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Keywords:

  • Tbx5;
  • Tbx4;
  • limb bud;
  • lateral plate;
  • somatic;
  • splanchnic;
  • chick

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Positioning of the limb is one of the important events for limb development. In the early stage of embryogenesis, the lateral plate mesoderm splits into two layers and the dorsal layer (the somatic mesoderm) gives rise to a series of distinct structures along the rostrocaudal axis, including the forelimb bud, flank body wall, and hindlimb bud. To determine whether positional information in the somatic mesoderm for regionalization along the rostrocaudal axis is also inherited by the ventral layer of the lateral plate mesoderm (the splanchnic mesoderm), experiments in which the splanchnic mesoderm was transplanted under the ectoderm in an in ovo chick system were carried out. Transplantation of the wing-, flank-, and leg-level splanchnic mesoderm resulted in the formation of wings, nothing, and legs, respectively. These results suggest that the splanchnic mesoderm possesses the ability to form limbs and that the ability differs according to the position along the rostrocaudal axis. The position-specific ability to form limbs suggests that there are some domains involved in the formation of position-specific structures in the digestive tract derived from the splanchnic mesoderm, and results of cell fate tracing supported this possibility. In contrast, analysis of shh expression suggested that the anteroposterior polarity in the limb region seems not to be inherited by the splanchnic mesoderm. We propose that the positioning of limb buds is specified and determined in the very early stage of development of the lateral plate mesoderm before splitting and that the polarity in a limb bud is established after the splitting of the mesoderm. Developmental Dynamics 233:256–265, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In vertebrate embryos in early developmental stages, the lateral plate mesoderm appears lateral to the paraxial mesoderm as a single layer of homogeneous mesenchymal cells. Then the plate splits horizontally into two layers, the somatic mesoderm and the splanchnic mesoderm. Limb bud mesenchyme is a major tissue derived from the somatic mesoderm. The forelimb and hindlimb buds, which are mostly composed of the somatic mesoderm-derived mesenchyme and the ectoderm-derived epidermis (termed somatopleure), arise at particular levels along the rostrocaudal axis of the embryo. Although it is not clear how the precise positions of the limb fields are determined, Hox genes seem to play roles in the determination of the limb-forming regions because some Hox genes exhibit region-specific expression in the lateral plate mesoderm and some Hox expressions in the paraxial mesoderm correlate with limb positioning (Burke et al., 1995; Cohn et al., 1997; Gaunt et al., 1999). On the other hand, recent studies have suggested that Tbx5 and Tbx4 genes are involved in this process. In normal development of vertebrates, Tbx5 and Tbx4 are expressed in the presumptive forelimb and hindlimb regions, respectively (Chapman et al., 1996; Gibson-Brown et al., 1996, 1998; Simon et al., 1997; Ohuchi et al., 1998; Logan et al., 1998; Isaac et al., 2000; Takabatake et al., 2000), and Wnt2b and Wnt8c are thought to be involved in the induction of region-specific expressions of Tbx5 and Tbx4, respectively (Kawakami et al., 2001; Ng et al., 2002). Several experimental studies have suggested that Tbx5 and Tbx4 are essential for limb initiation and limb-type identification (Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999, 2003; Ahn et al., 2002; Ng et al., 2002; Agarwal et al., 2003). We previously showed that the presumptive forelimb and hindlimb buds of the chick embryo begin to express Tbx5 and Tbx4 at stage 13 and that the presumptive forelimb and hindlimb buds at stage 9 were capable of expressing Tbx5 and Tbx4 autonomously in vitro, indicating that limb-type specification occurs before stage 9 (Saito et al., 2002). Splitting of the lateral plate mesoderm into the splanchnic mesoderm and the somatic mesoderm starts rostrally at stage 7/8 and moves steadily to completion at stage 13. Taken together with the observation that forelimb and hindlimb levels of the lateral plate mesoderm have not split at stage 9 (Funayama et al., 1999), these findings suggest that, at least in stage 9 chick embryos, the forelimb and hindlimb levels of the lateral plate mesoderm, which consist of the future somatic mesoderm and splanchnic mesoderm, possess the ability to express Tbx5 and Tbx4 genes and to form the forelimb and hindlimb, respectively. Regardless of this ability, only the somatic mesoderm gives rise to Tbx gene expression, resulting in the formation of the forelimb and hindlimb in normal development.

After determining limb position, the limb level of the somatopleure begins to outgrow as a limb bud, according to information given by organizing centers such as the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). The expression of the sonic hedgehog (shh) gene, which is thought to be the active agent of the ZPA, is localized in the posterior margin of the limb bud and plays a crucial role in patterning along the anteroposterior (AP) axis inside the limb bud (Riddle et al., 1993). Although shh begins to be expressed in the posterior limb bud immediately after the limb bud emerges, specification of the AP axis and ZPA activity (the ability to express shh) in the limb field occurs before swelling of the limb bud (Hamburger, 1938; Hornbruch and Wolpert, 1991), and the process of this specification involves several key genes such as Hoxb8, d-hand, Alx4, and Hoxd (Charite et al., 1994, 2000; Takahashi et al., 1998; Fernandez-Teran et al., 2000; Zakany et al., 2004). Although the mechanism and timing of ZPA specification remain unclear, it is known that the layer expressing shh in the limb field is only the somatic mesoderm.

If the specification of limb fields along the rostrocaudal axis occurs before the lateral plate mesoderm is divided into somatic and splanchnic mesoderm layers as described above, it is possible that these two layers inherit the same positional information along the rostrocaudal axis. This possibility could be examined by transplanting the splanchnic mesoderm under the lateral ectoderm, because it is known that the early splanchnic mesoderm can be re-specified as the somatic mesoderm under the influence of the lateral ectoderm (Funayama et al., 1999). Therefore, to examine the possibility of the splanchnic mesoderm sharing the same positional identity with the same level of the somatic mesoderm, we carried out experiments to determine whether the limb level of the splanchnic mesoderm has the ability to form the limb structure. We also carried out experiments to determine whether the location of the ZPA is specified before the splitting of the lateral plate mesoderm. Results of experiments in which the splanchnic mesoderm was transplanted under the host ectoderm suggested that the somatic mesoderm and splanchnic mesoderm share positional information for regionalization along the rostrocaudal axis but not for polarity in a limb bud. We also examined the possible involvement of this positional identity in defining the rostrocaudal pattern in derivatives of the splanchnic mesoderm. In normal development, the trunk (including the limb level) splanchnic mesoderm gives rise to mesenchymal components of the digestive tract. The digestive tract is initially formed as a simple tube of the embryonic gut, and then organs, including the esophagus, stomach, duodenum, small intestine, cecum, and large intestine, are created along the rostrocaudal axis. We traced the cell lineage of the same levels of somatic mesoderm and splanchnic mesoderm and determined the organs to which the forelimb-level splanchnic mesoderm and the hindlimb-level splanchnic mesoderm contribute. Our results suggest that the positional identity along the rostrocaudal axis in the splanchnic mesoderm is involved in the establishment of the rostrocaudal pattern in the digestive tract.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Splanchnic Mesoderm Has Limb-Forming Ability

The ability of the splanchnic mesoderm to form limbs and the region specificity of limb-forming ability were tested by implanting wing (forelimb) and flank and leg (hindlimb) levels of the splanchnic mesoderm under the host flank ectoderm (Fig. 1A). Because splitting of the lateral plate mesoderm is completed by the 20-somite stage (stage 13) of the chick embryo (Funayama et al., 1999), chick embryos at stages 13–17 were used as donors. When wing and leg levels of the somatic mesoderm were implanted as control experiments, an extra limb structure was formed in the host flank, and the limb types of those additional limbs were always wing and leg, respectively (Table 1). Whereas the transplanted splanchnic mesoderm gave rise to an ectopic limb in more than 30% of specimens (wing level, 24 of 78; leg level, 30 of 79; Fig. 1B–G; Table 1), flank-level transplants did not form any extra structures (0 of 34; not shown, Table 1). We hardly observed initial outgrowth from flank-level splanchnic grafts. Although wing/leg type in some extra limbs could not be identified by observation of their skeletal patterns, others had obviously formed limbs according to their original positions (Table 1); that is, discernible limbs derived from the wing-level splanchnic mesoderm had a wing-like phenotype (16 of 24; Fig. 1B,C,F), and leg-like limbs were formed only by implantation of the leg-level splanchnic mesoderm (20 of 30; Fig. 1D,E,G). For further confirmation of the identity of the extra limbs, the expressions of Tbx5 and Tbx4 were examined at 48 hr after manipulations. When the wing-level splanchnic mesoderm was transplanted, the extra limb buds expressed Tbx5 (2 of 4; Fig. 2A) but not Tbx4 (0 of 4). On the other hand, transplants derived from the leg level formed extra limb buds that expressed Tbx4 (3 of 4; Fig. 2B) but not Tbx5 (0 of 4). Two of the wing-level samples and one of the leg-level samples formed tiny limb buds and expressed neither Tbx5 nor Tbx4.

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Figure 1. A: A diagram showing the manipulation to implant the splanchnic mesoderm under the ectoderm. Mesodermal sheets from wing, flank, and leg levels of the splanchnic mesoderm were transplanted into the ectodermal pocket made in the host flank. B: Wing level of the splanchnic mesoderm formed a wing-type limb. C: Skeletal pattern of B. D: Leg level of the splanchnic mesoderm formed a leg-type limb. E: Skeletal pattern of D. F,G: Some extra limbs derived from the wing-level (F) and leg-level (G) splanchnic mesoderm had anteroposterior orientation, opposite to that the rostrocaudal axis of the host. W, wing; F, flank; L, leg. Numbers are digit number of the extra limbs.

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Table 1. Cartilage Patterns in the Extra Limb Buds Derived From The Somatic and Splanchnic Mesoderm
Donor Phenotype
WingUnidentifiedLegNo extra limbTotal
Somatic mesoderm     
 Wing levelSt.13–1515 (65%)  8 (35%)23
 Leg levelSt.13–15  11 (44%)14 (56%)25
Splanchnic mesoderm     
 Wing levelSt.137 (33%)2 (10%) 12 (57%)21
 St.148 (28%)5 (17%) 16 (55%)29
 St.15 1 (8%) 11 (92%)12
 St.161 (8%)  12 (92%)13
 St.17   3 (100%)3
 Total16 (21%)8 (10%) 54 (69%)78
 Flank levelSt.13   5 (100%)5
 St.14   10 (100%)10
 St.15   9 (100%)9
 St.16   9 (100%)9
 St.17   1 (100%)1
 Total   34 (100%)34
 Leg levelSt.13 1 (5%)3 (16%)15 (79%)19
 St.14 3 (8%)9 (25%)24 (67%)36
 St.15 3 (21%)6 (43%)5 (36%)14
 St.16 3 (37%)1 (13%)4 (50%)8
 St.17  1 (50%)1 (50%)2
 Total 10 (13%)20 (25%)49 (62%)79
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Figure 2. A,B: Expression pattern of Tbx5 (blue) and Tbx4 (red) mRNA in the implanted splanchnic mesoderm, detected by double-stained in situ hybridization. Extra limb bud derived from wing level of the splanchnic mesoderm expressed Tbx5 (A), whereas Tbx4 was expressed in extra limb bud derived from a leg level of the splanchnic mesoderm (B). C,D: The splanchnic mesoderm of a quail embryo was transplanted into a host chick embryo. Staining with chick-specific antibody (A223 in C) and quail-specific antibody (QCPN in D) revealed that the grafted quail tissue became the mesenchyme of an extra limb bud (D), whereas the ectoderm and muscle mass of extra limb buds were derived from the host chick tissues (C).

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Because transplanted donor grafts were inserted between the lateral ectoderm and somatic mesoderm in the flank region, it is likely that the grafted splanchnic mesoderm itself made the additional limb. However, the possibility that the grafted tissue induced the surrounding host somatic mesoderm to make the extra limb cannot be excluded. Chimera analysis using a quail graft and chick host, therefore, was performed (Fig. 2C,D). At 72 hr after the transplantation, the overlying ectoderm and migrating muscle precursor were positive to chick (host) -specific A223 antibody (Fig. 2C), but the mesenchyme in the extra limb bud was negative. When the same extra limb bud was stained by quail (donor) -specific QCPN antibody, the mesenchyme including the condensing cartilage primordia was recognized (Fig. 2D). These results revealed that the extra limbs were mainly composed of the transplanted splanchnic mesoderm except for some components such as muscle and vessels that had migrated from the host (Fig. 2C,D). Taken together, the findings indicate that the splanchnic mesoderm as well as the somatic mesoderm has the ability to form extra limbs in accordance with their position along the rostrocaudal axis, although the percentage (34% [54 of the 157 samples] Table 1) of extra limbs formed by the limb-level splanchnic mesoderm was lower than that (54% [26 of the 48 samples] Table 1) formed by the limb-level somatic mesoderm (Table 1).

AP Polarity of Extra Limbs Derived From the Splanchnic Mesoderm

When transplanting the limb-level splanchnic mesoderm, the AP skeletal pattern of the extra limbs sometimes showed the opposite orientation to the original AP axis of the transplant (Fig. 1F,G), suggesting that the limb-level splanchnic mesoderm may have insufficient information on AP axis formation of a limb bud. We therefore tried to determine the relationship between the AP axis of the donor graft and the rostrocaudal axis of the host by examining the expression pattern of shh in the extra limb buds. When transplants were inserted in the same direction as that of the host rostrocaudal axis (Fig. 3A), 16 of the 55 samples (29%) expressed shh (Table 2). The percentage of transplants that expressed shh was similar to the percentage of splanchnic mesoderm transplants that formed limbs (34%), suggesting that shh expression is correlated with outgrowth of the extra limbs. We found three types of shh expression pattern (Fig. 3B–D). In some samples, shh was expressed only in the anterior margin of the extra limb bud (9 of 16 [56%]; Fig. 3B and white bar in the right column of Fig. 3K). In some other samples, shh was expressed in both the anterior and posterior margins (3 of 16 [19%]; Fig. 3C and hatched orange bar in right column of Fig. 3K). The remaining samples (4 of 16, 25%) had broad expression of shh peripherally in the dorsal side of the extra limb bud (Fig. 3D and gray bar in the right column of Fig. 3K). On the other hand, when the limb-level somatic mesoderm was transplanted, shh expression was observed in 11 of 17 (65%) transplants (Table 2). The three types of shh expression pattern, i.e., only anterior side (4 of 11 [36%]), both sides (5 of 11 [45%]), and broad expression (1 of 11 [9%]), were observed. In addition, 1 of the 11 samples had shh expression only in the posterior margin of the extra limb bud.

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Figure 3. A: Schematic representation of insertion of transplants into the host flank in the same direction as that of the host axis. The expression of shh was examined 48 hr after manipulation. BD: When the splanchnic mesoderm was transplanted, the extra limb buds expressed shh in the anterior margin (B), in both the anterior and posterior margins (C), or broadly on the dorsal side (D). E–J: Chick–quail chimera analysis. E,H: The shh-expressing cells in the anterior margin (inside dotted line in E) were derived from the host chick cells (H). F,G,I,J: The shh-expressing cells in the posterior margin (F) and the dorsal region (G) were derived from the grafted donor cells (I,J). K: Histogram showing the proportions of samples that had shh expression on the anterior side (white), on the both sides (hatched orange), on the posterior side (solid orange), and broadly on the dorsal side (gray).

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Table 2. Expression Pattern of shh in the Extra Limb Buds Derived From the Somatic and Splanchnic Mesoderm
Donorshh expression of the extra limb
AnteriorPosteriorBothBroadNo expressionaTotal
  • a

    No outgrowth of the extra limb was observed in these cases.

Somatic mesoderm4151617
Splanchnic mesoderm90343955

These observations revealed that the extra limb buds tended to express shh in the anterior side, even though transplants were inserted in the same direction as that of the host rostrocaudal axis. The disharmonious expression of shh is possibly caused by the influence of the host flank tissue rather than the disposition of the grafted tissue. Previous studies showed that extra limb buds formed after FGF application into the flank region express shh in the anterior margin (Cohn et al., 1995; Crossley et al., 1996; Ohuchi et al., 1997). Moreover, it is known that the flank lateral plate mesoderm has ZPA activity and that the level of it in the flank is higher in the anterior region than in the posterior region (Yonei et al., 1995). These previous studies suggest that, after transplantation of the splanchnic mesoderm and somatic mesoderm into the flank, the host flank cells induce shh expression in the anterior transplants or invade into the extra limb buds to express shh. To investigate these possibilities, the origin of shh-expressing cells in the extra limb buds was ascertained by using a quail–chick chimera system. After transplantation of the quail splanchnic mesoderm into the chick flank, host flank cells were observed in the anterior margin of the extra limb buds in all samples in which shh expression was detected in the anterior side, and most of those host cells expressed shh (Fig. 3E,H). On the other hand, shh-expressing cells in the posterior margin and in the dorsal region of the extra limb buds were derived from grafted quail mesoderm (Fig. 3F,G,I,J). The same results were obtained by transplantation of the somatic mesoderm (data not shown). These results indicated that the host flank cells invaded into the anterior extra limb buds and gave rise to shh expression in the anterior margin of the extra limb buds and that the grafted donor cells were responsible for the shh expression only in the posterior and dorsal regions of the extra limb buds.

Thus, for considering the AP polarity in the grafted mesoderm with an shh expression pattern, we should ignore the expression in the anterior side of the extra limb bud. The broad shh expression is thought to represent the incorrect or strange AP polarity in the graft, and only the posterior-restricted expression of shh perhaps reflects the original information on AP polarity of the grafted mesoderm. Reconsidering the AP polarity of transplants in view of this point, only 19% (3 of 16) of the splanchnic mesoderm transplants possessed their original AP axis in the extra limb buds, whereas 55% (6 of 11) of the somatic mesoderm transplants retained the original polarity (solid and hatched orange in left bar of Fig. 3K). These findings suggest that the limb level of the splanchnic mesoderm has less information on the limb AP axis than does the same level of the somatic mesoderm.

Limb-Level Splanchnic Mesoderm Maps to a Particular Position in the Digestive Tract

The rostrocaudal level-dependent formation of a limb by the splanchnic mesoderm suggests that the limb-level splanchnic mesoderm inherits the same positional information as the information on limb formation and limb-type identity in the limb-level somatic mesoderm. It is possible that this positional information learned in the splanchnic mesoderm is used in the compartmentation of the embryonic gut along the rostrocaudal axis for determining the organ type of the digestive tract. To investigate this possibility, we generated a fate map of the limb-level lateral plate mesoderm (both somatic and splanchnic layers) by labeling the same positions of these two layers simultaneously with a lipophilic carbocyanine dye (1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate [DiI]) and tracking them in the limb bud (Fig. 4A,C) and the digestive tract (Fig. 4B,D). Forty-two positions were injected with DiI, and labeled cells were imaged at 4 days after injection. The results are summarized in Figure 4E,F. When the wing level of the lateral plate was tagged, labeled cells derived from the splanchnic mesoderm were always located in the rostral region of the small intestine. The rostral end of the labeled cell population never invaded into the duodenum, and the caudal end was limited to the yolk stalk (Fig. 4E-a,F; n = 13 of 13). On the other hand, the leg-level splanchnic mesoderm was distributed from the caudal region of the small intestine and cecum through to the large intestine, and the rostral end never extended to the rostral side over the yolk stalk (Fig. 4E-c,F; n = 24 of 24). The distribution of cells derived from the flank-level splanchnic mesoderm was mostly restricted to the area around the yolk stalk (Fig. 4E-b,F; n = 6/8), whereas some cells from the posterior flank level were observed in the caudal part of the small intestine (Fig. 4E-b,F; n = 2 of 8). These results indicate that (1) the wing-level and leg-level splanchnic mesoderm distributes at a certain level of the digestive tract; (2) cells in the wing-level and leg-level splanchnic mesoderm do not mix with each other in the digestive tract, and the boundary between them is at the yolk stalk; and (3) there is little expansion of the flank-level splanchnic mesoderm along the rostrocaudal axis in comparison with that of the limb-level splanchnic mesoderm.

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Figure 4. The same positions of the somatic mesoderm and splanchnic mesoderm were labeled by 1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate (DiI). AD: In one example, DiI-labeled cells were detected as a narrow belt along the proximodistal axis in the leg bud (A,C) and from the posterior part of the small intestine, by means of cecum, to the anterior large intestine in the digestive tract (B,D). A and B are brightfield images of C and D, respectively, and the head is at the top in A and C and on the right in B and D. Results of the experiment are summarized in E and F. E: Distribution of labeled cells from an embryo are shown by a single color within the digestive tract and within the limbs and flank. The illustration of the digestive tract is separated into three parts according to the positions of labeled cells: labeled cells in the somatic mesoderm of the same embryo (boxed illustration) are located in the wing bud (left), in the flank (middle), and in the leg bud (right). Distributional pattern of labeled cells along the radial axis in the digestive tract was omitted from the illustrations, and each colored line indicates only the rostrocaudal level of the distribution of labeled cells. F: Schematic drawing and table showing the number of cases where respective labeled cells were distributed.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Subdivision and Specification of the Splanchnic Mesoderm

In vertebrates, the lateral plate mesoderm is split by the coelom into the somatic mesoderm, later forming the body wall and limb buds, and the splanchnic mesoderm, forming the circulatory system, the respiratory system, and the digestive system. Funayama et al. (1999) demonstrated that the early splanchnic mesoderm, although morphologically separated and distinct from the somatic mesoderm, can be converted into the somatic mesoderm. In the present study, the properties of the wing, flank, and leg levels of the splanchnic mesoderm changed to those of the somatic mesoderm, and these levels of the splanchnic mesoderm then formed structures in accordance with their original levels, i.e., transplants of the wing-level and leg-level splanchnic mesoderm expressed Tbx5 and Tbx4 and formed wing-type and leg-type limbs, respectively, whereas transplants from the flank level failed to initiate outgrowth and never formed any skeletal structures. These findings using morphological phenotype as the limb cartilage pattern not only confirmed the results of a study by Funayama et al. (1999) but also demonstrated that region specificity exists along the rostrocaudal axis in the splanchnic mesoderm for making additional limb structures. Because Funayama et al. (1999) showed that the flank-level (23-somite) splanchnic mesoderm can also be redirected to the somatic mesoderm, the regional specificity would not be due to different abilities for splanchnic-to-somatic conversion but to regional difference in abilities to make limb buds. Therefore, information on regional difference to form an appropriate structure (i.e., wing, flank, or leg) seems to be given in the lateral plate mesoderm before its splitting into two layers and to be retained in both layers. This idea is supported by the results of our previous study, showing that wing and leg levels of the lateral plate mesoderm acquire the ability to express Tbx5 and Tbx4 before stage 9, a stage at which splitting of the lateral plate has not yet occurred (Saito et al., 2002). The limb-forming ability in the splanchnic mesoderm seems to have stage-dependency, and later-stage splanchnic mesoderm tends to form no limb structure (Table 1), suggesting that the information for regional specification that was inherited in the splanchnic mesoderm is getting lost as development proceeds.

If the same information as that given to the somatic mesoderm is also inherited by the splanchnic mesoderm, some questions arise: is this information used for subdivision of the splanchnic mesoderm into three distinct domains and what are the structures that each domain contributes to? The results of the DiI-labeling experiment (Fig. 4) revealed that the wing-level splanchnic mesoderm becomes the small intestine rostral to the yolk stalk and that the leg-level splanchnic mesoderm contributes to the formation of the caudal small intestine, cecum, and large intestine caudal to the yolk stalk. The flank-level splanchnic mesoderm participates in the formation of the small intestine around the yolk stalk. It should be noted that the flank-level splanchnic mesoderm accounts for a much narrower space than does the limb-level splanchnic mesoderm, although at stages 12–14 the wing (15–20 somites), flank (21–26 somites), and leg (27–32 somites) levels occupy areas of similar sizes. Similarly, the flank-level somatic mesoderm makes a relatively thin layer, but the limb-level somatic mesoderm outgrows to form a limb. The different states of extension of the limb region and flank region in both layers of the lateral plate reflect the position-specific properties along the rostrocaudal axis. Some Hox members have regionally restricted expression boundaries in the splanchnic mesoderm, demarcating morphological landmarks of the midgut. Chick Hoxc9 is expressed in the posterior midgut, and the anterior boundary of the gene expression in the splanchnic mesoderm is located at the yolk stalk (reviewed in Roberts, 2000). Of interest, the anterior boundary of Hoxc9 expression in the somatic mesoderm is at the anterior flank, and at later stages Hoxc9 is expressed in the hindlimb bud but not in the forelimb bud (Nelson et al., 1996; Cohn et al., 1997), indicating that the anterior border of Hoxc9 expression in the two layers of the lateral plate coincides with the boundary between the wing and flank subdivisions.

Because descendants of the flank-level splanchnic mesoderm were slightly mixed with cells derived from the wing and leg levels of the splanchnic mesoderm (Fig. 4E), it is not clear whether the boundary of each domain of the splanchnic mesoderm is established at stages 12–14 or not. During limb-developing stages, the anterior and posterior regions of the flank mesoderm are known to express Tbx5 and Tbx4, respectively. Therefore, the reason the wing/flank or flank/leg boundary is not clear might be that the wing and flank or the leg and flank share the competence to express Tbx5 or Tbx4. In any case, the wing and leg levels of the splanchnic mesoderm never mix with each other, suggesting that these levels are established as distinct domains.

The pattern formation along the AP axis in the limb bud is regulated by the ZPA. Hornbruch and Wolpert (1991) reported that slight ZPA activity in the lateral plate mesoderm first appears at stage 10 in chick embryos. The ZPA activity is almost ubiquitous at first, but in the presumptive wing region, the activity becomes localized in the posterior region around stage 12. On the other hand, it is not clear when ZPA activity emerges in the presumptive leg bud, although it appears in the posterior regions of leg buds at least by stage 16. The expression of shh, whose transcript is probably the actual morphogen from the ZPA, first appears in the ZPA regions of both wing and leg buds at stage 17, when limb buds start swelling (Riddle et al., 1993). Because the division of the lateral plate into the splanchnic mesoderm and somatic mesoderm is completed by stage 12 at the wing level and by stage 13 at the leg level (Funayama et al., 1999), region-specific ZPA activity and shh expression should be detectable after the lateral plate has been split. In this study, when the donor grafts from the splanchnic mesoderm and the somatic mesoderm were implanted in the same direction as that of the host rostrocaudal axis, 71% (39 of 55) of the splanchnic samples showed no shh expression, whereas 35% (6 of 17) of the somatic samples did not show shh expression, suggesting that it is much more difficult for the splanchnic mesoderm to express shh than it is for the somatic mesoderm. Moreover, it is likely that the splanchnic mesoderm implanted under the ectoderm tends to express shh more randomly than the somatic mesoderm (Fig. 3; Table 2). These results suggest that the splanchnic mesoderm has little information on shh expression. The AP polarity may be regulated after the somatic and visceral layers of lateral plate have split. In addition, it is known that the flank region has a very potent inverted ZPA-inducing gradient, as evidenced from the 100% inverted polarity of limb buds induced by FGF beads (Cohn et al., 1995), it could be that the variable polarity in the grafts is due to the competition between endogenous polarity and local effect of the flank host on the implanted mesoderm grafts. Therefore, it is also possible that the splanchnic mesoderm is susceptible to the potent ZPA-inducing ability in the flank.

Development of the Trunk Lateral Plate

Figure 5 shows a schematic representation of the hypothetical process of development of the trunk lateral plate. The single layer of the lateral plate mesoderm first subdivides into three distinct domains according to the positional information along the rostrocaudal axis. At this time, these three domains acquire the ability to form the wing, flank, and leg regions, respectively, as each distinct property. Then splitting of the lateral plate mesoderm occurs, and both layers retain the domain-specific property and form appropriate structures according to the secondary information in each domain. The domains of the somatic mesoderm form the wing, flank, and leg, respectively. In contrast, those of the splanchnic mesoderm change their property to “visceral” and form distinct parts of the small intestine, cecum, and large intestine. Specification of the AP polarity within the limb bud as regionalization of the ZPA occurs only in the somatic mesoderm after the split, to form the skeletal pattern of limbs along the AP axis. Probably, other modifications within each domain are also accomplished in each layer, resulting in the formation of appropriate organs along the rostrocaudal axis.

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Figure 5. A model of the process of trunk lateral plate development. During early developmental stages, the positional information along the rostrocaudal axis is given to the lateral plate mesoderm. On the basis of this information, the trunk lateral plate is subdivided into three domains (indicated in blue, gray, and pink), and then the lateral plate is split into two layers: the splanchnic mesoderm and somatic mesoderm. After splitting, each domain in each layer is modified to form distinct organs. For example, a modification along the anteroposterior axis (the zone of polarizing activity induction) occurs only in the limb region of the somatic mesoderm (indicated in green) after splitting. See text for details. LPM, lateral plate mesoderm; So-M, somatic mesoderm; Sp-M, splanchnic mesoderm.

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In conclusion, we propose that the lateral plate mesoderm has acquired information for subdividing the trunk lateral plate into three domains before splitting of the lateral plate occurs, meaning that the positioning of the limb is a very early event of development of the lateral plate. Although little is known about the molecular mechanism underlying the subdivision, it is possible that the median tissues, including the notochord, neural tube, and somites, may send out the information for subdividing the trunk lateral plate, because our previous study (Saito et al., 2002) suggested that median tissues are involved in restricted expression of Tbx5 and Tbx4 genes in the wing and leg buds. Another clue must be the Hox code. The Hox code in the paraxial mesoderm is known to be correlated with limb position (Burke et al., 1995; Gaunt et al., 1999). Limb-type–specific expression of Hox genes exists also in the lateral plate mesoderm (Nelson et al., 1996; Cohn et al., 1997). Furthermore, it has been shown that the axial position of the limb bud is shifted in Hoxb5-deficient mice (Rancourt et al., 1995). These facts suggest that the subdivision of the lateral plate mesoderm into three domains may occur according to the Hox code.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Experimental Manipulation and Skeletal Pattern Observation

Figure 1A shows a schematic representation of the transplant procedure. Fertilized chicken eggs (Gallus domesticus) and Japanese quail eggs (Coturnix coturnix japonica) were incubated at 39°C, and embryos were staged according to Hamburger and Hamilton (1951). Presumptive wing, flank, and leg regions of chick embryos were dissected at stages 12–17, and the somatic mesoderm and the splanchnic mesoderm were isolated as donor grafts using 0.5% trypsin in Tyrode at 4°C. The intermediate mesoderm was carefully excluded from the donor grafts. When it is necessary, the posterior–lateral corner of the grafts was clipped off to recognize the AP axis of the grafts. Chick embryos at stages 11–15 were used as hosts. The lateral ectoderm of the host embryo was cut at the somite 22/21 level (anterior margin of the presumptive flank) and partially peeled toward the somite 25 level (posterior margin of the presumptive flank) to make an ectodermal pocket into which the donor graft was inserted. After manipulations, the eggs were reincubated at 38°C for subsequent analyses. For skeletal pattern observation, embryos were fixed in 10% formalin 7 days after the operation and then stained with 0.1% Alcian blue in 70% acid alcohol, dehydrated in ethanol, and cleared in methyl salicylate.

Whole-Mount In Situ Hybridization and Immunohistochemistry

Whole-mount in situ hybridization was performed as described previously (Yonei et al., 1995; Saito et al., 2002). A digoxigenin-labeled RNA probe for chick shh (a kind gift from Dr. Tabin; Riddle et al., 1993) was detected by staining with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) for substrates of alkaline phosphatase. For double staining with Tbx4 and Tbx5 probes (kind gifts from Dr. Izpisua-Belmonte; Isaac et al., 1998), fluorescein-labeled (for Tbx4) and digoxigenin-labeled (for Tbx5) RNA probes were used, and they were detected by staining with Fast Red and BCIP for substrates of alkaline phosphatase, respectively.

For analyzing the distribution of grafted donor tissue, the splanchnic mesoderm taken from quail embryos was implanted in a chick embryo and then processed for immunohistochemistry as described previously (Yonei et al., 1995). At 48 or 72 hr after the manipulation, the hosts were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), embedded in O.C.T. compound (Miles), and serially sectioned at 10 μm. Primary antibodies used were mouse anti-QCPN (quail-specific; Developmental Studies Hybridoma Bank) and anti-A223 (chick-specific; Ide et al., 1994).

DiI Labeling

Chick embryos at stage 12–14 were used for fluorescent tracer dye labeling. The wing or leg level of the lateral plate was pierced vertically with a needle-shaped crystal of 1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate (DiI, Molecular Probes, Inc.) to mark the same positions of the somatic mesoderm and the splanchnic mesoderm. The DiI crystal was removed after 5 min, and the egg was sealed and reincubated. After a further 4 days of incubation, embryos were harvested and fixed in PBS containing 4% paraformaldehyde, rinsed with PBS twice, and analyzed using fluorescent microscopy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. Daisuke Saito for his technical assistance on double staining by whole-mount in situ hybridization.

REFERENCES

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  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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