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Home , Ectoderm, Neural crest, Notochord, Paraxial mesoderm, Somite, Surface ectoderm

Development 122, 231-241 (1996) 231 Printed in Great Britain © The Company of Biologists Limited 1996 DEV3253

The dorsal organizes the dermamyotome and induces axial myocytes in the avian

Martha S. Spence1, Joseph Yip2 and Carol A. Erickson1,* 1Section of Molecular and Cellular , University of California, Davis, CA 95616, USA 2Department of Neurobiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

Somites, like all axial structures, display dorsoventral reported previously to induce sclerotome. Thus, we have polarity. The dorsal portion of the forms the der- demonstrated that in the context of the embryonic envi- mamyotome, which gives rise to the and axial mus- ronment, a dorsalizing signal from the dorsal neural tube culature, whereas the ventromedial somite disperses to can compete with the diffusible ventralizing signal from the generate the sclerotome, which later comprises the . vertebrae and intervertebral discs. Although the neural In contrast to dorsal neural tube, pieces of ventral neural tube and notochord are known to regulate some aspects of tube, dorsal or cells, all of which this dorsoventral pattern, the precise tissues that initially have been postulated to control dermamyotome formation specify the dermamyotome, and later the from it, or to induce , either fail to do so or provoke only have been controversial. Indeed, dorsal and ventral neural minimal inductive responses in any of our assays. However, tube, notochord, ectoderm and neural crest cells have all complicating the issue, we find consistent with previous been proposed to influence dermamyotome formation or to studies that following ablation of the entire neural tube, regulate myocyte differentiation. In this report we describe dermamyotome formation still proceeds adjacent to the a series of experimental manipulations in the chick embryo dorsal ectoderm. Together these results suggest that, to show that dermamyotome formation is regulated by although dorsal ectoderm may be less potent than the interactions with the dorsal neural tube. First, we demon- dorsal neural tube in inducing dermamyotome, it does strate that when a neural tube is rotated 180¡ around its nonetheless possess some dermamyotomal-inducing dorsoventral , a secondary dermamyotome is induced activity. from what would normally have developed as sclerotome. Based on our data and that of others, we propose a model Second, if we ablate the dorsal neural tube, dermamy- for somite dorsoventral patterning in which competing dif- otomes are absent in the majority of . Third, if we fusible signals from the dorsal neural tube and from the graft pieces of dorsal neural tube into a ventral position notochord/floorplate specify dermamyotome and sclero- between the notochord and ventral somite, a derma- tome, respectively. In our model, the positioning of the myotome develops from the sclerotome that is proximate dermamyotome dorsally is due to the absence or reduced to the graft, and myocytes differentiate. In addition, we also levels of the notochord-derived ventralizing signals, as well show that myogenesis can be regulated by the dorsal neural as to the presence of dominant dorsalizing signals. These tube because when pieces of dorsal neural tube and unseg- dorsal signals are possibly localized and amplified by mented paraxial are combined in culture, binding to the of the ectoderm, where they can myocytes differentiate, whereas mesoderm cultures alone signal the underlying somite, and may also be produced by do not produce myocytes autonomously. In all of the exper- the ectoderm as well. imental perturbations in vivo, the dorsal neural tube induced dorsal structures from the mesoderm in the Key words: dermamyotome, myogenesis, neural tube, , avian presence of notochord and floorplate, which have been embryo

INTRODUCTION lack obvious morphological polarity (Bellairs, 1963; Christ et al., 1972; Mestres and Hinrichsen, 1976). Within a few hours, The first arises by ingressing through the however, morphogenetic movements generate regional histo- to form a uniform band of tissue that flanks logical differences along the dorsal-ventral axis of each somite the neural tube. Although initially unsegmented, the paraxial (Mestres and Hinrichsen, 1976; reviewed by Stern et al., 1988; mesoderm soon becomes partitioned in an anterior-to-posterior Ordahl, 1993). Cells comprising the ventromedial portion of wave to form the somites, which are epithelial in nature and the somite undergo an epithelial-to-mesenchymal transforma- 232 M. S. Spence, J. Yip and C. A. Erickson tion to form the sclerotome, which lies lateral to the neural tube that dorsal fate is a default pathway in the absence of a ven- and notochord (Hay, 1968). In contrast, cells in the dorsal tralizing signal from the neural tube and notochord (Pourquié portion of the somite maintain an epithelial organization and et al., 1993), and therefore that dorsal-specifying signals may constitute the transient dermamyotome. Cells at the cranial not be necessary at all. edge of the dermamyotome later give rise to the more ventrally To characterize the cellular interactions that signal situated myotome, while the remaining dermamyotome cells formation of the dermamyotome as well as specification of become (Kaehn et al., 1988; Tosney et al., 1994). myocytes, we have employed the techniques of experimental Although the derivatives of the somites (i.e. dermatome, . Because most of our experiments attempt to myotome and sclerotome) are positioned with obvious dorsal- identify and characterize various signaling centers in the ventral polarity, such regionalization is not intrinsic to either context of the embryo, the environmental conditions and the unsegmented paraxial mesoderm or the most recently spatial organization of inducing and responding tissues are formed somites. This absence of regional specification at early close to normal, in contrast with previously employed tissue stages is revealed in experiments in which 180¡ rotation of the culture assays where these signals may be lost or altered. paraxial mesoderm or early somites has no effect on the sub- Results from a series of manipulations including dorsoventral sequent dorsal-ventral patterning (Aoyama and Asamoto, rotation of the neural tube, extirpation of the dorsal neural tube 1988). Similarly, when the dorsal half of an early somite is and grafting of putative inducing tissues ventrally adjacent to replaced with a ventral half, the grafted ventral tissue the sclerotome together support a model in which the dorsal undergoes normal myogenesis (Christ et al., 1992). These neural tube induces the formation of the dermamyotome. In studies suggest that dorsal-ventral pattern within each somite contrast, , ventral neural tube and neural crest is established through interactions with surrounding axial cells appear to have only modest, direct signaling roles, or do structures during and subsequent to . not participate in the induction of the dermamyotome. Fur- Several axial tissues could potentially contribute to somite thermore, our study suggests that the signal emanating from patterning. For example, the neural tube and notochord are the dorsal neural tube is a diffusible molecule that can compete, known to play a role in the development of the sclerotome. Co- in the embryo, with the ventralizing signal from the notochord. cultures of somite with neural tube and notochord produce an abundance of , a sclerotome derivative (Lash et al., 1957; Lash, 1968; Kosher and Lash, 1975). Moreover, when a MATERIALS AND METHODS notochord is grafted ectopically to dorsal regions of the somite, the sclerotome expands at the expense of the dermamyotome Embryo culture (Pourquié et al., 1993; Bober et al., 1994; Goulding et al., White Leghorn embryos were used for all in vivo manipula- 1994). Finally, early removal of the notochord enhances the tions (Avian Sciences Department, University of California-Davis or development of the dermamyotome and results in the absence Western Scientific, Sacramento, CA). Some embryos were treated and of sclerotome (van Stratten and Hekking, 1991; Rong et al., maintained in ovo. On the day before the operation, a small hole was 1992; Goulding et al., 1993, 1994). Recent studies reveal that cut into the egg shell above the embryo, the window sealed with cel- is produced by the notochord and floor plate lophane tape and the egg returned to the incubator. Just prior to any at the correct time to induce sclerotome (Johnson et al., 1994) experimental manipulation, a drop of 0.02% neutral red in Locke’s saline was applied to the to stain the embryo and and, furthermore, that heterologous cells expressing Sonic reveal the vitelline membrane, which was subsequently slit with a hedgehog can induce sclerotome formation, as assessed using tungsten needle to access the embryo. After surgery, the eggs were molecular markers, both in culture assays (Fan and Tessier- resealed and incubated at 38¡C and 70% humidity until the embryos Lavigne, 1994) and in vivo (Johnson et al., 1994). were fixed. Unlike the sclerotome, candidate molecules that regulate the Alternatively, some embryos were cultured in vitro after surgery. formation of dermamyotome or the myotome from it have not Embryos were cut from the and transferred to a 100 mm been identified. Indeed, our understanding of how these tissues Petri dish coated with a layer of 2% agar. The vitelline membrane was develop is further complicated by persistent controversy con- teased away, the experimental manipulation performed (see below) cerning which neighboring tissues exert inductive influences and the embryo cultured according to previously published methods on the paraxial mesoderm. The neural tube has been demon- (Erickson and Goins, 1995). Briefly, the embryo was oriented ventral side up, the saline removed so that the embryo was flat and stretched, strated repeatedly to play a role in myogenesis (e.g. Watterson and a filter paper ring (Whatman #1) centered over the embryo to keep et al., 1954; Vivarelli and Cossu, 1986; Kenny-Mobbs and the embryo fully spread. A Nuclepore filter (8 µm pore size, Costar Thorogood, 1987; Christ et al., 1992; Rong et al., 1992; Corp., Pleasanton, CA) was then layered over the embryo and ring, Borman and Yorde, 1994; Buffinger and Stockdale, 1994; which served to support the embryo during later culture. The whole Stern and Hauschka, 1995; Münsterberg and Lassar, 1995), assemblage was then turned so that the embryo was dorsal side up and there is some evidence that a signal is produced specifi- and suspended over a well in an organ culture dish (Falcon #3037) cally by the dorsal neural tube (Fan and Tessier-Lavigne, containing 1.5 ml Liebowitz L-15 medium supplemented with 10% 1994). Yet several studies also indicate that dermamyotome- fetal calf serum, 2 mM glutamine and 1% penicillin-streptomycin (all and myocyte-inducing molecules are produced by other from GIBCO, Grand Island, NY). The moat surrounding the well was tissues, including the surface ectoderm (Christ et al., 1972; filled with sterile double-distilled water and the embryo returned to the egg incubator at 38¡C and 70% humidity. Rong et al., 1992; Fan and Tessier-Lavigne, 1994; Kuratani et al., 1994), the notochord or ventral neural tube (Buffinger and Neural tube rotations and ablations Stockdale, 1994; Stern and Hauschka, 1995; Münsterberg and To discern whether the neural tube influenced the polarity of adjacent Lassar, 1995) and neural crest cells (Christ et al., 1992; axial structures, the orientation of the neural tube was experimentally Borman and Yorde, 1994). Finally, some evidence suggests altered. Segments of neural tubes were excised with tungsten needles, Dorsal neural tube induces the dermamyotome 233 leaving the notochord in place, as described previously (Yip, 1990). Co-culture studies The neural tube was then rotated 180¡ around the dorsal-ventral axis To assay the ability of various embryonic tissues to induce myocytes and immediately repositioned in the embryo (Fig. 1). Embryos were from unspecified paraxial mesoderm, the trunk regions (encompass- incubated for another 9-26 hours and fixed. ing the last 9 somites plus segmental plate) of stage 13-15 quail The dorsal neural tube was ablated from some embryos to embryos (Coturnix coturnix japonica) were removed with tungsten determine if dermamyotome development was inhibited. The dorsal needles and subsequently cut into 3-somite-length pieces (300 µm in portion of the neural tube of stage 12-14 embryos was suctioned off length), including a piece comprising the entire segmental plate. The using a mouth-controlled micropipette with a 0.1 mm tip diameter. pieces were digested briefly with full-strength Pancreatin at 37°C until Embryos were reincubated until they reached stage 16-18 and then the tissues could be separated easily from each other using tungsten fixed. needles. For each axial level, the neural tube, notochord, ectoderm and mesoderm (either somites or segmental plate) were collected sep- Grafting experiments arately and stored in complete F12 medium until used. The ability of various embryonic tissues to induce an ectopic der- A piece of segmental plate approximately 300 µm in length was mamyotome from the ventral somite was assessed by grafting these introduced into a small culture well (8-chamber culture slides, Lab- tissues into the space between the notochord and segmental plate. Tek, Nunc, Naperville, IL) in complete F12 medium. A potential Embryos were cut from the blastoderm and transferred, ventral side inducing tissue was also added to each well and the tissues up, to an agar-coated Petri dish filled with warm Locke’s saline. A maneuvered with a blunt tungsten needle until they touched. Cultures small slit was made in the between the neural tube and were incubated at 37¡C in 5% CO2 for 4 days and the medium was paraxial mesoderm just posterior to the last-formed somite. The replaced every other day. The presence of myocytes was determined piece of donor tissue was lightly stained with neutral red and by immunocytochemistry (see below). maneuvered into the slit with a tungsten needle. The embryos were Because ectoderm does not readily spread on a planar substratum, then incubated dorsal side up for another 18 to 22 hours (until stage we also employed gels to immobilize and culture the tissues. 16-18) and fixed. Collagen solution was prepared as described previously (Tucker and Embryonic tissues tested for their ability to induce the formation Erickson, 1984) and 50 µl droplets gelled in 35 mm plastic Petri of a dermamyotome included dorsal, lateral and ventral neural tube, dishes. The mesoderm and potential inducers were pipetted onto this as well as ectoderm and neural crest cells. To obtain these tissues, base and covered with a second layer of collagen so that the tissues trunks at the axial level of the unsegmented mesoderm of stage 13- were completely enveloped. Cultures were incubated for 4 days. 15 embryos were excised with tungsten needles and digested with Pancreatin (GIBCO) at 37¡C until the tissues began to separate. The Immunocytochemistry trunks were then transferred to cold Hank’s balanced salt solution We used a variety of techniques to identify myocytes and neural crest (HBSS) and the various tissues separated by manual dissection. At cells, both in embryo sections and in culture. For most of our studies, this time, the neural tube was cut longitudinally into dorsal, ventral we employed whole-mount labeling (see Erickson et al., 1992; Tosney and lateral segments. The isolated tissues were incubated in F12 et al., 1994 for details). A polyclonal antibody against the intermedi- medium supplemented with 10% fetal calf serum, 2 mM glutamine, ate filament (1:200, Biogenex, San Ramon, CA) was used to 3% chick embryo extract and 1% penicillin-streptomycin solution identify myocytes (Erickson et al., 1987; Kaehn et al., 1988; Tosney (complete medium) for 2 hours to allow the cells to re-express pro- et al., 1994). In some cases, operated embryos were labeled with the teolytically digested surface proteins. Neural crest cells were obtained HNK-1 antibody to visualize the distribution of neural crest cells. from 24-hour-old neural tube cultures, as described previously (e.g. HNK-1-producing hybridoma cells were obtained from the American Loring et al., 1981; Erickson and Goins, 1995). The tissues were Type Culture Collection and culture supernatant produced in our facil- washed in HBSS and stained with neutral red prior to grafting. In ities. After tissues were immunolabeled, they were embedded in some cases, the tissues were labeled for 2 hours in a 50 µg/ml solution Spurr’s resin, sectioned at 3 µm on a Sorvall MT-2 microtome using of wheat germ lectin conjugated with fluorescein (Sigma, St Louis, a diamond histoknife (Diatome) and viewed with a Leitz Diaplan MO) to identify unequivocally the graft in tissue sections. microscope equipped with epifluorescence. The color micrographs in

Somite

Fig. 1. Diagrammatic representation Neural crest cells of the neural tube rotation experiments. A segment of the neural tube from the level of somites 16 to 21 was excised, rotated dorsoventrally Ectoderm from Notochord and replaced. The embryo was dorsal surface reincubated for another 9 to 26 hours. Note that these rotations were performed at an axial level where the dorsal-ventral axis of the somite was already apparent. Even so, a secondary dermamyotome develops from the sclerotome. 234 M. S. Spence, J. Yip and C. A. Erickson

Fig. 4 were captured using a cooled CCD C72 MTI camera and NIH ventrally, we fixed 6 embryos 7-10 hours after the rotation. No Image software and merged in Photoshop. obvious clusters of cells could be detected adhering to the Cultures were labeled with the monoclonal antibody 13F4 to rotated neural tube, suggesting that the ectopic dermamyotome identify myocytes (Rong et al., 1987). In our hands this antibody was not derived from dorsal somite. produced less background fluorescence in cultured tissue than the desmin antibody. Cultures were fixed in 4% paraformaldehyde for 20 Dorsal neural tube ablations minutes, washed in PBS, blocked in 0.1% BSA/PBS and incubated in 13F4 (1:100 dilution in 0.1% BSA/PBS; Developmental Studies To test further the hypothesis that the dorsal neural tube Hybridoma Bank) for 2 hours. The cultures were washed with PBS induces dermamyotome formation, we ablated the dorsal (2×), blocked in 0.1% BSA/PBS and incubated in secondary antibody portion of the neural tube, along with the narrow strip of (goat anti-mouse conjugated with FITC or RITC, 1:100; Cappel) for ectoderm that adheres to it, in stage 12-14 embryos. Manipu- one hour. The cultures were then washed, coverslipped and viewed lated embryos were allowed to develop for an additional 18- as described above. In some cases, cultures were doubled-labeled with 20 hours and subsequently analyzed for the presence of a der- the HNK-1 antibody and the desmin antibody. mamyotome at the level of ablation. When the dorsal neural tube was removed at the level of the unsegmented mesoderm (n=16), 87.5% of the embryos RESULTS displayed either a complete absence (10 out of 16; 62.5%) or Nomenclature In this study, we use somite pairs as a convenient marker for Fig. 2. identifying and reporting axial positions. Traditionally, the first Histological formed somite pair is designated as ‘1’, the next most posterior sections through pair as ‘2’, and so on. Our study focuses on the thoracic level, embryos whose which encompasses somite pairs 20-27. neural tubes (nt) Regardless of the stage of development, the relative maturity were rotated and of each somite is consistent from one embryo to the next at the then fixed 26 thoracic level when measured with reference to the most hours later. (A,B) Phase recently formed somite (Loring and Erickson, 1987; Ordahl, contrast and 1993; Tosney et al., 1994). Consequently, we also use a fluorescence posterior-to-anterior staging scheme employed previously (see micrographs of an Loring and Erickson, 1987; Tosney et al., 1994), in which the HNK-1-labeled most recently formed pair of somites is designated as ‘−1’, the embryo. HNK-1 next most anterior pair as ‘−2’, etc. immunoreactivity identifies early Neural tube rotations migratory neural Since there is reason to believe that the dorsoventral pattern of crest cells. The the somites is established through interactions with the neural notochord (n) was left in its normal tube (summarized in INTRODUCTION), we rotated the neural position, although tube around its dorsoventral axis, leaving the notochord in its a small piece normal position, to test whether this could produce a corre- remains attached sponding inversion of paraxial mesoderm pattern. The neural to the former tube was excised at the level of somites 16-21 in stage 17-20 ventral surface of chick embryos, rotated 180¡ and the embryos reincubated for the neural tube. A 9-26 hours (Fig. 1). Prior to surgery, the ventral portion of the dermamyotome somites at the axial level of rotation had begun to disperse to (dm) developed in form the sclerotome while the dermamyotome was morpho- a normal position. logically distinct in the dorsal somite. After rotation of the Also an ectopic dermamyotome- neural tube and further development, however, the ventrome- like dial margin of the somite was no longer mesenchymal, but developed from rather was epithelial in character, resembling the derma- what would have myotome that persisted dorsally (n=5; Fig. 2). This was par- been sclerotome ticularly noteworthy since the sclerotome had already begun to (arrowheads). The disperse at the time of the operation. Proximity to the dorsal vesicle attached to neural tube appears to be important for the induction of a the dorsal neural second dermamyotome, because in one embryo in which the tube is probably a neural tube was oriented tangentially, the length of the ectopic remnant of dermamyotome that formed from each somite pair corre- ectoderm (e). (C) Fluorescence micrograph of a section taken through a region where the neural tube is tangential with respect to sponded to the extent of the dorsal neural tube with which the the embryonic dorsal-ventral axis. The size of the ectopic somite was in contact (Fig. 2C). dermamyotome (limits marked by arrowheads) is proportional to the To examine whether the secondary dermamyotome could extent of dorsal neural tube in direct contact with the somite, have developed from pieces of dorsal somite that may have suggesting that close contact is necessary for signaling to occur. s, adhered to the neural tube and been subsequently displaced sensory ganglion. Scale bar, 50 µm. Dorsal neural tube induces the dermamyotome 235

Fig. 3. Sections through 6 embryos whose dorsal neural tubes were ablated at the level of the segmental plate. The degree to which dermamyotome formation was affected is variable. (A,B) In most cases, there was a complete absence of the dermamyotome. Note that there are no neural crest cells in these sections (as assayed by HNK-1 immunoreactivity), indicating that the dorsal neural tube has been removed and has not regenerated. (C,D) In a smaller number of instances, remnants of the dermamyotome develop (arrowheads). Generally these are found in lateral positions in the embryo, as seen in C. Rarely, small epithelial vesicles are observed (D). (E) Although the dorsal portion of the neural tube has been successfully ablated in this embryo, as evidenced by the lack of neural crest cells, normal dermamyotomes are found. Possibly enough neural tube with inducing ability remains to stimulate dermamyotome development. (F) In this instance, where the entire neural tube was removed, one large dermamyotome forms dorsally. nt, neural tube; n, notochord; dm, dermamyotome. Scale bar, 50 µm. a significant loss (4 out of 16; 25%) of the dermamyotome at Embryonic tissues grafted medial to the ventral the level of ablation (Fig. 3A-D). The remaining embryos (2 somite out of 16; 12.5%) revealed no apparent effect on derma- The previous two experiments demonstrated a role for dorsal myotome morphology (Fig. 3E). These results suggest that axial tissues in the induction of the dermamyotome. However, the dorsal neural tube, or perhaps the narrow strip of these could not rule out the possibility that dermamyotome- ectoderm covering it, specifies dorsal somite structures. The inducing signals are produced either by the medial ectoderm appearance of part or all of the dermamyotome in some cases adhering to the neural tube or by neural crest cells emigrating may be due to the variability in the amount of neural epithe- from the dorsal neural tube. To test potential roles for these lium that was removed from each embryo. Alternatively, tissues and further investigate the influence of the neural tube, several studies show that the lateral dermamyotome develops we used two additional experimental strategies. independently of the cues that regulate formation of the First, pieces of isolated embryonic tissues were grafted medial dermamyotome (Bober et al., 1994; Pourquié et al., ventrally into the space between the notochord and paraxial 1995), which may explain why when remnants of dermamy- mesoderm at the axial level where somites have not yet formed otome are found, they are positioned laterally (e.g. Fig. 2C). to determine what embryonic tissues are capable of stimulating Curiously, when the entire neural tube was removed in one ectopic dermamyotome formation. The presence of a derma- individual, leaving an intact notochord in place, a fused myotome was assayed by morphological criteria. We also used somite with a dorsally positioned dermamyotome resulted an antibody against the intermediate filament protein desmin in (Fig. 3F). order to immunocytochemically detect myocytes. Out of 37 The paraxial mesoderm apparently responds to a dorsalizing embryos in which pieces of dorsal neural tube were grafted cue associated with the dorsal neural tube quite early and irre- ventrally (Fig. 4A,B), 49% displayed either the presence of a versibly, because when the dorsal neural tube is ablated at pro- morphologically distinct dermamyotome (6 out of 37) or gressively more anterior levels (i.e. embryologically older desmin-positive cells proximal to the graft (12 out of 37). In the regions), dermamyotome formation is less affected. Specifi- remainder of the embryos that showed no obvious ectopic der- cally, when the dorsal neural tube is ablated at the more mamyotome, the graft was quite distant from the somite, sug- anterior level of somites −1 to −3 (n=7), only 43% (3 out of 7) gesting that the grafted tissue must be close to the mesoderm to of the embryos exhibit a loss of the dermamyotome. There is exert an effect. Pieces of lateral neural tube also induced either no loss of the dermamyotome when the neural tube is removed an organized epithelium in the ventral somite or desmin-positive from the axial level of somites −4 to −6 (n=5). cells (Fig. 4C,D), although the response was less robust. 236 M. S. Spence, J. Yip and C. A. Erickson

Fig. 4. Pieces of isolated embryonic tissues were labeled with fluorescein- conjugated lectin (green labeling), grafted between the notochord and somite, and the presence of myocytes assessed by immunoreactivity with an antibody to desmin (red labeling). (A,C,E) Fluorescence images; (B,D,F) corresponding phase images. (A,B) A piece of dorsal neural tube was grafted ventrally and the embryo fixed 24 hours later. Myocytes are distributed ventrally (arrowheads) proximate to the grafted tissue. In a phase micrograph of the same section (B), note also the epithelial arrangement of the somitic cells (indicated by arrowheads) associated with the grafted piece of dorsal neural tube. Normal myotome structure and position is observed on the contralateral side. (C,D) A piece of lateral neural tube was grafted between the notochord and somite. A few myocytes arise in contact with the graft (arrowheads), but these cells are not organized into an epithelium. (E,F) A piece of ventral neural tube was grafted into a ventral position but, although it is in intimate contact with the somite, no myocytes have differentiated. Two small areas of rhodamine fluorescence (arrowheads) dorsal to the graft are blood vessels. Scale bar, 50 µm.

In contrast, when ventral neural tube pieces were grafted in mesoderm in vitro. Such studies allowed us to better control an identical fashion (n=14), the ventral somite cells were not the size of tissue pieces used and quantitate the inductive organized in an epithelium in any of the embryos, and only 7% response. of the embryos (1 out of 14) displayed desmin-positive cells Dissected neural tubes approximately 300 µm in length were next to the graft (Fig. 4E,F). This difference between the dorsal taken from the axial level of the segmental plate, and cut and ventral grafts is statistically significant (Chi-square longitudinally into 3/4 dorsal and 1/4 ventral pieces, or alter- analysis, P=0.05). natively, into 3/4 ventral and 1/4 dorsal pieces (Fig. 6A). These When pieces of ectoderm were grafted adjacent to the neural tube pieces were then cultured individually with a piece notochord (n=10), 90% of these embryos displayed either a of segmental plate (approximately 300 µm in length). The vague epithelial arrangement of somite cells (5 out of 10; Fig. extent of myogenesis was assessed by quantifying the number 5A) or a few desmin-positive cells (4 out of 10). The response of 13F4-positive cells per culture. Because the density of over- was not nearly as dramatic as that generated by dorsal neural lapping cells precluded accurate counting, we categorized the tube pieces, however (Fig. 5B). cultures as: <10 13F4-positive cells; 10≥100 cells; and >100 Finally, we tested the role of neural crest cells in derma- cells (Fig. 7). Control cultures of paraxial mesoderm alone myotome formation by grafting clusters of neural crest cells produced no myocytes (n=29 cultures). The results of co- isolated from 24-hour-old cultures between the somite and culture treatments are summarized in Fig. 6B. notochord. In 12 embryos, none showed any evidence of der- The greatest number of 13F4-immunoreactive cells was mamyotome formation proximate to the graft (data not shown). found in the 1/4 dorsal neural tube cultures (n=39 cultures; over 90% of all cultures contained more than 10 myocytes). Co-culture studies Fewer immunoreactive cells were found in cultures that To further distinguish between the relative roles of the dorsal, contained either 3/4 dorsal or 3/4 ventral neural tube pieces. ventral and lateral neural tube in the induction of myogenesis, The smallest number of immunoreactive cultures resulted from we co-cultured portions of the neural tubes with paraxial 1/4 ventral neural tube explants (n=56 cultures; 55% of these Dorsal neural tube induces the dermamyotome 237

Fig. 5. (A) When pieces of ectoderm (e) are grafted ventrally, cells that should have been mesenchymal, as on the contralateral side, are organized into a thin epithelium (indicated by arrowheads). This is not as robust a response as seen when pieces of dorsal neural tube (dnt) are similarly grafted (B). Note in B the epithelial organization of the ventral somite (arrowheads) despite its proximity to the notochord (n). The grafted tissue is not acting as a physical barrier between the somite and notochord and thereby blocking ventralizing signals. nt, neural tube. Scale bar, 50 µm. contained no myocytes), demonstrating that the dorsal neural Co-culture of dorsal neural tube and paraxial mesoderm in tube has the greatest ability to induce myocyte formation under collagen gels still yielded a dramatic response (>100 these culture conditions. myocytes/culture) in all cultures (n=4), as expected from the Neural crest cells derived from 24-hour-old cultures and co- planar culture assay. cultured with paraxial mesoderm (n=11) resulted in no We also assessed the relative ability of progressively older myocytes in 55% (6 out of 11) of the cultures, or a moderate neural tubes to induce myogenesis (Fig. 8). Neural tubes from level (10≥100 myocytes) of myogenesis in 45% (5 out of 11) 4 different axial levels Ð unsegmented mesoderm (n=11), of the cultures. somite level −1 to −3 (n=11), −4 to −6 (n=10), and −7 to −9 We found it difficult to establish cultures of dorsal ectoderm (n=12) Ð were cut in half longitudinally and the dorsal halves on planar substrata and therefore could not assess their ability co-cultured with segmental plate. This experiment did not to induce myogenesis using this assay. However, we repeated reveal statistically significant differences in the inductive some of these experiments by culturing tissues in collagen gels, effects of dorsal neural tube from different axial levels. Stern where adhesion to the substratum was not essential and where and Hauschka (1995) also reported that rostral and caudal the tissues could be forced to stay together. Under these con- neural tubes are equivalent in their capacity to promote myo- ditions, two or three pieces of ectoderm co-cultured with a genesis. This result is not surprising in light of the neural tube piece of unsegmented mesoderm (n=14) produced no response rotation experiments described earlier, in which all levels of in 43% (6 out of 14), a minimal response in 43% (6 out of 14), the rotated piece could induce a secondary dermamyotome. and a substantial response in 14% (2 out of 14) of the cultures. Our culture system also allowed us to evaluate at what axial

A B

75 < 10 myocytes ≥ 10 100 myocytes 24 > 100 myocytes 31 50 32 Neural Tube Pieces + SP 27 20 19 3/4 D + 18 11 14 25 1/4 V + Percentage of Cultures 7 6 4 1/4 D + 0 + 3/4 V 1/4 V 3/4 V 3/4 D 1/4 D Neural Tube Pieces

Fig. 6. (A) Diagrammatic summary of co-culture experiments in which paraxial mesoderm derived from the axial level of the segmental plate (SP; indicated by arrow) was combined with dorsal or ventral pieces of neural tube for 4 days and the differentiation of myocytes assessed by immunoreactivity with the 13F4 monoclonal antibody, which is a marker for myocytes. Cultures were characterized as: containing <10 myocytes; 10≥100 myocytes or >100 myocytes. (B) Percentages of cultures in each category are summarized in the bar graph. Total number of cultures for each category are indicated at the top of each bar. 238 M. S. Spence, J. Yip and C. A. Erickson

epithelial structure resembling the dermamyotome forms. Also, when the dorsal neural tube is ablated at a posterior axial level, no dermamyotome develops in the majority of cases. Finally, when pieces of dorsal neural tube are co-cultured with paraxial mesoderm either in tissue culture or in the embryo, an ectopic dermamyotome forms and myocytes differentiate from the unspecified mesoderm. Since no dermamyotome forms or muscle cells differentiate in the absence of the dorsal neural tube in all our experimental conditions, our results further suggest that a dorsalizing signal is required to specify the der- mamyotome, rather than dorsal structures simply arising by default. Although we demonstrate that the dorsal neural tube can induce dermamyotome formation and myocyte differen- tiation, and is also the most potent inducer in all of our assays, Fig. 7. Examples we have not ruled out the possibility that redundant or over- of cultures lapping cues emanate from other tissues. For example, immunolabeled ectoderm has been proposed to signal dermamyotome with the 13F4 formation directly (Christ et al., 1972; Fan and Tessier- antibody, which reveals myocytes, Lavigne, 1994; Kuratani et al., 1994). In support of a role for and rated as >100 ectoderm is the observation that in the absence of the entire myocytes (A), neural tube, but in the presence of a notochord, the somites 10≥100 myocytes fuse in the midline and one large overarching dermamyotome (B) and <10 develops (our results; Christ et al., 1972; Bober et al., 1994). myocytes (C). Thus, in the absence of dorsal neural tube cues, a normal der- Note in A how far mamyotome develops in association with the ectoderm and the myocytes are suggests that redundant cues are produced by ectoderm and distributed from neural tube. If the ectoderm does produce such a cue, it is the piece of dorsal difficult to explain why dermamyotomes do not develop when neural tube. i, putative inducer. the dorsal neural tube is ablated but the ectoderm over the Scale bar, 100 µm. somites remains intact. Moreover when pieces of ectoderm are grafted ventrally in the embryo adjacent to the ventral somite, only a very minimal response is observed (see also level myocytes were specified. As noted above, unsegmented Kenny-Mobbs and Thorogood, 1987; Fan and Tessier- mesoderm cultured alone produced no myocytes, as measured Lavigne, 1994) compared with the dramatic response to the by 13F4 immunoreactivity (n=29). In contrast, somites −1 to dorsal neural tube. Given that a putative inducing factor −3 cultured alone (n=15) produced myocytes in 60% of the produced by the dorsal neural tube appears to be diffusible cultures and somites −4 to −6 (n=10) and somites −7 to −9 (Fan and Tessier-Lavigne, 1994; see below), another expla- (n=12) produced myocytes 100% of the time. These results are nation for the weak inductive activity displayed by the identical to other studies that analyze myogenesis in stage 13 ectoderm is that such a factor could diffuse from the dorsal embryos using culture assays (Stern and Hauschka, 1995) or neural tube and bind to the associated in vivo experimental manipulations (Aoyama and Asamoto, with the basal lamina of the ectoderm. 1988). Thus, myocyte specification occurs within a few hours Another possibility that our study cannot rule out is that the of somite segmentation. dorsal neural tube may signal the overlying ectoderm to acquire dermamyotome-inducing ability. There is precedent for such a mechanism being involved in ventral somite speci- DISCUSSION fication, since Sonic hedgehog produced by the notochord induces formation of the neural tube floor plate, which in turn Our results, derived from both in vivo and in vitro analysis, produces more Sonic hedgehog and is apparently responsible suggest that factors from the dorsal neural tube induce der- for both stimulating sclerotome formation as well as repress- mamyotome formation and myocyte differentiation, that these ing dermamyotome formation (Johnson et al., 1994; Fan and factors are diffusible over a short range and compete with ven- Tessier-Lavigne, 1994). Thus when the dorsal neural tube was tralizing factors, and that specification of dorsal fate occurs ablated in our studies, it is conceivable that we may have inter- shortly after somite segmentation. fered with signaling to the ectoderm. Nevertheless, one addi- tional observation argues against this possibility. Bober et al. Dorsal neural tube is an inducer (1994) report that, when one lateral half of a neural tube is Many different embryonic tissues have been proposed to removed, the medial portion of the dermamyotome on the specify dorsal somite structures, but our results indicate that depleted side does not form, even though the other half of the the dorsal neural tube is the most potent of these. When the neural tube is still intact, including its dorsal component. The neural tube is rotated so that the dorsal surface abuts the ventral remaining neural tube could presumably still signal the somite, sclerotome dispersion is inhibited locally and an overlying ectoderm just as effectively. Thus we suggest that if Dorsal neural tube induces the dermamyotome 239

Dorsal neural tube from different axial levels co-cultured with segmental plate (SP) DNT 75% positive (n=12) SP

DNT 40% positive (n=10) -7 to -9 SP

-4 to -6 Fig. 8. Summary of co-culture experiments in which the myocyte-inducing ability of the dorsal neural tube (DNT) from increasingly -1 to -3 DNT anterior axial levels was assessed. The dorsal 64% positive (n=11) half of the neural tube was isolated from four SP Segmental different axial levels and then combined with Plate pieces of paraxial mesoderm isolated from the level of the segmental plate (SP). There is no DNT significant difference in the inducing ability of 72% positive (n=11) the neural tube as it ages (i.e. from the more SP anterior axial levels). the ectoderm plays a role in dermamyotome development, it is dorsal portion of a somite represses dermamyotome formation likely to be redundant. (Pourquié et al., 1993; Goulding et al., 1994) and that Neural crest cells have also been proposed to induce the notochord-derived Sonic hedgehog competes with the dorsal formation of the myotome as well as stimulate myogenesis, but signal to turn off dermamyotome-specific gene expression we view this possibility to be unlikely. First, neural crest cells (Johnson et al., 1994; Fan and Tessier-Lavigne, 1994). In grafted ventrally adjacent to the sclerotome fail to induce der- addition, when the notochord is removed, resulting in the dor- mamyotome formation. Similarly in co-culture studies, few or salization of the ventral neural tube (van Straaten and Hekking, no myocytes arise in the presence of pure populations of neural 1991; Goulding et al., 1993), dermatome-specific gene crest cells. The moderate number of myocytes observed in expression and myotome formation is extended ventrally, some cultures can be explained by the fact that purified crest again supporting the model that the notochord and ventral cells must be harvested from neural tube cultures, which may neural tube suppress the development of the dermamyotome. contaminate them with a dorsal neural tube-derived inducing A definitive explanation of these conflicting results must await factor. Finally, myotome formation occurs concomitant with the identification of the inducing factors. However, one possi- the invasion of neural crest cells into the somite at the thoracic bility is that the neural tube reciprocally signals the notochord level (e.g. Loring and Erickson, 1987; Tosney et al., 1994) but, and that, in the absence of neural tube influence (as would be at more posterior levels, crest cells invade the somite many the case in Rong et al., 1992; Buffinger and Stockdale, 1994; hours prior to the appearance of the myotome (our unpublished Stern and Hauschka, 1995), the notochord no longer produces results). Thus, there is no strict correlation between contact factors that repress dermamyotome formation, thereby with neural crest cells and the appearance of the myotome. revealing myogenic factors that it also generates. The obser- There are several reports suggesting that the notochord may vation that ectopically expressed Sonic hedgehog alone in the induce dermamyotome formation and muscle differen- dorsal somite results in the expansion of the myotome rather tiation. First, as already discussed, when the entire neural tube than its repression is evidence of a notochord-derived is ablated but the notochord in left in place (Christ et al.,1972; myogenic inducer (Johnson et al., 1994). Bober et al., 1994; and this study), a single large derma- myotome spans the space where the neural tube would be The dorsal neural tube product is diffusible normally. Second, the notochord apparently has a myocyte- It is likely that the inducing signals derived from the dorsal inductive capability in the absence of the neural tube in the neural tube are diffusible, although the distance over which embryo. For example, Rong and co-workers (1992) removed they can diffuse is difficult to discern from our current obser- the neural tube and notochord along the entire embryonic axis vations. We noted from our co-cultures on planar substrata that and then replaced these axial structures with 2 or 3 exogenous the dorsal neural tube had to be placed in close proximity to . Under these circumstances muscle cells differen- the mesoderm (but not necessarily touching) to see strong tiate. Finally, when paraxial mesoderm is co-cultured with induction but, in this assay, we were not able to quantitate notochord, muscle cells differentiate (Buffinger and Stockdale, precisely this distance. Stern and Hauschka (1995) also noted 1994; Stern and Hauschka, 1995; Münsterberg and Lassar, the need for close positioning. Under our culture conditions, a 1995; our unreported results). It is difficult to reconcile these diffusible factor might fall to very low levels a short distance observations with the fact that grafting a notochord into the from the dorsal neural tube. In fact, co-cultures embedded in 240 M. S. Spence, J. Yip and C. A. Erickson collagen gels (Fan and Tessier-Lavigne, 1994), where the environment. A complete dermamyotome rarely forms when diffusing molecule may be concentrated or even bound to the the dorsal neural tube is ablated at the level of the segmental gel matrix, suggest effects of over 100 µm. We also noted that, plate, suggesting that specification has not yet occurred. In although the inducing and responding tissues needed to be contrast, dorsal neural tube ablation at the level of the three close, myocytes appeared widely throughout the paraxial last-formed somites results in dermamyotome development in mesoderm (>100 µm from the inducing source) and not just in 50% of the cases, while somites −4 to −6 apparently no longer the contact zone (see Fig. 8A), suggesting that this factor can need the influence of the dorsal neural tube since ablation has diffuse through and perhaps be concentrated in the tissue envi- no effect on dermamyotome differentiation. Aoyama and ronment of the paraxial mesoderm. We did not rule out directly Asamoto (1988) also showed that myocytes are specified by that an inducing signal in our system is propagated from one somite −3 in stage 13 embryos by rotating a somite around its myocyte to the next. Such propagation seems unlikely, dorsoventral axis, which then produced an inverted dorsoven- however, because we might have observed similar numbers of tral pattern. Interestingly, the somites remain responsive to myocytes arising in our cultures of somites at different ages signaling from the neural tube after segmentation (see also but, instead, we found that greater numbers of myocytes dif- Stern and Hauschka, 1995) and the dorsal neural tube itself ferentiated from progressively older somites. retains inducing properties at more anterior axial levels, pre- Our in vivo studies also cannot reveal how far such a factor sumably many hours after they are needed. Such precision in may diffuse since tests of induction were always done in com- identifying the timing of specification should be useful in elu- petition with the host notochord or floorplate, which compli- cidating the molecular basis for the various steps in the speci- cates the interpretation. Nevertheless myocytes often appeared fication, determination and differentiation of myocytes and >50 µm from the closest edge of the dorsal neural tube dermal precursors. fragments. Any further tests of how far such an inducer could act must clearly be carried out in the embryo since rates and Model for dorsal-ventral pattern distances of diffusion will depend upon the ability to move Our data, along with a wide array of previously published through and bind to the extracellular matrix or through other studies, suggest the following model for the establishment of tissues. dorsal-ventral pattern in the somite. Competing diffusible Our studies, as well as those of others, suggest that dorsal signals from the dorsal neural tube and notochord/floorplate neural tube-derived factors can compete with ventralizing specify dermamyotome and sclerotome, respectively. The signals. Fan and Tessier-Lavigne (1994) elegantly demon- dorsal neural tube signal is proposed to diffuse broadly but in strated such competition when they co-cultured dorsal neural ventral regions is out-competed by Sonic hedgehog and tube and Sonic hedgehog-producing heterologous cells with perhaps other ventral signals. Thus, the spatial positioning of unspecified mouse presomitic mesoderm. Similarly in our the dermamyotome dorsally is due to the absence or low con- study, when 3/4 dorsal neural tubes, which are likely to contain centration of notochord-derived signals, as well as to the ventralizing signals, are cultured with paraxial mesoderm, presence of dorsal signals. It is possible that these latter fewer myocytes differentiate compared to 1/4 dorsal neural signals are localized and amplified by binding to the ectoderm tubes, again suggesting antagonizing signals. We have been or that the ectoderm also produces dorsalizing factors. An able to demonstrate directly that such a competition occurs in alternative possibility is that the dorsal neural tube signals the embryo as well. In two different experimental paradigms Ð only the medial edge of the somite, and this dermamyotome- either when the neural tube was rotated 180¡ around its dorsal- specifying signal is propagated laterally in the plane of the ventral axis or when pieces of dorsal neural tube were grafted somitic epithelium. This signal, if propagated ventrally, ventrally Ð host notochord was always left in its normal would be inhibited by ventralizing signals from the floorplate position. Under these circumstances, dorsal structures (either a and notochord. Identification of the dorsal signaling dermamyotome or myocytes) differentiated in response to a molecules awaits further experimentation, but particularly dorsalizing cue despite their proximity to the host notochord. attractive candidates are members of the Wnt family, which This competition is not due simply to the grafted tissue phys- are diffusible signaling factors produced in the neural tube ically blocking a ventralizing signal from the notochord, since that act over both a short and long range (Parr et al., 1993). the dorsalizing effect of the grafted dorsal neural tube was Finally, it is likely that several dorsalizing signals are noted even when the notochord had unimpeded simultaneous necessary, including those that maintain the epithelial contact with the somite (Fig. 5B). structure of the dermamyotome, as well as additional factors that may dictate specific cell fate. Timing of specification Several recent studies where progressively older somites were We thank Charles Ordahl and David McClay for insightful placed in culture suggest that, within only a few hours after comments concerning and signaling mechanisms and separation from the segmental plate, the myocytes are already Mark Reedy, David Parichy and Tuan Duong for careful scrutiny of determined and will later differentiate, even in the absence of the manuscript. Two anonymous reviewers contributed substantially to the tightened focus and balance of the Discussion, for which we the neural tube (Vivarelli and Cossu, 1986; Kenny-Mobbs and thank them. This research was supported by NIH grants DE 05630 to Thorogood, 1987; Buffinger and Stockdale, 1994; Stern and C. A. E. and NS 23916 to J. Y. Hauschka, 1995). 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