Tendon morphogenesis in the developing avian limb: plasticity of fetal tendon fibroblasts

Abstract

The differentiation and patterning of tendon fibroblasts is at present a poorly understood aspect of musculoskeletal development in the vertebrate limb. Precursors of tendon fibroblasts originate in the somatic mesoderm adjacent to the early limb bud and gradually become incorporated into the limb mesenchyme as development proceeds. It is unclear whether these progenitor cells are committed to the tendon lineage at this early stage, or whether cells become committed only as they are incorporated into a developing tendon. Following a review of our current knowledge of early tendon development, we present recent results from our preliminary studies looking at tendon cell commitment. Using a lacZ encoding replication-deficient retrovirus, we have mapped regions of the early limb bud that contain presumptive tendon progenitor cells, and later used these sites for implanting labelled fetal tendon fibroblasts into developing limbs. Following implantation, we found that these cells successfully re-incorporated into developing proximal and distal tendons, but also surprisingly contributed to other tissue lineages within the limb. Our results suggest that fetal tendon fibroblasts may not be irreversibly committed to a tendon cell fate in the limb and may be somewhat plastic in their ability to integrate into other tissue lineages during development.

Fig. 1

Schematic diagram summarizing the steps performed in preparing fetal tendon fibroblasts for subsequent injection into early hind limb buds. Tendon was dissected from 8-day chick hind limbs (step 1) and cut into small fragments to aid cell dissociation (step 2). The resultant fetal tendon cells were resuspended and cultured in the CXL viral supernatant for 24 h (step 3). Following addition of fresh growth medium cells were cultured for specific periods of time before being resuspended in fresh growth medium at a cell concentration of 1 × 10⁷ cells mL⁻¹ (step 4) and injected into tendon-forming regions of the hind limb (step 5).

Fig. 2

Typical labelling observed in hind limbs following injection of lacZ encoding retrovirus into presumptive tendon-forming regions of early limb buds. (A) Labelled cells present within tendons along the dorsal surface of the shank (S) (white arrow) and also in the dermis underlying the dorsal surface of the limb in the region of the shank-foot junction (black arrow). (B) Closer inspection shows that labelled cells run in longitudinal rows along the tendon. (C) Histological analysis reveals that labelled cells are present within the common tendon of gastrocnemius (t. GE/GI) at the distal region of the shank. Other tissue lineages were labelled as a result of the viral injections, including skeletal muscles, dermis and other connective tissues. Histological sections illustrated here reveal labelled cells within the muscle of flexor perforatus 4 (FP4) in the shank (D), dermis of the foot (E), periosteum of the developing tibiotarsus bone (TT) within the shank (F) and within connective tissue layers of a branch of the peroneus communicans nerve within the ventral shank (G). (H) Specimen showing a small discrete cluster of individual labelled chondrocytes within the tibiotarsus (TT) of the shank. (I) Labelled cells revealed within feather bud dermis on the dorsal surface of the shank. Scale bar = 100 µm.

Fig. 3

Fetal tendon fibroblasts transfected with the lacZ gene and maintained in culture are able to re-incorporate into developing tendons following injection of these cells into early tendon-forming regions. (A) Labelled cells running in a longitudinal orientation along the proximal portion of the ventral foot (arrow) below the tarsometatarsus (TM). (B) Histological analysis of A reveals that cells are incorporated within tendon on the ventral side of the foot (*). Cells are also revealed within the connective tissue layers of surrounding muscles (arrows). (C) Labelled cells running longitudinally within the common tendon of gastrocnemius (black arrow) of the dorsal shank of another specimen and also cells incorporated into the connective tissues of the gastrocnemius muscle (white arrow). Scale bar = 100 µm.

Fig. 4

Fetal tendon fibroblasts transfected with the lacZ gene and maintained in culture are also found within a number of other tissue lineages following injection into early tendon-forming regions of developing limbs. (A) Embryo showing labelled cells incorporated in the dorsal thigh region. (B) Higher magnification of A reveals the labelled cells are within the dermis (*) and the mesenchyme of the feather buds (arrow). (C) Histological section showing labelled cells within the connective tissue layers of a superficial ventral nerve in the distal limb. (D) Labelled cells within the ridges of the tarsometatarsus (TM) of the foot (arrow). Histological analysis reveals that labelled cells are present within the periosteum (E – arrows) and also within the cartilage (F – arrows). (G) Labelled cells running longitudinally along the anterior aspect of the shank (S) (arrow). Histological analysis of G reveals that labelled cells are present within the fibrocollagenous layers of the tibialis anterior artery (a. TA) (H – arrows) and within the connective tissue layers of the tibialis cranalis (TC) muscle (I – arrow). Scale bar = 100 µm.

Fig. 5

Fetal tendon fibroblasts, transfected with the lacZ gene and cultured for minimal periods of time are found in a number of different tissue types following injection into early tendon-forming regions of developing limbs. (A) Labelled cells incorporated deep within the thigh (T) just below the developing femur (F) within the main neurovascular bundle (black arrow). Labelling also present within the dermis on the dorsal surface of the shank (S) (white arrow). (B) Higher magnification of A reveals that the labelled cells are present along the length of the nerves within this region, the main ischiadicus nerve (n. I), and its branches, the peroneus communicans nerve (n. PC) and the tibialis nerve (n. T). Histological analysis reveals that the cells are incorporated into the connective tissue layers of these nerves. (C) Labelled cells within the tibialis nerve (arrow). (D) Labelled cells within the peroneus communicans nerve (arrow). (E) Labelled cells are also revealed within the fibrocollagenous layers of the iliaca artery (a. I) (arrow), which is also a constituent of this neurovascular bundle. (F) Labelled cells are present along the length of the artery and following its bifurcation in the distal region of the thigh. Scale bar = 100 µm.