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Specification of pharyngeal endoderm is dependent on early signals from axial mesoderm

Department of Biological Sciences, University of Denver, Denver, CO 80208, USA
Present address: Department of Cellular and Structural Biology, Campus Box B111, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver, CO 80262, USA

View larger version (37K): [in a new window]   Fig. 1. Diagrams of experimental manipulations of axolotl gastrulae. (A) A tilted dorsal vegetal view of an intact axolotl embryo at stage 10 reveals the state of the bottle cell apices (arrows) at the time of microsurgical manipulation. (B) Lateral view of the axolotl early gastrula fate map according to Pasteels (Pasteels, 1942). Dorsal is right. An, animal; bp, blastopore; ec, ectoderm; en, endoderm; lp, lateral plate mesoderm; nc, notochord; ne, neurectoderm; pcm, prechordal mesoderm; pe, pharyngeal endoderm; s, somitic mesoderm. (C) Dorsal view of the axolotl early gastrula fate map (Pasteels, 1942). Colors indicate tissues as in B. The broken black lines divide the spherical embryo at 30° intervals, both in latitude and longitude. The broken white line indicates the region of the explants shown in Ea, Eb and Ec, extending from 45° (the level of the blastopore is bp) to 110° (the animal extent of the axial mesoderm), and 30° bilaterally from the midline. (D) Keller sandwiches were constructed from paired dorsal marginal zones and allowed to develop intact until the hatchling larval stage (a). Keller endomesodermal isolates were generated (b) by removing the converging and extending endomesoderm (pink and blue regions below broken line) at early (b1) and later (b2) stages of gastrulation. (Ea) Single dorsal lip explants were trimmed to the size of the presumptive axial mesoderm (pink) and pharyngeal endoderm (blue) according to published fate maps. The predicted endodermal and mesodermal regions were then separated microsurgically (see Materials and Methods section for details). (Eb) Keller sandwiches were constructed, as in D, trimmed to the size of the future axial mesoderm and pharyngeal endoderm, and subdivided into suprablastoporal endoderm (blue) and more animal axial mesoderm (pink). (c) Fused dorsal lip explants were created by first trimming Keller sandwiches, and then subdividing them into even thirds. The most animal axial mesoderm and suprablastoporal endoderm were either raised separately (c1) or allowed to fuse (c2). The intermediate portions, containing the imprecise border between endoderm and mesoderm, were also raised separately (c1,c2).

View larger version (89K): [in a new window]   Fig. 2. Taste buds form normally in exogastrulae and endomesodermal isolates of exogastrulae. (A) A transverse section through the endomesodermal region of an intact exogastrula has been immunostained for calretinin to reveal the distribution of differentiated taste buds, which are present in large number. The arrow indicates the apical region of the taste bud shown at higher magnification in B. (C) This CR-IR taste bud found in an intact exogastrula displays the characteristic accumulation of CR-IR material in the apical microvilli (*), which is commonly seen in vivo (Barlow et al., 1996). (D) Numerous CR-IR taste buds were found in this endomesodermal isolate from a stage 11.5 exogastrula. The arrowhead indicates the apical region of the taste bud shown in E at higher magnification. (E) Again, this taste bud has intense CR-IR in the apical microvilli, throughout the cytoplasm of numerous fusiform cells within the bud. (F) Another CR-IR taste bud from an endomesodermal isolate, illustrating cytoplasmic immunostaining within fusiform cells, as is the case in vivo. Scale bars: 150 µm in A; 100 µm in B,D; are 25 µm in C,E,F.

View larger version (20K): [in a new window]   Fig. 3. The percentage of Keller isolates with taste buds differs from that of exogastrula isolates. Virtually all intact Keller sandwiches (white bars) and exogastrulae (black bars) produced taste buds. By contrast, while most Keller isolates, regardless of the stage of isolation, generated taste buds, the percentage of exogastrula isolates with taste buds declined as the presumed endomesoderm was removed at progressively earlier stages. (nd, not done; sample sizes are above histogram bars.)

View larger version (68K): [in a new window]   Fig. 4. Both Keller sandwiches and endomesodermal isolates thereof give rise to CR-IR taste buds. (A) Live Keller sandwiches 24 hours after construction have undergone normal convergence and extension of the mesoderm (arrows) and neurectoderm (asterisks). (B) This transverse section through an intact Keller sandwich explant at stage 41 or hatching was immunostained for calretinin (green) to show the presence of taste buds, and parvalbumin (red), which reveals the location of muscle. Notochord (noto) is also easily recognize via its distinct morphology. (C) Numerous multicellular CR-IR taste buds (arrow) are evident in most Keller sandwiches. (D) Immunostained sections of hatchling stage Keller sandwich endomesoderm isolated at stage 13 also revealed CR-IR (green) taste buds with apical processes (arrow), and parvalbumin-IR muscle (red). (E) At higher magnification, each taste bud is clearly multicellular, comprising fusiform, CR-IR cells. Scale bars: 100 µm in B; 50 µm in C,D,E.

View larger version (95K): [in a new window]   Fig. 5. Presumed mesodermal portions of dorsal lip explants make taste buds, whereas isolated endodermal components do not. (A) Small endodermal dorsal lip explants (Endo30s) persist as round balls of cells through to hatchling stages. (B) Cryosections of Endo30 explants were devoid of CR-IR; neither CR-IR taste buds nor solitary CR-IR cells were evident. However, yolk granules, indicative of endodermal tissue, autofluoresce in the green channel. Nuclei are stained with Hoechst (blue). (C) Mesodermal explants undergo substantial morphogenesis, and possess melanin granules typical of ectoderm (arrows). (D) Taste buds with numerous fusiform, CR-IR cells (green, * marks apical region of each taste bud) are present in this section of a mesodermal dorsal lip explant counterstained with Hoechst (blue). Parvalbumin-IR muscle (red-mu) is also present in this section. (E) The taste bud on the right in D is shown at higher magnification to illustrate the fusiform nature of CR-IR (green) cells within the bud, as well as the characteristic apical processes of these cells (*). (F) A few solitary, irregularly shaped, CR-IR cells (green) are shown in this cryosection of an Endo50 endodermal dorsal lip explant counterstained with Hoechst (blue). This explant did not develop taste buds, however. Scale bars: 100 µm in B; 50 µm in D,F; 25 µm in E.

View larger version (22K): [in a new window]   Fig. 6. Mesodermal dorsal lip explants generate taste buds and mesodermal tissues, whereas endodermal dorsal lip explants are generally devoid of both. Mesodermal dorsal lip explants (Meso70, animal 70% of dorsal lip explant; Meso70KS, Keller sandwich Meso70 explant) typically possess taste buds (dark-gray bars), notochord (pale-gray bars) and muscle (black bars). Endodermal isolates (Endo30, suprablastoporal 30% of dorsal lip explant; Endo50, suprablastoporal 50% of dorsal lip explant; Endo30KS, Keller sandwich Endo30 explant) generally do not contain any of these tissues. A smaller percentage of 50% mesodermal explants (Meso50) generate taste buds than do the larger Meso70 explants, consistent with the inclusion of less endoderm in the Meso50 explants.

View larger version (103K): [in a new window]   Fig. 7. Dorsal lip endoderm generates taste buds only when fused with dorsal lip mesoderm. (A) Dorsal lip endodermal explants remained as rounded balls throughout the culture period. (B) Mesodermal explants underwent extensive morphogenesis, and developed obvious notochords (arrows), and rough regions of muscle (arrowheads). (C) Fused explants again underwent morphogenesis, but the notochord and muscle elements were typically not as apparent, which is probably due to the presence of the endodermal epithelium. (D) No taste buds are evident in this typical section of an En30 explant immunostained for calretinin. (E) In this cryosection of an animal mesoderm dorsal lip explant, while CR-IR taste buds are absent, notochord (not) and parvalbumin-IR muscle (red) are clearly present. In addition, adjacent tissue has been neuralized, as evidenced by the presence of a neural tube (nt) with CR-IR neurons and axons (green), as well as of the otic vesicle (ov) with parvalbumin- and CR-IR hair cells (arrow; yellow). (F) This micrograph shows a higher magnification view of the parvalbumin- and CR-IR hair cells shown in E. (G) A section through a fused mesoderm-endoderm explant reveals 3 CR-IR taste buds (green; arrows), as well as notochord (not) and parvalbumin-IR muscle (red). (H) This micrograph is a high magnification view of the three taste buds shown in G. Although the plane of section is not optimal, these organs clearly comprise multiple CR-IR cells. Scale bars: 200 µm in C,D,E; are 25 µm in F,H.

View larger version (30K): [in a new window]   Fig. 8. Taste buds only form when presumptive pharyngeal endoderm is fused with presumptive axial mesoderm. Endoderm alone (Endo) is devoid of taste buds and differentiated mesoderm. Mesoderm alone (Meso) lacks taste buds (striped bars), but develops notochord (gray bars) and muscle (black bars). When these two regions are fused (Endo+Meso), now all explants generate all three differentiated tissues. Intermediate regions (Int) cultured alone typically generated mesodermal derivatives, and reasonably frequently gave rise to taste buds. Sample sizes are above histogram bars.