Download identification of tendon and ligament specific genes

IDENTIFICATION OF TENDON AND LIGAMENT SPECIFIC GENES
+*Archambault, JM, *Jelinsky SA, *Saraf K, *Brown EL, *Seeherman H, *Wozney J
+*Wyeth Discovery Research, Cambridge, MA
[email protected]
INTRODUCTION
Mesenchymal stems cells have a demonstrated ability to differentiate
into muscle, bone and fat. These cells should also be able to differentiate
into tendon or ligament fibroblasts, but the conditions necessary for this
differentiation are not yet known. Also, there are few definitive markers
available to verify that the stem cells have differentiated into tendon or
ligament cells. The objective of this study was to identify genes that are
specific to tendons and ligaments when compared to all the other
musculoskeletal tissues, and that could be used as phenotype markers.
METHODS
Tissues were harvested from 10 male, 18 week-old Sprague-Dawley
rats using a protocol approved by our institution’s IACUC. The
following musculoskeletal tissues were harvested from each animal:
Tendon (Achilles and patellar), Paratenon (from the Achilles tendon),
Ligament (MCL and LCL), Meniscus (lateral and medial), Muscle
(quadriceps), Fat (inguinal area), Cartilage (femoral groove and tibial
plateau), Bone (Femur), Bone Marrow (flushed from the femur).
The tissues were snap frozen in liquid nitrogen, except for the bone
marrow, which was immediately mixed with lysis buffer then frozen.
The tissues were freeze-fractured then extracted using TRIzol reagent
(Invitrogen). RNA was isolated from the aqueous phase of the extract
using an RNeasy kit (QIAGEN). The bone marrow RNA was extracted
directly with the RNeasy kit. RNA concentrations were determined with
a fluorescent dye and RNA standard (PicoGreen kit, Molecular Probes).
Transcriptional profiling to monitor the expression level of more than
30,000 transcripts was performed with an Affymetrix rat genome 230
2.0 array. All array images were visually inspected for defects and
quality. Arrays with high background, low signal intensity, or major
defects were eliminated from further analysis. Signal values were
determined using Gene Chip Operating System 1.0 (GCOS, Affymetrix).
For each array, all probe sets were normalized to a mean signal intensity
value of 100. The default GCOS statistical values were used for all
analysis. A gene was considered detectable if the mean expression in
any tissue was greater than 100 signal units and the percentage of
samples with a Present (P) call as determined by GCOS default settings
was greater than or equal to 66%. Normalized signal values were
transformed to the log base10 and an ANOVA analysis was performed.
A gene was considered to be differentially expressed if the p-value based
on an ANOVA test was <0.0005 and the difference between tissues was
at least four-fold. The qualifiers that met these conditions were used for
further analysis. Data for each qualifier were normalized to a mean of
zero and a standard deviation of 1 (z-score normalization). Hierarchical
clustering was performed with the GeneCluster software and visualized
using Treeview application.
Confirmation of the selectivity of the expression of candidate genes
was done using real-time quantitative RT-PCR with primer/probe sets
from Applied Biosystems. Musculoskeletal tissues from two rats that
were not part of the transcriptional profiling analysis were assessed,
along with testis, heart, lung, liver and bladder tissues. Expression of the
genes in question was normalized to GAPDH expression.
RESULTS
The yield on average was 0.20 ug RNA per mg of wet tissue mass,
with bone having the lowest yield (0.05) and fat the highest (0.33). For
each tissue type, 4-10 samples were part of the final analysis.
Approximately 5900 genes were identified with an expression level that
differed at least four-fold between tissues.
Genes were labeled as either tissue selective or tissue specific. A gene
was considered tissue selective if the corresponding qualifier was
detectable in the tissue of interest and the mean expression of the
qualifier in the tissue of interest was two-fold higher (p <0.05) than the
mean expression in any of eight other tissues. A tissue selective gene
was also called tissue specific if it was not detected in any of the eight
other tissues. Thus tissue specific genes are a subset of tissue selective
genes that are unique to that tissue.
Very few tissue specific genes were identified in tendon, paratenon,
ligament, meniscus, cartilage, bone or bone marrow (n<10). However, a
large number of specific genes were identified in muscle (n=102) and fat
(n=77). Only 4 genes showed tissue selective expression in patellar
tendon, 3 genes in ligament and 6 genes in paratenon. In contrast, a large
number of tissue selective genes were identified in muscle (n=632), fat
(n=348), bone marrow (n=325), cartilage (n=157) and bone (n=32).
Cartilage selective transcripts included aggrecan, type II collagen, sox-9
and matrilin. Bone selective transcripts included WISP1, protocadherin
7, CD52 antigen and dentin matrix protein 1.
Hierarchical clustering revealed the relationship between individual
tissues. Ligament and tendon tissue were found to have highly similar
expression profiles, with paratenon tissue being closely related (Figure
1). This close relationship would explain why few specific or selective
genes were found in these tissues. Based on this observation, tendon,
ligament and paratenon tissues as a group where compared to the
remaining 6 tissues. Thirty-nine genes were specifically expressed in
tendon or ligament, and 298 genes were selectively expressed.
Examples of known genes are tenomodulin, thrombospondin 4,
follistatin, neuropeptide Y receptor, laminin beta 2, integrin beta 4, IGF
binding protein 6, and mesenchyme homeobox 2. RT-PCR confirmed
that the expression of tenomodulin was limited to tendons and ligaments
(Figure 2).
Men
BM Bone Cart
Tendon
Lig
Para
Fat
Musc
Figure 1: Dendogram of differentially expressed genes within all
musculoskeletal tissues. Hierarchical clustering was used to group the 61
samples from the nine distinct tissues types.
Figure 2: Confirmation of tendon and ligament specific tenomodulin
expression by RT-PCR.
DISCUSSION
This analysis of the expression profile of all the musculoskeletal
tissues in the rat sought to identify genes that were specific to tendons
and ligaments. A very small number of genes such as tenomodulin [1]
were expressed in tendons and ligaments, but not in the other
musculoskeletal tissues. The widely used tendon-specific gene scleraxis
[2] was not present on the rat array, so its specificity could not be
confirmed. The genes identified in this experiment could be used to
establish that treated mesenchymal stem cells have differentiated into
tendon or ligament cells. The analysis will be extended to additional
species such as mouse and human to verify the tendon and ligament
specific expression of the genes described here.
REFERENCES
[1] Shukunami et al: BBRC, 280: 1323-7, 2001
[2] Schweitzer et al: Development, 128: 3855-66, 2001
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 1103