describe modifications made to leverage existing ISH tools such that they can be used in tendon tissue with a high degree of specificity and sensitivity. Since the tendon is a highly matrix-dense tissue, protocol adjustments must often be made to achieve similar degrees of probe penetration and specificity. These specific permeabilization methods and signal amplification strategies of the tendon tissue are integral to improving the efficacy of the ISH protocols discussed. Without these steps, it is chal…

Materials

1 M triethanolamine buffer
10% Formalin solution
10% Tween-20
20x Saline Sodium Citrate buffer
4% PFA
ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V2	ACD	323100
Acetic Anhydride
Axio Imager Microscope	ZEISS
C57BL/J6 mice		JAX ID: 000664
Coverslips	Fisher	12-541-042
ddH2O
ETDA	Thermofisher	AM9262
EtOH
Glucose	VWR Chemicals BDH	BDH9230-500G
HCR RNA-FISH Bundle	Molecular Instruments Inc.
HybEZ II Hybridization System	ACD
Immedge Barrier Pen	Vector Laboratories	H4000
Leica SPE Confocal Microscope	Leica
Parafilm	Fisher
Phosphate-buffered saline (PBS, 1x)	Invitrogen	AM9625	Dilute 10x PBS in milli-Q water to get 1x solution
Protease IV
Proteinase K	Roche	3115836001
RNAscope H2O2 and Protease Reagents	ACD	PN 322381	Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V3
RNAscope Multiplex Fluorescent Detection Kit	ACD	PN 323110	Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V2
RNAscope Target Retrieval reagents	ACD	322000	Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V4
RNAscope Wash Buffer	ACD	PN 310091	Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V5
RNAscope Probe Diluent	ACD	300041
Slide holder	StatLab	4465A
Staining Dish with Lid	StatLab	LWS20WH
Superfrost Plus Microscope slides	Fisher	1255015	treated, charged slides
Tris-HCl
Xylene	Sigma-Aldrich	534056-4L