Department of Anatomy and Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA 94143
*Author for correspondence (e-mail: ordahl{at}itsa.ucsf.edu)
Accepted 22 May 2002
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SUMMARY |
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Key words: Myotome, Muscle patterning, Chick/quail, Experimental embryology, Somite, Dermatome, Competence, Confocal microscopy, Epaxial, Stem cell, Progenitor cell, Asymmetry
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INTRODUCTION |
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One unresolved issue is the extent to which intrinsic or extrinsic influences govern the initiation and maintenance of early muscle development. The results of tissue recombination experiments in vitro and in vivo have led to proposals that myogenic fate is initially induced in naïve somite cells and later maintained by extrinsic signals emanating from tissues surrounding the somite (reviewed by Borycki and Emerson, 2000; Hirsinger et al., 2000
). Prospective candidates for the initial inductive signals include sonic hedgehog (Shh) from the neural tube/notochord complex (Borycki et al., 1998
; Borycki et al., 1999
) and Wnt signals from the dorsal neural tube and adjacent dorsal ectoderm (Stern and Hauschka, 1995
; Tajbakhsh et al., 1998
; Wagner et al., 2000
). Subsequent maintenance of myogenic patterning and differentiation has been attributed to the BMP4 antagonist noggin (Hirsinger et al., 1997
; Reshef et al., 1998
) and members of the TGF, bFGF and IGF families (Stern et al., 1997
; Pirskanen et al., 2000
).
Alternatively, exogenous sonic hedgehog has been proposed to serve as a survival/proliferation factor for early myotomal progenitor cells rather than as an inducer per se (Teillet et al., 1998; Cann et al., 1999
; Marcelle et al., 1999
). This is consistent with the finding that myogenically competent cells (cells that are capable of generating differentiated muscle in an ectopic, permissive environment in vitro or in vivo) are present within the primitive streak and node as well as in the pre-gastrula blastoderm (Krenn et al., 1988
; George-Weinstein et al., 1994
; Stern and Hauschka, 1995
). The presence of such cells is further supported by the demonstration that messenger RNAs encoding myogenic determination factors (MDFs), such as MyoD, can be detected in the blastoderm, primitive streak and node prior to the appearance of paraxial mesoderm (Gerhart et al., 2000
; Kiefer and Hauschka, 2001
). Finally, determined myogenic progenitor cells (cells that are capable of creating muscle tissue in an antagonistic signalling environment in vivo) are present in the pre-segmented mesoderm and newly formed somites prior to the onset of overt myogenic differentiation (Williams and Ordahl, 1997
). Thus, myogenically competent cells appear prior to and maintain their state of determination in the absence of signals that putatively induce them.
Another issue concerns the location and timing of asymmetric cell division in the formation of the myotome. Clonal analysis of myotome development in mouse embryos shows that myotomal myocytes in multiple adjacent segments along the body axis arise from mitotic division of individual ancestral cells in the primitive node and streak or even earlier blastoderm (Nicolas et al., 1996; Eloy-Trinquet et al., 2000
). The conserved lateral-to-medial position of such myocytes within multiple segments has led these workers to hypothesise that myotome progenitor cells are mediolateral pre-patterned by the time the somite buds off the rostral end of the pre-somitic mesoderm (Eloy-Trinquet and Nicolas, 2002
). In this latter hypothesis, mitotic activity of myogenic progenitor cells after somite formation would serve to expand the existing pre-pattern but repeated asymmetric division of such cells could not generate, de novo, the medio-lateral position of cells within each myotome segment.
This conclusion is inconsistent with recent results from our laboratory indicating that both primary myotome and dermomyotome cells emerge from the dermomyotome dorsomedial lip (DML) in a time- and position-dependent fashion (Denetclaw et al., 1997; Denetclaw et al., 2001
). Furthermore, surgical transplantation experiments showed that replacement of quail DML with that of chick (or vice versa) resulted in medially located myotome and dermomyotome cells of exclusively donor origin and sharply demarcated from laterally located host cells (Ordahl et al., 2001
). These results indicate that mediolateral position within the myotome (and dermomyotome) is a time- and position-dependent function of DML activity, rather than pre-patterned position that is already established at the time of somite formation.
That such a small portion of tissue, and therefore a limited number of cells, can generate both myotome and dermomyotome leads to the hypothesis that the DML contains a pool of self-renewing stem-like cells, herein referred to as progenitor cells. Division of these progenitor cells produces daughters that ultimately emerge from the DML as myotome and/or dermomyotome cells. According to this hypothesis, asymmetric cell division and/or timing of differentiation within the DML, rather than pre-patterned cell location in the newly formed somite, is responsible for the lateral-to-medial position of myotome cells. In the present report, we use experimental embryological methods to test these hypotheses. The results support the conclusion that asymmetry within the DML is responsible for the growth and patterning of the primary myotome and indicate further that DML activity is required throughout the embryonic period for the complete development of each myotomal segment. In addition, the capacity to form primary myotome is restricted to the DML very early in development.
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MATERIALS AND METHODS |
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Immunohistochemistry and microscopy
Embryo whole-mount specimens and tissue sections were processed for immunohistochemistry as described previously (Ordahl et al., 2001). Primary antibodies used were mouse anti-qcpn, 1:50 (Developmental Studies Hybridoma Bank), rabbit anti-desmin, 1:500 (Sigma) and mouse anti-BrdU, 1:500 (Caltag). Whole-mount embryos were mounted in PBS in well slides and sections mounted in glycergel (Sigma). Confocal microscopy was carried out using a Nikon E600FN microscope fitted with a PCM 2000 laser-scanning unit (Nikon) and C-Imaging Simple 32 software (Compix). Optical sections were collected at between 1 and 5 µm intervals. Individual and partial z-series were examined and compiled using NIH image 1.61ppc and Adobe Photoshop 5.5.
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RESULTS |
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Sclerotome has been previously shown to have myogenic capacity at later embryonic stages (Dockter and Ordahl, 1998). All nine cases in which DMLs were replaced with sclerotome fragments resulted in truncated myotomes and no donor derived myocytes (Table 1, 3.01-3.09). Additionally, donor tissue expanded within the host somite boundaries comparable with growth of neighbouring somites (not shown).
Paraxial mesoderm transplants to the DML position
Transplant and tracing experiments have shown dermomyotome tissue lateral to the DML has myogenic potential (Williams and Ordahl, 2000) and contributes fibres to the myotome (Denetclaw et al., 1997
; Kahane et al., 1998a
; Huang and Christ, 2000
; Kahane et al., 2001
). The central region of quail dermomyotome sheet was transplanted to replace the DML in a chick host (Fig. 3A,B). In each case (Table 1, 4.01-4.05), the donor tissue was able to give rise to myotomal muscle that occupied the medial-most myotome position, was axially aligned and restricted to the host somite segment (Fig. 3B). Additionally, myocyte nuclei were arranged along the centres of the myotome fibres (Fig. 3A, arrow). Finally, the cut edge of the transplanted dermomyotome tissue positioned adjacent to the neural tube had reformed an epithelial DML. Thus, the central dermomyotome sheet is able to functionally replace an ablated DML.
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The competence of presomitic mesoderm (PSM) to respond to the DML environment was similarly tested using somite sized pieces of rostral, mid or caudal regions of PSM [or classified according to Pourquié and Tam (Pourquié and Tam, 2001)], transplanted into a DML ablated somite. In all rostral and mid-level PSM transplants (Table 1, 6.01-6.05) donor-derived myotome was produced after overnight incubation, transplanted tissue remained within the host somite boundaries and formed a DML-like structure.
Caudal PSM was used to replace the DML in two cases (Fig. 3E,F; Table 1, 6.06, 6.07) and in both cases grew within the somite boundaries of the host. One transplant formed a structured DML and produced myocytes, although a gap was evident between the host myotome and the donor-derived myocytes (Fig. 3F, arrows). The second caudal PSM transplant neither formed a DML-like structure nor produced myocytes (Table 1 and data not shown).
Time and position dependent entry of newly replicated DML cells into the myotome
DML transplants were carried out on E2 embryos as described previously and exposed to a single pulse of BrdU either 2 or 4 hours before harvest after overnight reincubation. Cryosections of 2 hour BrdU-treated DML transplant embryos (three out of three cases) reacted with anti-quail antibody revealed a double layer of quail nuclei within the dermomyotome and underlying myotome (Fig. 4A,D). High-density BrdU labelling was evident in the dermomyotome (Fig. 4B,C) but the quail-derived myotome was almost completely devoid of nuclei that had incorporated BrdU. A few quail nuclei in the myotome layer were labelled with BrdU [Fig. 4C (arrows), Fig. 4D (black dots)]. The nuclei at the extreme medial edge of the dermomyotome (see Fig. 4D) had not incorporated BrdU (Fig. 4C). Sections from other levels of this somite and other somites showed cells in comparable locations that had incorporated BrdU.
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After a second overnight re-incubation, in the secondary host, the transplanted DML had again expanded dorsomedially in concert with its neighbours (Fig. 6C). The dorsomedialward growth of the myotome is indicated by the extent of quail nuclei in the operated somite. The DML and medially located myotome comprised quail cells (Fig. 6C,D), as did the corresponding overlying dermomyotome (data not shown). The presence of quail nuclei within myocytes was observed within individual optical sections. In contrast to the primary host, donor-derived myocyte nuclei in the second host were not arranged centrally within the somite. Therefore, throughout the time of the serial transplant the cells of the DML retained their competence for myotome and dermomyotome growth and morphogenesis.
DiI injections at the axial midpoint of the donor DML [Site B according to Denetclaw et al. (Denetclaw et al., 1997)] gave rise to DiI-labelled myofibres in the myotome of the primary host after overnight re-incubation (data not shown) and as described previously (Denetclaw et al., 1997
). A second DiO injection at the time of the second transplant showed that myofibres continued to be deposited from the donor DML in the myotome of the secondary host (Fig. 6E). The presence of DiI labelled fibres in the myotome of the secondary host (Fig. 6E) showed that labelled cells from the original, DiI labelled, donor DML continued to generate myocytes in both the primary and secondary hosts and therefore throughout the 2-day period of the serial transplant experiment.
Longevity of DML myogenic output
To examine the longevity of DML myogenic output through later stages of development, embryos were re-incubated for longer periods after DML transplants at E2. Re-incubation of a DML transplanted embryo for 2 days, through to E4, after surgery showed that the entire medial region of the myotome continued to be derived from the transplanted quail DML (Fig. 7A). Myocytes spanned a single segment with centrally aligned nuclei. The confocal image in Fig. 7A shows the myotome layer of the operated somite; therefore, dermis that originates from the transplanted DML is largely omitted. However, some dermis nuclei can be seen at the caudal part of the operated somite and in the middle of the next caudal somite (Fig. 7A, arrowheads). Therefore, upon de-epithelialisation of the dermomyotome, DML-derived dermal cells migrate past their original axial somite boundaries.
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The desmin labelling in E7 embryos resulted in brighter fluorescence intensity at the medial part of the muscle (Fig. 7B, arrowheads), reminiscent of the pattern noted in younger embryos where the youngest (dorsomedial) primary myotome fibres were more intensely labelled than older, laterally situated counterparts (Ordahl et al., 2001). If the unisegmental medial muscle was the most recently laid down, then a DML ablation carried out in older embryos should result in absence of the medial muscle. To test this hypothesis, DML ablations were carried out at low cervical or wing-level of E4 embryos and embryos reincubated for 2 or 3 days until E6 or E7, respectively. An example of an embryo that had a DML ablated from the rostral wing-level and was re-incubated until E7 is shown in Fig. 8A, where normal muscle pattern is indicated by the unoperated left-hand side of the embryo. By contrast, at the operated level the medial muscle is absent (Fig. 8A,B, filled bar) and some disruption to the laterally situated multisegmental muscle is evident. Analysis of individual optical scans confirmed the absence of the deeper, unisegmental, medial muscle and that it was the more superficial, multisegmental muscle that was pulled slightly towards the medial direction. Control embryos, which were treated similarly except that the DML was not ablated, retained the medialmost muscle (Fig. 8C).
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DISCUSSION |
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First, DML transplanted at E2 is sufficient to complete the epaxial myotome from the point at which the host myotome is truncated as a consequence of host DML ablation (Fig. 7). The fact that donor-derived myotome cells occupy the entire medial position relative to that start point is consistent with previous work showing that primary epaxial myotome expansion proceeds in a lateral-to-medial direction (Denetclaw et al., 1997; Venters et al., 1999
; Denetclaw and Ordahl, 2000
; Ordahl et al., 2001
). In addition, even at E7, new myocytes laid down at the extreme dorsomedial limit of the myotome are of donor DML origin. Taken together, these findings indicate that the DML retains potency to generate all of the cells comprising the primary epaxial myotome throughout the entire embryonic period.
Second, a prediction that devolves from the above conclusion is that DML ablation at any point during myotome formation should cause cessation of primary myotome development from that point onwards. Consistent with this prediction, ablation of the DML at E4 resulted in medially restricted truncation of the myotome that was evident at the end of the embryonic period (E7). Therefore, progressivity of primary myotome development is dependent upon an intact DML.
Third, the DML retains its potency to generate new myotome even after serial transplantation through two host embryos. Therefore, the DML continues to be the source of new cells entering the myotome, either directly or indirectly via DML-derived dermomyotome sheet, from the point of transplant.
Finally, it is mitotic activity in the DML that results in new cells entering the myotome because BrdU-labelled nuclei are chased into the myotome layer after a BrdU pulse administered to the embryo. Moreover, the appearance of BrdU-labelled myotome nuclei is time-dependent as few, if any, BrdU-labelled cells enter during a 2 hour pulse-chase experiment but many labelled myotome cells are seen after a 4 hour pulse-chase in accord with earlier studies (Langman and Nelson, 1968). Labelled myotome cells are predominantly restricted to the dorsomedial aspect of the myotome. Such distribution of BrdU-labelled myotome nuclei highlights the flow of daughter cells from mitotic DML progenitors to the dorsomedial aspect of the myotome.
The few BrdU-labelled myotome cells that appear during a 2 hour pulse-chase are scattered randomly throughout the primary myotome layer. This random positioning indicates that these cells did not arise directly from the DML during the 2-hour pulse chase, but either entered the myotome earlier (where they remained both undifferentiated and mitotically quiescent) or were derived from mitotically active precursors in the dermomyotome sheet and entered from the cranial and/or caudal lips of the dermomyotome (Marcelle et al., 1995; Kahane et al., 1998b
; Kahane et al., 2001
). In either case, these few cells represent the beginnings of secondary myotome formation (Kahane et al., 1998a
), whose precursors were derived from the DML at an earlier stage of myotome formation.
The results presented here are also consistent with previous transplant and dye studies, indicating that the epaxial dermomyotome epithelium is itself DML derived. The above conclusions regarding morphogenetic potential of primary myotome progenitor cells in the DML are also valid for both myogenic and dermal, and possibly other, precursor cells contained within DML-derived dermomoyotome sheet.
Permanence of DML myogenic progenitor cell pool
Recent analysis of myotome formation in mouse embryos (Eloy-Trinquet and Nicolas, 2002) concluded that there is no permanent stem cell population that gives rise to the myotome, a conclusion in apparent conflict with that reached from the experiments reported here. The notion of permanence, in this instance, implies that once a stem cell begins producing myotomal daughters it must continue to do so throughout the period of myotome development. There is, however, no reason to rule out the possibility that the DML contains stem cells with varying longevity [see fig. 4 in Denetclaw et al. (Denetclaw et al., 2001
)]. Thus, while some DML stem cells may give rise to myotome daughter cells during the entire embryonic period (and thus be permanent in the strict sense outlined above), the results of Nicolas and co-workers show that such stem cells are probably rare, while most generate myotomal daughters over a limited period of time and space (Eloy-Trinquet et al., 2000
; Eloy-Trinquet and Nicolas, 2002
). Predictions that opposing stem cell systems in the DML and its ventrolateral counterpart would result in an increase in labelled progeny at medial and lateral myotome extremities, respectively, do not take into account the dynamic nature of dermomyotome expansion demonstrated here. The expansion of these lips in opposing directions results instead in a corresponding deposition of myocytes in progressively medial, or lateral, positions. Therefore, such coherent opposing growth vectors (from dorsomedial and ventrolateral dermomyotome lips) may play an active role in maintenance of a proposed active clonal boundary between epaxial and hypaxial myotome clones (Eloy-Trinquet and Nicolas, 2002
). Because DML transplant analyses involve tracking daughters of the entirety of the progenitor cell pool, the absolute contribution of individual cells is impossible to predict. We propose, therefore, that the progenitor cells in the DML constitute a dynamically stable stem cell pool where the majority of mother cells may produce myogenic daughters for limited but overlapping periods during the development of the entire myotome. We refer to them as embryonic progenitor cells because they are restricted to development of a limited range of tissue and cell types, and in order to distinguish them from embryonic stem cells (which have much broader developmental potential).
Dynamic aspects of the inter-relationships among the progenitor cells within the DML and their daughters entering the myotome and dermomyotome remain undefined. For example, although many or all of the DML-derived dermomyotome epithelial cells remain both mitotically active and pluripotent the presence of such cells within the myotome can, at present, only be speculated upon. While differentiated myotome cells do not incorporate BrdU, the possibility that mitotically quiescent, undifferentiated cells may reside in the myotome cannot be ruled out. If such cells are generated during DML activity, they could undergo mitoses at a later point to generate muscle tissue. In addition, the ability of a given DML progenitor cell to give rise to both myotome and dermomyotome daughters remains undefined. Thus, any given DML cell may give rise to only one type of daughter (e.g. myotome) for a limited period and to the other type of daughter (dermomyotome) during other periods. Finally, some progenitor cells may remain quiescent (giving rise to neither type of daughter) for unknown periods, while being carried along with actively dividing neighbours during the dorsomedial expansion of the DML. Taken together, these considerations suggest that the DML progenitor cell population is complex and contains cells with various and possibly changeable phenotypic and mitotic potentialities.
Myogenic competence is restricted to paraxial mesoderm
Previous work from numerous laboratories has shown that competence for myogenic development is restricted to the paraxial mesoderm (Christ et al., 1979; Wachtler et al., 1982
) and shown a role for neighbouring structures in promoting somitic myogenesis (reviewed by Borycki and Emerson, 2000
; Hirsinger et al., 2000
). We therefore used non-paraxial mesoderm tissue as a control donor tissue to assess the extent to which the DML environment could induce growth and myogenicity. As predicted from those previous experiments, no myogenic potential was ever observed in donor tissue from the lateral plate mesoderm. In addition, CPM transplanted into the DML position also lacked myogenic potential. This was unexpected as CPM does form muscle tissue in the head (Noden, 1983
; Couly et al., 1992
; Trainor et al., 1994
). However, the retention of cranial muscle in Myf5/Pax3 null mice (Tajbakhsh et al., 1997
) and recent work describing discrete control of head versus body musculature (Mootoosamy and Dietrich, 2002
) argue for inequivalence between precursors of these populations. It remains possible, however, that longer re-incubation would yield a myogenic response by CPM in a DML position, simply reflecting the intrinsic differentiation schedule of cranial musculature (Noden et al., 1999
).
An interesting finding, however, was that all of the donor tissues that were negative for myogenic potential were able to respond to the DML environment by vigorously growing (expanding) in concert with adjacent, unoperated host paraxial mesoderm tissue. We conclude from this that the growth factor environment surrounding the DML is sufficient to support division and hypertrophy of many cell and tissue types, as suggested for sonic hedgehog during somitic myogenesis (Teillet et al., 1998; Charrier et al., 2001
; Kruger et al., 2001
). However, the growth factor environment is not sufficient to impart myogenic potential to non-paraxial mesoderm tissue.
The ventrally located sclerotome was also found to be non-myogenic in the test used here. Classical somite rotation experiments (Aoyama and Asamoto, 1988) showed that the ventral, pre-sclerotome regions of newly formed somites (ssI-ssII) are capable of forming normal myotomes while those from ssIII and older somites were not. The sclerotome transplant experiments reported here employed donor somites ssIV and older and so these conclusions are consistent with those of earlier workers. Other sclerotome transplantation experiments in which donor tissue fragments were placed dorsal to the DML (Dockter and Ordahl, 1998
) showed that sclerotome from somites older than ssIII retains myogenic competence, but only after E4 during the period of multi-segmental muscle formation. Thus, the sclerotome loses capacity for primary myotome growth and morphogenesis, while retaining capacity to participate in later muscle patterning and growth.
The dermomyotome lateral to the DML retains myogenic competence and is able to form primary myotome when placed in the DML position. Because cells in this region of the dermomyotome are derived from the DML (Denetclaw et al., 1997; Ordahl et al., 2001
) they represent symmetric daughters of the DML stem cell pool. These DML daughters within the dermomyotome retain latent myogenic competence for primary myotome formation that can be initiated when these cells are placed in the DML position. Finally, myogenic progenitor cells from the VLL of wing-level somites are similarly competent to mimic the DML role in primary epaxial myotome formation. Interestingly, older stage VLLs are unable to fulfil such a role, suggesting depletion of myogenically competent cells with migration from the somite into the limb field (Chevallier et al., 1977
; Christ et al., 1977
; Jacob et al., 1978
; Jacob et al., 1979
) (for a review, see Birchmeier and Brohmann, 2000
).
In conclusion, the results of the studies conducted here are consistent with the hypothesis that the dermomyotome DML contains a population of self-renewing cells, which simultaneously give rise to daughters that contribute to the myotome and dermomyotome. These daughters emerge from the DML in a topologically asymmetrical fashion, whereby mother cells are displaced medially in a process that results in the lateral-to-medial expansion of both the myotome and dermomyotome epithelium. Asymmetric division of mother cells and/or asymmetric timing of terminal differentiation of daughters combined with morphogenetic movements of cells emerging from the DML accounts for the growth and morphogenetic patterning of the myotome, and is also likely to govern patterning of other dermomyotome-derived tissues such as the dermis. Earlier in vitro experiments also implicated asymmetric, stem-cell-like divisions in the development of hypaxial muscle lineages (Quinn et al., 1984; Quinn et al., 1985
). DML-directed growth and morphogenesis bears similarity to the blastematic growth of regenerating limb mesenchyme. A clear difference between the regenerating limb blastema and the DML is that the latter is epithelial, although serial transplanation experiments reported here indicate that the epithelial nature of the DML may not be necessary for proper growth and morphogenesis. We therefore propose that the DML is an epithelial blastema, which governs unidirectional growth and patterning through asymmetrical division of progenitor cells within the DML and asymmetrical morphogenetic cell movements of their daughters.
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ACKNOWLEDGMENTS |
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