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Persistent myogenic capacity of the dermomyotome dorsomedial lip and restriction of myogenic competence

Department of Anatomy and Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA 94143

View larger version (56K): [in a new window]   Fig. 1. Transplant scheme and DML evaluation. (A) Schematic representation of the tissues used to replace an ablated DML. (B) Confocal image stack of an E2.5 quail embryo immunolabelled with anti-desmin (red) and qcpn (green) antibodies. (C) Schematic of B showing the DML as a cluster of quail epithelial cells at the dorsomedial limit of the somite, which are also lightly labelled by the desmin antibody. (D) Transverse section of an E3 chick somite that received a quail DML transplant 24 hours prior to harvest. Desmin immunoreactivity marks the myotome and some cells within the DML and lateral dermomyotome. The bar indicates the extent of donor-derived myotome as determined by the presence of quail nuclei in an adjacent serial section (shown in Fig. 4). (E) Schematic of D showing the position of the myotome, dermomyotome and DML with areas comprising quail nuclei shaded. Abbreviations: CPM, cranial paraxial mesoderm; dm, dermomyotome; DML, dorsomedial lip of the dermomyotome; LPM, lateral plate mesoderm; M, medial; mt, myotome; NT, neural tube; PSM, presomitic mesoderm; R, rostral; VLL, ventrolateral lip of the dermomyotome. The rostral direction is towards the right in B,C and in subsequent whole-mount images.

View larger version (148K): [in a new window]   Fig. 2. Transplant of non-dermomyotome tissues to a DML position. (A-D) Confocal z-series images immunolabelled with anti-desmin and qcpn antibodies to label the myotome (red) and quail (green), respectively. (A,B) Transplant of LPM from the level of the rostral PSM (Table 1, 1.07). (A) The expansion of the transplanted quail tissue is comparable with that of neighbouring myotomes. The quail tissue remained within the rostral and caudal boundaries of the host somite (outlined with a broken line) and expanded dorsomedially, but no epithelial DML structure was apparent. (B) Desmin labelling shows truncation of the myotome in the operated somite from the level of the DML ablation (arrowheads). Note robust growth of the neighbouring myotomes. (C,D) CPM transplant (Table 1, 2.04). (C) The quail tissue expanded dorsomedially in concert with the neighbouring somites and has largely remained within the somite boundaries. Arrows indicate encroachment of quail nuclei in cells that have crossed somitic boundaries. (D) The myotome of the operated somite is truncated in comparison to neighbours corresponding to the site of the DML ablation (arrowheads).

View larger version (89K): [in a new window]   Fig. 3. Transplant of somitic and presomitic tissue to replace the DML. (A-F) Confocal z-series of transplanted embryos immunolabelled for desmin (red) and quail (green). (A,B) Transplant of the dermomyotome sheet (Table 1, 4.01). (A) Composite through the myotome region shows dorsomedial expansion of the transplanted tissue and centrally aligned nuclei in quail derived myotome (arrow). The arrowheads indicate the level of the DML ablation. An epithelial DML has reformed at the correct position and is composed of quail cells. (B) The desmin immunoreactivity of A showing the medially located, donor-derived, myotome has grown to a similar extent as its neighbours. (C,D) Transplant of the VLL (Table 1, 5.02). (C) A single optical section in the plane of the myotome of the operated somite shows quail nuclei associated with donor derived myotome. The transplanted tissue has remained within the segment boundaries, a few cells at the medial limit have extended past the segment boundaries. In this example, quail myocyte nuclei (arrows) are not centrally localised but spread along the axial myotome length. (D) The desmin channel of C. The growth of the donor myotome is consistent with that of neighbours (bar) and an epithelial DML structure is evident in the correct location. (E,F) Transplant of caudal PSM (Table 1, 6.06). (E) The quail tissue has expanded in concert with the neighbouring somites and formed a DML. (F) The desmin channel of E highlights the DML structure and shows myofibres situated just lateral to the DML. There is a gap (bar) between the host and the older, laterally situated, donor myofibres (arrows). Donor fibres correspond to the axial length of the transplanted fragment.

View larger version (104K): [in a new window]   Fig. 4. BrdU incorporation into the epaxial myotome (2 hours post administration). (A-C) Transverse cryosections through an embryo that received a DML transplant from ssXIII of a 25 somite quail embryo to ssXI at 26 somites and was harvested the following day, 2 hours after BrdU administration. (A) Section through the axial mid region of the recombinant somite at the plane of the myocyte nuclei reacted to label quail cells. Two layers of quail nuclei can be seen; the medialmost (lower) layer corresponds to the myotome (mt) and the more superficial layer the dermomyotome (dm). (B) The same section as A showing BrdU-labelled cells. (C) A composite of A and B, showing BrdU co-localised with quail immunoreactivity in the dermomyotome and predominantly non-BrdU-labelled quail nuclei in the myotome layer. A few BrdU-labelled quail cells are present in the myotome layer (arrows). (D) Schematic representation of the section shown in A-C. The extent of non-BrdU-labelled donor-derived nuclei in the myotome layer are indicated (bracket, A). Comparable data were obtained in three out of three treated embryos. DML, dorsomedial lip; NT; neural tube.

View larger version (129K): [in a new window]   Fig. 5. BrdU incorporation into the epaxial myotome (4 hour post administration). (A,B) A section from a chick embryo that received a ssXV DML from a 30 somite quail embryo transplanted at ssXIV (23 somites) after overnight re-incubation and harvesting 4 hours after BrdU administration. The section is through the plane of the myocyte nuclei, a single layer at the inner extent of the quail nuclei. The slightly oblique plane of section results in the myotome layer being depicted truly as a single cell layer while the dermomyotome appears stratified. (A) Quail derived dermomyotome and myotome labelled with qcpn antibody. (B) Same section as A showing BrdU-labelled nuclei. Broken line outlines the neural tube. (C) Composite image of A,B. BrdU-labelled nuclei (red) and quail nuclei (green). The asterisk marks the medial part of the myotome comprising dual labelled nuclei and the arrow marks the medial boundary of quail myonuclei that have not incorporated BrdU. One or two quail nuclei are present in the dermis overlying the lateral donor-derived dermomyotome. (D) Schematic interpretation of the section in A-C highlighting the medial position of BrdU-labelled quail myocyte nuclei (bracket, B) and the non-BrdU labelled quail myotome nuclei located laterally (bracket, A). The same distribution of BrdU and/or quail nuclei was seen in three out of three embryos. DML, dorsomedial lip; NT; neural tube.

View larger version (80K): [in a new window]   Fig. 6. Serial DML transplant. (A) Schematic outline of the serial lip experimental procedure. The example shown is embryo s-03 (Table 2). (B-D) Composite confocal images of whole-mount embryos labelled with anti-desmin antibody (red) and anti-quail antibody (green). (B) Primary host after reincubation. The operated segment is highlighted by the broken line that indicates the position of the quail DML prior to harvest for transplantation into the second host. DML harvest caused the apparent disruption to somite morphology. The operated somite developed normally with a clear boundary between host and donor myotome (asterisks); centrally aligned quail nuclei are evident in the medial myotome. Some quail cells are apparent in adjacent somites. This is an artefact resulting from the harvest of the quail DML. (C) The secondary host after overnight re-incubation following DML transplant. The operated somite myotome grew medially to a similar extent as its neighbours and quail cells are evident at the DML and within the medial myotome. The quail-derived myocytes are not running completely parallel to the body axis, although they still remain within the somitic boundaries. (D) A higher magnification of the secondary host somite showing individual quail nuclei in myocytes (arrows). (E) A composite image of fluorescent dye labelling of transplanted tissue. Dye-labelled fibres in the myotome of the secondary host resulting from donor DML dye injections of DiI and DiO prior to transplant in the primary and secondary hosts, respectively. Some DiI-labelled fibres (arrowheads) are evident medial to DiO-labelled fibres (arrows) with some overlap.

View larger version (44K): [in a new window]   Fig. 7. Continuing development of the epaxial musculature. (A) A composite of four consecutive z-scans through the plane of the myotome of an E4 embryo transplanted at ssXV at 20 somites with the ssXIV DML of a 21-somite quail embryo. Extensive dorsomedial expansion of quail myotome is evident (bar) and a dense cluster of quail nuclei are located at the DML position. The myotome has retained its uni-segmental organisation and central myocyte nuclei alignment. Some quail dermis cells (arrowheads) are present over the neighbouring somite and at the caudal edge of the transplanted somite. (B) Dorsal view of desmin immunoreactivity composite of a z-series through an embryo that received a DML transplant of ssX at 20 somites with a ssXV DML from a 25 somite quail and was re-incubated for 5 days. Broken bars mark, and join, corresponding neighbouring segments. The unbroken bar joins the transplanted segment (asterisk) to its contralateral counterpart. Medially, the musculature is unisegmental while the overlying more laterally positioned muscle extends over several segments. Arrowheads indicate more intensive immunolabelling at the medial limit of the musculature. (C) A higher magnification view of the medial limit of the transplanted segment. Quail nuclei are evident within myofibres (arrows). bv, blood vessels.

View larger version (123K): [in a new window]   Fig. 8. Late ablation of the DML. (A) Confocal image of a desmin-labelled embryo in which a DML at the wing-level was ablated at E4 and the embryo reincubated for 3 days. Bars extend between corresponding segments on opposing sides of the embryo, the unbroken bar marks the DML ablated segment (asterisk) and broken bars run between non-operated neighbours. The medial unisegmental muscle is absent at the level of the DML ablation when compared with the unoperated control side. (B) A higher magnification of the operated area from the embryo shown in A. (C) An embryo in which a wing-level DML was exposed and treated with enzyme but not ablated at E4, and the embryo reincubated for 3 days. Corresponding segments are marked as above. The medial segmented muscle is present, some disruption is evident in the more superficial, lateral multisegmental muscle at the operated site. The mock-operated control embryo shown in C is slightly older than that shown in A,B. (D) High-magnification view of the medial limit of one segment of the embryo shown in C. A mononucleate myocyte (an arrow marks the nucleus and arrowheads mark its rostral and caudal ends) is present at the medial extremity and multinucleate myotubes are apparent laterally.