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Changing the axis changes the perspective

2002; Wiley; Volume: 225; Issue: 4 Linguagem: Inglês

10.1002/dvdy.10198

1097-0177

John C. Gerhart,

Neurogenesis and neuroplasticity mechanisms

Among the studies reported in this issue are ones by Lane and Sheets (2002) and by Kumano and Smith (2002) that concern the reassignment of dorsoventral and anteroposterior axes on the fate map of the Xenopus embryo. The two studies complement one another, with Lane and Sheets (2002) emphasizing the assignment of the anteroposterior axis to what had previously been called the dorsoventral axis and with Kumano and Smith (2002) emphasizing the assignment of the dorsoventral axis to a dimension not previously assigned, although aligned with the animal–vegetal axis. They dwell mostly on axis designations for the mesoderm rather than for ectoderm or endoderm, on the grounds that mesoderm determines the fates and axes of the other two germ layers. This is true in two ways. (1) Within the mesoderm is Spemann's organizer, which is composed of a head organizer (the prospective prechordal plate/head mesoderm territory) and a trunk–tail organizer (the prospective notochord territory). During gastrulation and neurulation, the ectoderm is induced by the organizer to form the central nervous system. Recent work on Bmps, Bmp-antagonists, and the default pathway for neural development has illuminated the neural–epidermal bipotentiality of the ectoderm and the essentiality of the organizer for neural fates, their orientation, and their placement in the embryo. Later, the organizer induces the floor plate of the neural tube, which is a source of signals for the dorsoventral patterning of the tube. Mesoderm itself is induced by the organizer to form somites running the length of the body and to form the heart and tail bud. Later, the organizer induces the sclerotome portion of the somites. Endoderm is induced to form anterior gut regions and the hypocord. Most aspects of the dorsoventral and anteroposterior axis of the embryo depend on the organizer's inductions and, therefore, its location. And (2) the organizer mesoderm has a major role in axis formation by way of its morphogenetic activities, particularly the convergent extension morphogenesis by cells of the prospective notochord territory, and the migration of cells of the head mesoderm territory. Thus, if researchers want to emphasize the developmental mechanisms of axis formation, they should focus on the mesoderm, the germ layer most involved, and they should name its axes as accurately as possible. I support the efforts of Lane and Sheets (2002) and Kumano and Smith (2002) to make the designations useful and accurate rather than historical. It will take many of us, myself included, a while to speak of “anterior and posterior” marginal zones rather than “dorsal and ventral” marginal zones, or to speak of cortical rotation and beta-catenin accumulation establishing the anteroposterior axis rather than dorsoventral axis, or to designate various treated embryos as “posteriorized” rather than “ventralized,” and “anteriorized” rather than “dorsalized,” or to rename Xvent genes as Xpo genes. The old dorsoventral axis designation may retain some usefulness in nonmechanistic circumstances. For example, if you mark the egg surface with a spot of Nile blue dye halfway from the animal pole to the equator on the same side as the dark sperm entry point, the mark will generally show up in the ventral epidermis of the tadpole. If you apply the spot at the same level in the animal hemisphere on the side opposite the sperm entry point, the mark will in general show up as a streak in the centerline of the neural plate. Because the neural tube is dorsal and the belly epidermis ventral, this makes a dorsoventral axis for the ectoderm. Fate maps of the ectoderm (not mesoderm) confirm this assignment. We and others have used such convenient marking of fertilized eggs for years to predict where the dorsal midline will be in the tadpole. Much of the old work with marking was of this gross predictive sort. But making convenient predictions about the embryo's final orientation is a long way from making analytical statements about mechanisms of axis formation. Lane and Sheets (2002) review the designations of axes in the old literature. The old descriptive predictive language, which was suitable for ectoderm, did not change as mechanistic interpretations moved to mesoderm formation and the inductive-morphogenetic activities of mesoderm. The fate map of mesoderm is oriented differently from that of ectoderm, and so a different set of axes is needed. Figure 1B is meant to help the reader visualize the new axes in the mesoderm of the Xenopus late blastula (10,000 cells), just before gastrulation begins. In Xenopus the mesodermal fate map is spread onto an annulus or band of cells located just inside a surface layer of prospective endoderm cells (gut roof and walls) and just outside a central core of yolky endoderm. Because the annulus is located at the margin (i.e., boundary) of the animal and vegetal hemispheres, it has long been called the “marginal zone.” The organizer occupies a 60- to 90-degree sector of the circumference of the zone. One axis will run vertically across the zone (top to bottom, as drawn in Fig. 1) and the other will run horizontally, from one side to the other, passing through the organizer. The axes reflect the placement of particular territories in the marginal zone, namely, those for prospective notochord, somites, lateral plate, heart, head mesoderm, and blood. Although the anatomic placement of these structures in the tadpole is clear (provided one defines the dorsal–ventral and anterior–posterior dividing planes), there is some disagreement about where the territories are in the marginal zone of the late blastula. The authors take pains to go through all the data, arriving at two revisions, as mentioned below, both important for their new designation of axes. In excellent current textbooks, for example, the 2nd edition of Wolpert et al. (2002, page 93), the marginal zone map of mesoderm is drawn in a way consistent with a dorsoventral rather than anteroposterior progression of territories in the circumference of the marginal zone (see Fig. 1A), but the authors argue that such locations and inferred axes are incorrect. Old and new axes. The marginal zone mesoderm of the late blastula is shown as an annulus. A: The old axes are applied—the dorsoventral axis is shown in the horizontal dimension. The prospective territories are shown with a right to left progression of dorsal to ventral structures. The tadpole is shown in a related orientation. B: The new axes are applied, with the dorsoventral in the vertical direction and the anteroposterior horizontal as drawn. The marginal zone has two bands, dorsal (upper, animal) and ventral (lower, vegetal). The tadpole is shown in the related orientation. A, anterior; AP, animal pole; AS, anterior somites; BI, blood islands; D, dorsal; H, heart; HM, head mesoderm; LP, lateral plate; N, notochord; P, posterior; PS, posterior somites; V, ventral. First, they call attention to blood islands, which are the most ventral of fates in the tadpole. The prospective territory for the islands was previously placed on the fate map at the side of the marginal zone opposite the organizer. This placement would make the contraorganizer side the ventral pole of a dorsoventral axis across the diameter, with the organizer as the dorsal pole. Lane and Smith (1999), though, have reported that blood islands arise from cells located at the vegetal edge of the entire marginal zone, a very different location. The ventral pole would then be at the bottom of the annulus. Second, they note that the contraorganizer site of the marginal zone is occupied by prospective posterior somites (those of the posterior trunk and tail), which are dorsal or dorsolateral mesoderm, not ventral mesoderm. The authors look closely at other territories, as shown in Figure 1B, and present the fate map of the marginal zone mesoderm in a new way—it contains two circumferential bands: one animal (prospective dorsal) and one vegetal (prospective ventral). The dorsal band contains territories of the notochord and somites. These are dorsal fates. The ventral band contains territories of the lateral plate, heart, blood, and prechordal plate/head mesoderm. These are ventral fates. In general their assignment fits the new data and the old Nile blue fate maps of Keller, and are consistent with the lineage tracer injection maps of Dale and Slack, with some provisions the authors explain. The only territory that seems questionable to me is the prechordal plate/head mesoderm. Is it really ventral or dorsal or both? It depends exactly where one defines the dividing plane between dorsal and ventral. Lane and Sheets (2002) suggest a dividing plane running through the mouth, gut, and anus, in which case the head mesoderm would have to be ventral to the mouth. But this can be remedied with a small shift of the plane. The entire mesodermal annulus will invert itself during gastrulation, so that what was closest to the vegetal pole before gastrulation will be closest to the animal pole afterward. When Lane and Sheets (2002), and Kumano and Smith (2002), draw the dorsal to ventral axis of the marginal zone as being in the animal-to-vegetal direction, they mean its orientation before gastrulation. For example, the prospective heart mesoderm, which initially lies at the vegetal edge of the zone, will come to lie near the animal pole. This new axis would be orthogonal to the new dorsoventral axis. With the territory of posterior somites at the contraorganizer position, a posterior pole is fixed at one side of the dorsal (animal) band of the marginal zone. As the authors note, the somite territory is broad (270–300 degrees of circumference), and the anterior somites derive from positions close to the organizer, so this progression of somites fits into the new anteroposterior axis. In the ventral (vegetal) band, the territories are also located in a posterior to anterior order: the prospective lateral plate is at the posterior pole (under the posterior somites), then comes the heart, and then the prechordal plate/head mesoderm at the organizer side, the anterior pole. Even the blood island territory at the vegetal edge of the marginal zone seem to have an anteroposterior correspondence, for as Lane and Smith (1999) have noted, anterior blood islands come from the organizer side and posterior islands from the contraorganizer side. It is only the notochord territory that may present a problem: it is at the anterior pole of the dorsal band, but is it really an anterior structure? It will eventually run as a rod from the hindbrain to the tail tip. Although not as anterior as the prechordal mesoderm, its territory certainly contains anterior-fated cells. However, it also contains posterior-fated ones as well, because all levels of the notochord come from this 60- to 90-degree-wide trapezoidal territory. As Lane and Sheets (2002) point out, the developing notochord pushes posteriorly by convergent extension from an anterior position. The extension of notochord cells (the trunk–tail organizer) through nearby tissues such as prospective somite mesoderm is important for their anteroposterior patterning. How does this reassignment of axes help us think about mechanisms of axis formation? Fates, after all, do not reveal mechanisms but just the regular outcomes of unperturbed development. Kumano and Smith (2002) point out that one is led to think about the setting up of the dorsoventral axis in the marginal zone as a modification of endoderm–mesoderm induction, which establishes the marginal zone mesoderm in the first place. In situ staining of the expression of specific genes shows early differences of the animal and vegetal bands. Kumano and Smith (2002) discuss evidence for dorsoventral patterning by differential responses to secreted nodal (transforming growth factor-beta) signals from vegetal cells and of secreted fibroblast growth factor (FGF) signals from animal cells. Indeed, when eggs fail at FGF signaling, due to the presence of introduced dominant negative FGF receptors, the embryos developed from them lack the notochord and somites, but contain head and heart mesoderm (Amaya and Kirschner, 1991). The involvement of the marginal zone mesoderm with mechanisms of anteroposterior patterning is revealed by an intriguing spectrum of phenotypes that have been produced by various simple chemical and physical treatments in Xenopus embryos in the past 20 years and more recently in zebrafish by various mutations. The naming of these phenotypes has always been a problem. Stanley Scharf and I, following on the work of Philip Grant and George Malacinski, thought of these as “ventralized” phenotypes (Scharf and Gerhart, 1980). All treatments block cortical rotation in the first cell cycle (ultraviolet irradiation of the vegetal pole, hydrostatic pressure, brief cold exposure, or brief nocodazole treatment). All interfere with microtubule formation. According to the current understanding, a thin layer of unidirectional parallel microtubules forms in the first cell cycle just interior to the egg cortex and materials are transported along the tubules from the vegetal pole to the marginal level on one side of the egg. The tubules disappear by the end of the first cell cycle. These transported materials stabilize beta-catenin locally, giving the egg a lasting asymmetry across its marginal (equatorial) level. When endoderm–mesoderm induction occurs later, that side of the marginal zone becomes the organizer. Without microtubules in the first cell cycle, the organizer fails to form in the late blastula. The fertilized egg remains cylindrically symmetrical and develops without one axis. Which one? We used to say the dorsoventral axis was impaired and that the early beta-catenin stabilizing asymmetry marked the prospective dorsoventral axis. Indeed, we had some justification, for without an organizer, the final embryos lacked the notochord and neural tube, two flagrantly dorsal structures. But they also lacked the heart and gill slits, two ventral structures. They also had an excess of lateral plate (coelomic) mesoderm, red blood cells, and ciliated epidermis (like belly epidermis), all ventral structures. Cooke and Smith (1987) reported that such embryos develop 10–20× more blood than do normal embryos. The short gut was identified as posterior gut. With the new axis designation, should we say they are posteriorized embryos and that the early asymmetry after cortical rotation reflects the prospective anteroposterior axis? Probably so. The ventralized embryos are limit forms of a continuous series of deviations from normal patterning, clearly affected in the anteroposterior axis. The intermediate forms have partial organizers, due to only partial cortical rotation, or partial beta-catenin stabilization, or partial removal of the organizer by surgery in the late blastula. As Stewart and Gerhart (1990) found, the amount of organizer in the late blastula is related to the eventual anteroposterior completeness of the body axis. With a slight loss of the organizer, the head is missing in the eventual tadpole; with more loss, the head and trunk are missing (only the tail present), and with total loss, no body axis is formed at all. The series of truncated intermediates fully fits the expectation that the anteroposterior axis, not the dorsoventral axis, has been set up by the early asymmetry, as Lane and Sheets (2002) suggest. In such axis-deficient forms, the newly assigned dorsoventral axis would still be formed across the animal–vegetal dimension of the marginal zone, and the evidence supports this. The ventralized appearance of the limit form may be misleading, according to the explanation of Kumano and Smith (2002). Somites do not develop from the somite territory unless induced by organizer signals. Results from in vitro experiments identify these as Bmp antagonists such as noggin, chordin, and xnr3. Any part of the 270- to 300-degree-wide territory can form anterior or posterior somites, depending on the timing and intensity of organizer signals. But if the territory does not make somites at all due to lack of Bmp antagonists, what does it make? It is not known. Does it lapse by default into ventral (vegetal) band mesoderm, eventually differentiating excess lateral plate and blood islands? If so, the mesoderm indeed became “ventralized” in the limit forms, but by a means uninformative for the naming of axes. The assignment of the anteroposterior axis by Lane and Sheets (2002) and by Kumano and Smith (2002) raises this issue. It is still unknown how the anteroposterior truncation series depends on the completeness of the organizer, as an aspect of the mechanism of axis formation. The other extreme of the spectrum of phenotypes is also interesting. Kao and Elinson (1988) produced these by exposing blastulae briefly to lithium ion, which leads to the ubiquitous stabilization of beta-catenin. Scharf et al. (1989) produced them by briefly exposing fertilized eggs to D2O, after which they form a precocious and random microtubule array, probably transporting vegetal materials (which stabilize beta-catenin) to all meridians of the equator (Rowning et al., 1997). These forms have expanded organizers. Indeed Kao and Elinson (1988) showed that organizer transplants could be made from opposite positions of the marginal zone of the dorsoanteriorized limit forms. Organizer-specific genes, which are expressed in the 60- to 90-degree-wide sector in a normal early gastrula, are expressed 360 degrees around the marginal zone of lithium treated embryos. After development, the limit form is cylindrically symmetric. It contains excess notochord (indicating the presence of dorsal mesoderm), a large central heart (indicating the presence of ventral mesoderm), circumferential eye pigmentation, and adhesive organ (indicating neural induction of the ectoderm). Their marginal zone probably has a normal dorsoventral dimension but only the anterior end of the anteroposterior axis, because the zone is composed entirely of organizer cells. They should probably be called “anteriorized” rather than “dorsalized” or “dorsoanteriorized” embryos. Intermediate forms lack tail and trunk while still developing the head, the reverse of the other truncation series. The mechanism by which anteriorization results from an oversized organizer is not known, except to say that uninduced tissues are absent. The analysis of the series would illuminate understanding of the development of the normal body axis that has proportionate anterior and posterior levels. In summary, the authors have made persuasive arguments, to my mind, for assigning prospective axes to the marginal zone mesoderm, in an orientation with the dorsoventral axis in the animal–vegetal dimension and the anteroposterior axis along the circumference (across the diameter) of the zone. As they note, amphibia will now look more like other chordates in their axis orientation at the late blastula/early gastrula stages, whereas they did not previously. Still, the reader should not get too carried away in expecting prospective axes to contain opposing materials at discrete poles. The anteroposterior axis may be more aptly described as a single pole set in a widespread nonpolar background, with the definitive axis to be worked out later during the inductions and morphogenesis of gastrulation and neurulation. Also the prospective axes may not have a Cartesian straightness and orthogonality. They may be bent, for example in the head mesoderm–notochord territories. “Axes” are an abstraction of the organization of the mesoderm, and it is best ultimately to know the locations of all the territories. Will other amphibia look like Xenopus, in these regards, including species with very large yolky eggs or with their prospective mesoderm on the surface rather than inside? I do not know, but I suspect that the new axes will offer a more incisive perspective on their organization than did the old.