Lane MC and Smith WC (1999), The origins of primitive blood in Xenopus: impl...

XB-ART-13732

Development 1999 Feb 01;1263:423-34. doi: 10.1242/dev.126.3.423.

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The origins of primitive blood in Xenopus: implications for axial patterning.

Lane MC , Smith WC .

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The marginal zone in Xenopus laevis is proposed to be patterned with dorsal mesoderm situated near the upper blastoporal lip and ventral mesoderm near the lower blastoporal lip. We determined the origins of the ventralmost mesoderm, primitive blood, and show it arises from all vegetal blastomeres at the 32-cell stage, including blastomere C1, a progenitor of Spemann's organizer. This demonstrates that cells located at the upper blastoporal lip become ventral mesoderm, not solely dorsal mesoderm as previously believed. Reassessment of extant fate maps shows dorsal mesoderm and dorsal endoderm descend from the animal region of the marginal zone, whereas ventral mesoderm descends from the vegetal region of the marginal zone, and ventral endoderm descends from cells located vegetal of the bottle cells. Thus, the orientation of the dorsal-ventral axis of the mesoderm and endoderm is rotated 90( degrees) from its current portrayal in fate maps. This reassessment leads us to propose revisions in the nomenclature of the marginal zone and the orientation of the axes in pre-gastrula Xenopus embryos.

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Species referenced: Xenopus laevis
Genes referenced: dio3 igf2bp3 kcnt1

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	Fig. 1. Summary tailbud fate maps of the marginal zone and pertinent Nieuwkoop/Faber (N/F) stages for Xenopus laevis. (A) The summary fate map for stage 6-30 of Dale and Slack (1987a,b) taken from Smith(1989). (B) The composite fate map of the marginal zone. In the ventral marginal zone, a small amount of somitic mesoderm is present, while lateral plate mesoderm, which includes blood, predominates. This composite map, shown in textbooks, review articles and seminars is an amalgamation of many fate maps superimposed onto the boundary lines of the original fate map (stage 10-22) compiled by Keller (1976). See text for further description. (C) Keller’s revised map (1991). The ventral marginal zone includes significant prospective somitic tissue (shown in red on the ventral side), while leading edge mesoderm (orange), which includes lateral plate, intermediate and head mesoderms, resides in the vegetal portion of the marginal zone. (D) N/F stage 30 tailbud. (E) N/F stage 6 (32-cell stage), with the blastomere nomenclature of Nakamura and Kishiyama (1971) indicated. (F) N/F stage 41 tadpole. A, anterior; ap, animal pole; vp, vegetal pole; D, dorsal; LEM, leading edge mesoderm; LP,= lateral plate; N, notochord; P, posterior; S, somites; V, ventral.
	Fig. 2. Tailbud and tadpole stage, whole-mount embryos, labeled with RDA at stage 6, photographed under combined epifluorescence and epi-illumination to reveal labeled and unlabeled tissue. Dorsal is up and anterior to the left in all images. Faint red is the embryo; bright red is RDA label. (A,B) B4- and C4-labeled, stage 28-30 embryos. No label is present in the head. Trunk somites (s), lateral plate and the tailbud anlagen are heavily labeled. The region including the anterior VBI (abi) is unlabeled. (C) B4-labeled, stage 41 tadpole. Trunk/tail somites, tail epidermis and the proctodeum are heavily labeled. The anterior VBI (abi) is unlabeled. (D) C4-labeled, stage 41 embryo. Trunk/tail somites and epidermis are labeled. The region including the posterior VBI (pbi) and posterior lateral plate is labeled. Labeled blood cells are fixed in the heart (asterisk) of the C4-injected embryo.
	Fig. 3. Primitive blood in stage 39 tadpoles. Dorsal is up and anterior to the left in all images. (A) GFP-expressing blood cells in the tail of a C1-injected embryo. Three fluorescent images captured with a SIT camera, superimposed on a phase image of the embryo. The first fluorescent exposure was used for background subtraction, the second pseudo-colored red and the third pseudo-colored green. The image shows individual, labeled cells circulating in a tail vein. Arrows of different shapes indicate individual cells at the two time points. One labeled blood cell (asterisk) moves out of the field of view between the two time points. (B) C1- injected embryo. Notochord, somites, head mesoderm and the anterior, ventral region (arrow) including the region of the anterior VBI (vbi) are labeled. (C) Tail of C1-injected embryo. GFP-expressing, circulating blood in the caudal artery and vein. (D) B1-injected embryo. Notochord, brain, floorplate and somites are labeled. The VBI region (arrow) and leading edge mesodermal derivatives are unlabeled. (E) Tail of B1-injected embryo. Exposure time is the same as in C. No labeled blood circulates in the vasculature. The notochord and floorplate are labeled. (F) A4-injected embryo. Progeny include tail somites and ventral epidermis. (G) A4- derived, labeled blood circulates in the contralateral gill arches of the embryo shown in F. Combined epifluorescence and epi-illumination is used in images in B, D and F. The images in C, E and G are epifluorescence only.
	Fig. 4. Circulating, fluorescent cells are erythrocytes. (A) A control embryo, stage 40, stained for hemoglobin with benzidine. Vessels such as the vitelline veins (arrow) are heavily labeled. (B) Circulating cells collected by tail bleed from a C1-injected embryo. (C) A subset of collected cells express GFP. All GFP-positive cells stain with benzidine (D), indicating they contain hemoglobin.
	Fig. 5. In situ hybridization for aT3 globin at stage 40. (A) Control embryo. Cells within the vasculature and the posterior VBI express globin. The anterior VBI has already disintegrated. (B) Li+- generated, DAI 9-10 embryo expresses globin. (C) D2O-generated, DAI 10 embryo, with its radial heart (ht), expresses globin.
	Fig. 6. (A). Provisional Xenopus laevis tadpole fate map for mesoderm, projected onto stage 6. In the absence of a complete fate map for stage 41, our results for somites and primitive blood are combined with data for other mesodermal derivatives (e.g. notochord, head and heart mesoderms) from Moody’s stage 6-34 fate map (1987b). Somites (S, red) map to the animal region of all sectors of the marginal zone (blastomeres C1-C4, B1-B4 and A1-A4). Codistribution of somitic mesoderm (red) with notochordal mesoderm (N, reddish brown – blastomeres C1, C2, B1, B2, B3, A1, A2 and A3) is indicated by the mixed coloring of the notochordal territory. Blood arises from leading edge mesoderm (orange), situated in the vegetal region of the marginal zone, with a variable contribution from blastomeres A4 and A3, indicated by orange asterisks. Anterior leading edge mesoderm, including head and heart mesoderms (yellow asterisks) and the anterior portion of the ventral blood islands (abi), arises from C1, C2, D1 and D2 blastomeres. Posterior leading edge mesoderm, including the posterior region of the ventral blood islands (pbi) and probably the pronephros, arises from blastomeres C3, C4, D3 and D4. As there is no clear evidence at present that leading edge mesoderm cells are specified as particular cell types (e.g., blood) while residing in the vegetal region of the marginal zone of pre-gastrula embryos, the abbreviations for tissues shown within the LEM territory are meant to indicate general, not exact, distributions of the prospective tissues. For example, the precise relationship between the prospective head and heart mesoderms is unknown. They lie near the upper blastopore lip, but whether both mesoderms reach the median, or whether head mesoderm lies at the median while heart mesoderm resides more laterally, is not known. (B) Morphogenesis of the leading edge mesoderm (after Keller, 1991), including the prospective blood islands. Only the marginal zone is shown, from stage 10 to ca. stage 12.5. The animal pole (AP) is held in a fixed position. Dorsal mesoderm is red and leading edge mesoderm (comprised of head, intermediate and lateral plate mesoderms) is orange. Migration of the leading edge mesoderm and involution of the dorsal mesoderm begin at the upper lip around stage 10, and spreads to the lower lip by stage 10.5. Asterisks mark the future position of the ventral blood islands at the ventral midline, where the vanguard of the migrating leading edge coalesces into the blood islands. See text for fuller explanation. abi, anterior ventral blood islands; AP, animal pole; UL, upper lip; LL, lower lip; Hd, prospective head mesoderm; Ht, prospective heart mesoderm; pbi, posterior ventral blood islands; Pn, prospective pronephros.