Download Flexor Digitorum Sublimis Tendon Development Depends on

Flexor Digitorum Sublimis Tendon Development Depends on Muscle Translocation
1
Huang AH1; Riordan TJ1; Hershko S2; Yumoto N3; Burden SJ3; Zelzer E2;+Schweitzer R1
Shriners Hospital for Children, Portland, OR 97209 USA; 2Weizmann Institute, Rehovot, Israel; 3NYU Medical School, NY, NY 10011 USA
[email protected]
INTRODUCTION In many vertebrate species, including mouse and
human, the flexor muscles that govern motor control of the hand
(autopod) are composed of two distinct functional groups: (1) extrinsic
muscles that reside exclusively within the forearm (zeugopod) and
connect to the long tendons of the autopod and (2) intrinsic muscles that
are localized completely within the autopod with their attaching tendons
[1]. The muscles and tendons of the first type include the flexor
digitorum sublimis (FDS). In the current study, we traced the remarkable
development of the FDS tendons and muscles and showed that the
sublimis muscles first form as differentiated myofibers in the autopod
before moving to their final location in the zeugopod. This finding is
exceptional as there are no reports in the literature of muscles moving as
multi-nucleated, differentiated fibers during development [2]. We also
demonstrated that FDS tendon and muscle development is tightly
coupled; the formation of tendon is dependent on active translocation of
its muscles and muscle translocation in turn, is dependent on tendon.
The development of the sublimis tendon and muscles has clinical
significance as there are several pathologies involving ectopic FDS
muscles within the wrist or attached to tendons [3], giving rise to hand and
wrist pain. Understanding the genetic basis of these developmental
processes may inform clinical treatments.
MATERIALS AND METHODS Existing mouse lines used in this
study were previously described: ScxGFP [4], HB9Cre;Ist2DTA [5], mdg
[6], ScxNull [7]. Whole mount myosin staining, in situ hybridization, and
immunostaining were performed as previously described [7].
RESULTS While almost all of the tendons of the forelimb, including
the FDP tendons, were fully formed at E14.5, the FDS tendons were not
completely formed until E16.5 [1]. At E14.5, only a segment at the
metacarpophalangeal joint was present; the digit and metacarpal
segments were entirely absent. Instead, three unidentified muscles were
observed in place of the metacarpal tendons (Fig 1). At this stage, these
muscles were intrinsic (restricted to the autopod) and did not extend past
the wrist. To determine the identity of these unexpected muscles, we
followed their fate through E14.5-E16.5. Whole mount myosin staining
showed that during these stages, the muscles elongated and translocated
into the zeugopod as differentiated muscles (Fig 2A), with the FDS
tendons forming in their wake (Fig 2B). By E16.5, the FDS tendons
were fully formed, extending from the autopod to the zeugopod, with the
FDS muscles exclusively localized within the zeugopod.
At E14.5, only a small segment of the FDS tendon was present at the
metacarpophalangeal joint [1]. As this structure was formed before the
initiation of muscle translocation, we wondered whether interaction
between these tissues was important for muscle movement. We therefore
examined muscle translocation in the ScxNull mutant, in which the
metacarpal FDS tendon segments do not form [7]. Myosin staining
showed a failure of FDS muscle translocation at E16.5 in the ScxNull
(Fig 3), suggesting that tendon is required for muscle movement.
Fig. 3 (A) Whole mount myosin staining of ScxNull mutants show that in the absence
of the FDS tendons, the FDS muscles (outlined) do not translocate (E16.5).
One interesting structural feature of the FDS muscles at E14.5 was the
presence of motoneurons within each muscle (not shown). Their
intriguing presence prompted us to determine whether muscle activity
was required for translocation. We examined the muscles and tendons of
two mutants lacking muscle contraction: HB9Cre;Ist2DTA and mdg.
While the muscles and tendons of the HB9Cre;Ist2DTA mice appeared
normal (albeit smaller) at E16.5 (Fig 4), the metacarpal tendons of the
mdg mice failed to form; instead the FDS muscles remained localized
within the autopod, fusing and crossing the wrist as a single muscle (Fig 4).
Fig. 3 In situ hybridization for Col1a1 (tendon) followed by myosin immunostaining
in transverse sections at the (left, right) metacarpal and (middle) wrist levels
(E16.5). Yellow and blue triangles indicate tendon and muscle, respectively.
Fig. 1 (A) Schematic showing the FDS tendons and muscles at E14.5 and E16.5.
Transverse sections at the metacarpal level (dashed line) of ScxGFP forelimbs stained
with myosin (MHC) for muscle at (B, B’) E14.5 and (C, C’) E16.5. FDS tendon and
muscle are indicated by yellow and blue triangles, respectively.
Fig. 2 (A) Whole mount myosin staining from E14.5 to E16.5 show FDS muscle
translocation from autopod to zeugopod (arrows). Solid line represents wrist level. (B)
Sagittal sections of ScxGFP forelimbs immunostained with myosin show FDS tendon
(yellow triangle) forming as the muscle (blue triangle) moves.
DISCUSSION The conflicting phenotypes between HB9Cre;Ist2DTA
and mdg were unexpected given that muscle contraction was impaired in
both mutants. The mechanisms by which muscle contraction was disrupted
in the two mouse strains were distinct however. In the HB9Cre;Ist2DTA,
the lack of muscle contraction was caused by loss of motoneurons. In mdg,
the lack of excitation-contraction coupling was due to a loss-of-function
mutation in calcium signaling [8]. Therefore, the sublimis phenotype in
mdg may be due to a failure in calcium signal propagation within these
muscles (independent of muscle contraction). While the presence of
motoneurons within these muscles was suggestive, results from the
HB9Cre;Ist2DTA mouse indicate that muscle contraction is not a
requirement for FDS muscle translocation. The role (if any) of these
neurons therefore remains to be elucidated. In the absence of tendon,
muscle translocation did not occur; however, whether the effect of tendon
on muscle movement was direct or indirect is unclear and will be the focus
of future studies.
ACKNOWLEDGEMENTS This work was supported by NIH grant
R01AR055973 from NIAMS.
REFERENCES [1] Watson+ Dev Dyn 2009 [2] Evans+ Anat Embryol
2006 [3] Elliot J Hand Surg 1999 [4] Pryce+ Dev 2007 [5] Yang+ Neuron
2001 [6] Pai Dev Biol 1964 [7] Murchison+ Dev 2007 [8] Chaudhari J
Biol Chem 1992.
Paper No. 0007 • ORS 2012 Annual Meeting