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Phosphatized Polar Lobe-Forming Embryos from the Precambrian of Southwest China

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Science  16 Jun 2006:
Vol. 312, Issue 5780, pp. 1644-1646
DOI: 10.1126/science.1125964

Abstract

In developing embryos of some extant spiralian animals, polar lobe formation is one of the symmetry-breaking mechanisms for segregation of maternal cytoplasmic substances to certain blastomeres and not others. Polar lobe formation leads to unique early cleavage morphologies that include trilobed, J-shaped, and five-lobed structures. Fossil embryos similar to modern lobeforming embryos are recognized from the Precambrian Doushantuo Formation phosphates, Weng'an, Guizhou Province, China. These embryos are abundant and form a developmental sequence comparable to different developing stages observed in lobe-forming embryos of extant spiralians. These data imply that lobe formation is an evolutionarily ancient process of embryonic specification.

The Weng'an fossil fauna from Precambrian Doushantuo phosphates in Weng'an, Guizhou Province, China contains likely fossil representatives of the oldest known metazoans (1, 2). The fossil-bearing interval of the Doushantuo has been dated as at least 580 million years old (3, 4). Previous studies have presented fossil evidence of sponges (5), cnidarians (6, 7), and possible bilaterians (810) in this formation. In addition, a large number of embryos (1113) display a variety of developmental patterns and different morphological types, implying that metazoans may have been diverse 40 million years before the Cambrian (1). The affinities of these embryos, however, remain enigmatic. We present fossil evidence of developing embryos that resemble the different developmental stages of lobe-forming embryos, which in modern fauna are found in spiralians including many extant mollusks and some annelids (14, 15).

We studied material from the gray facies of the Weng'an Phosphate Member of the upper Doushantuo Formation at Wusi, Baisaikang, and Nanbao quarries, Weng'an county, Guizhou (1). Rock samples were treated with a 10% acetic acid solution for removal of specimens from the matrix. Fossil embryos were recovered from the acid residues, coated with gold, and observed and photographed with a Hitachi S2600N scanning electron microscope (SEM); we identified 248 embryos with morphological characters like those of lobe-forming embryos common in many modern mollusks (1618). A few selected samples were examined by synchrotron radiation microtomography (SR-μCT) at both the European Synchrotron Radiation Facility (ESRF) and the Taiwan Synchrotron Radiation Research Center (NSRRC).

Lobe formation is a sequential process of dynamic change, which occurs by the protrusion and then absorption of a cytoplasmic lobe called a polar lobe. The lobe protrudes from the vegetal pole of the embryo at each round of cytokinesis, leading to the formation of dumbbell, three-fold (trefoil), J-shaped, and five-lobed morphologies (Fig. 1). The polar lobe (PL) superficially resembles a blastomere, but the lobe is anucleate and is connected to the CD or D blastomere by a deep constricted neck. Fossil PLs can be recognized by their size and connecting necks and by the complementary relation between their size and that of the CD or D blastomere from which they arose.

Fig. 1.

Schematic diagrams showing changes in shape of the fertilized egg before cleavage and embryos during cleavage of the PL-forming species in some extant mollusks and annelids. (A) Egg with the first lobe; (B) trefoil stage; (C) two-cell stage; (D) J-shaped embryo; (E) five-lobed embryo.

In the modern mud snail (Nassarius obsoletus) the first PL is protruded soon after fertilization, leading to the formation of a calabash-shaped structure: a large ball attached to a small one (16). Similar structures occur in many of our samples from the Weng'an. In N. obsoletus, the fertilized eggs at the first-lobe stage bear a superficial resemblance to unequally cleaving two-cell embryos, but they differ in that the embryo consists of a rather large uncleaved fertilized egg with a smaller anucleate lobe. After first cleavage, the larger blastomere (CD cell) in the lobe-forming two-cell embryos protrudes a lobe from its vegetal pole, forming a trilobed structure as seen in modern mollusks (termed a trefoil stage). A total of 94 specimens with similar structures are recognized in the Weng'an fossil assemblage. A large number of them are encased partially or entirely within an envelope, rendering unlikely the possibility that the interpreted PLs are merely adventitious extrusions of cytoplasm due to damage (19). The diameter of the Weng'an eggs ranges from 0.2 to 1.2 mm. The inferred fossil embryos show variable arrangements with the blastomere and PL in loose contact in many specimens and in tight contact in other specimens.

On the basis of the nature of cleavage, two different groups of embryos are recognized: equal division (Fig. 2, A to K) and unequal division (Fig. 2, L to V). The CD cell (plus PL) in equal-division embryos is the same size or slightly larger than the AB cell, whereas in unequal-division embryos it is typically twice as large as the AB cell (Table 1 and Fig. 3). This implies the existence of at least two different groups of organisms within which there may have been more than one species.

Fig. 2.

Lobe-forming embryos from the Precambrian Doushantuo Formation. (A to K) Lobe-forming embryos with equal division both in trefoil stage [(A) to (H)] and J-shaped stage [(I) to (K)]. (L to V) Lobe-forming embryos with unequal division in trefoil stage [(L) to (U)] and J-shaped stage (V). (W to Z) Five-lobed embryos. Panels (A′), (B′), and (L′) are dissecting images of (A), (B), and (L), respectively, showing preservation of a connected neck (black arrow); (B″) is a magnified image of (B′). Abbreviations: MT, biogenetic microtunnel; FF, diagenetic fissure filling. Images are SEM [(C) to (K), (M) to (Z)] or SR-μCT [(A), (B), and (L)]. Scale bar, 250 μm.

Fig. 3.

Values of the ratios PL/AB (y axis) plotted against values of PL/(PL + CD) (x axis) from the measurements in Table 1. The data were fit by linear least squares, with the result that the upper plot of points (open squares) from unequal-division embryos fits 1.9706 (±0.0046), with a correlation coefficient of 0.9173; the lower plot of points (solid squares) from equal-division embryos fits 1.0472 (±0.0013), with a correlation coefficient of 0.933.

Table 1.

Measurements of the volume of AB cells, CD cells, and PLs of the embryos illustrated in Fig. 2, with relative volumes of different combinations of these components.

SampleAB cell (mm3)CD cell (mm3)PL (mm3)CD + PL/ABPL/CD + PL (%)PL/AB (%)CD/AB (%)
Embryos with equal division
1 (View inline) 0.035 0.033 0.006 1.1 15% 17% 94%
2 (View inline) 0.018 0.014 0.004 1.0 22% 22% 78%
3 (View inline) 0.064 0.050 0.020 1.1 29% 31% 78%
4 (View inline) 0.050 0.035 0.015 1.0 30% 30% 70%
5 (View inline) 0.006 0.004 0.002 1.0 33% 33% 67%
6 (View inline) 0.022 0.016 0.010 1.2 38% 45% 73%
7 (View inline) 0.006 0.004 0.003 1.1 39% 42% 65%
8 (View inline) 0.033 0.020 0.014 1.0 41% 42% 60%
9 (View inline) 0.082 0.042 0.036 1.0 46% 43% 51%
10 (View inline) 0.050 0.025 0.025 1.0 50% 50% 50%
11 (View inline) 0.012 0.007 0.007 1.2 50% 58% 58%
Embryos with unequal division
12 (View inline) 0.020 0.030 0.008 1.9 21% 40% 150%
13 (View inline) 0.008 0.011 0.004 1.9 27% 50% 138%
14 (View inline) 0.028 0.034 0.016 1.8 32% 57% 121%
15 (View inline) 0.004 0.006 0.003 2.2 32% 70% 150%
16 (View inline) 0.019 0.027 0.014 2.2 34% 74% 142%
17 (View inline) 0.022 0.028 0.017 2.0 38% 77% 127%
18 (View inline) 0.012 0.015 0.011 2.2 42% 92% 125%
19 (View inline) 0.026 0.027 0.021 1.8 44% 81% 104%
20 (View inline) 0.030 0.030 0.026 1.9 46% 87% 100%
21(View inline) 0.067 0.064 0.063 1.9 48% 93% 95%
22 (View inline) 0.024 0.025 0.023 2.0 48% 96% 104%

The three lobes are noticeably different in size in these specimens. SR-μCT examination of the trefoil fossils displaying equal (Fig. 2, A and B) and unequal division (Fig. 2L) suggests that these three lobes are homologous with the CD cell, AB cell, and PL of trefoil embryos, respectively. Among the three lobes in the equal-division embryos (Fig. 2, A and B), the second largest one is interpreted as the CD cell because it is separated completely from the likely AB cell (the largest lobe) by a thin membrane but is connected to the likely PL (smallest lobe) by a narrow cytoplasmic neck (Fig. 2, A′, B′, and B″). In Fig. 2A, the PL, AB cell, and CD cell volumes are 0.01 mm3, 0.022 mm3, and 0.016 mm3, respectively; in Fig. 2B these volumes are 0.036 mm3, 0.082 mm3, and 0.042 mm3, respectively (Table 1).

The relative sizes of the PL and CD cells differ among the embryos, but measurements from the specimens illustrated in Fig. 2 and listed in Table 1 reveal a complementary relationship between the volumes of the PL and CD cells (Fig. 3). In different specimens of a given species of PL embryo at the trefoil stage, the volume of the lobe plus the CD cell (PL + CD) should bear a constant relation to the volume of the AB cell, whether the lobe is just beginning to be protruded, is fully extended, or is in the process of retraction. For the analysis in Fig. 3, we normalized the size data for the different embryos by plotting the volumetric ratios PL/AB versus PL/(PL + CD) for each embryo.

The results show that the sum (PL + CD) has a constant relationship to the size of AB. This is expected because in a lobe-forming embryo, the AB blastomere is not affected by PL extrusion. There are two populations of fossil embryos in our study: those in which PL/AB = PL/(CD + PL), and those in which PL/AB = 2PL/(CD + PL)—that is, the equal and unequal embryo types referred to above. The high correlation coefficients for the two curves demonstrate that each is a statistically robust relation that could not have resulted from the random aggregation of three spheroidal bodies developmentally unrelated in origin. The complementary relation of the PL with the CD volumes directly supports the homology of the three lobes in these fossils with the PL, the CD cell, and the AB cell, respectively, of modern trefoil embryos. The constant relationship between PL + CD and AB volumes implies that the PL volume is a dynamic variable, as required for a cytoplasmic lobe undergoing active protrusion and resorption.

As first cleavage is completed in modern lobe-forming embryos, the neck of the PL rapidly increases in diameter, leading to the formation of a J-shaped embryo until the CD cell finally absorbs the PL. About 39 J-shaped lobe-forming embryos can be identified on the basis of fossil shape. These are represented mainly by two different groups related to equal (Fig. 2, I to K) and unequal trefoil embryos (Fig. 2V), respectively. The smallest among the three parts of each embryo typically represents the PL. It can also be recognized by the presence of the PL constriction (PLC), which is relatively deeper than the cleavage furrow (CF) (Fig. 2I). The fully protruded lobe (Fig. 2K) is nearly equal in size to the CD cell.

During second cleavage of modern lobe-forming embryos, the last phase of lobe protrusion leads to the formation of a five-lobed morphology, which comprises the first four blastomeres of equal or subequal size and a PL protruding from the D cell. About 17 embryos are recognized to be fossil representatives of such five-lobed embryos (Fig. 2, W to Z). The lobes in these embryos are mostly uncompacted and nearly equal, with the smallest one corresponding possibly to the PL.

Lobe formation and retraction constitutes a symmetry-breaking device for the segregation of polar material to only one blastomere, an alternative to asymmetric cleavage. This mechanism is used today in disparate groups of mollusks including polyplacophorans, gastropods, scaphopods, and bivalves (14, 2022), as well as a few types of annelids (Chaetoplerus) (14). The PL appears also in polyclad turbellarian flatworms (Hoploplana inquilina)(23, 24), which are widely accepted as a basal group of spiralians. In spiralian embryogenesis, the constituents of PL are required for specification of mesodermal structures and secondarily of other embryonic parts that are induced by mesoderm (15). That is, the blastomeres that receive the PL cytoplasm become mesoderm founder cells.

These fossil embryos may not be spiralians or spiralian ancestors, and indeed none of the later cleavage forms that have been recovered in the same deposits convincingly display the characteristic patterns of spiral cleavage (1, 8, 11). The most widespread and basal mode of embryogenesis in bilaterians operates by means of cleavage stage specification of blastomere fate, followed immediately by establishment of differential gene expression in the cell lineages descendant from these blastomeres (25, 26). The diverse territories of the embryo then soon express specific gene batteries generating a mosaic of differentiated cell types. This general mechanism is used in both direct and indirect developing bilaterian forms. PL formation is only one of a great variety of mechanisms whose essential function is the asymmetric delivery of maternal components of regulatory importance to specific blastomeres (25, 26). In the case of the lobe-forming embryos studied here, the second cleavage lobe indicates that these embryos will specify one particular blastomere out of four that will be distinct in subsequent regulatory states. Only bilaterian embryos proceed in such a manner, but this is a typical bilaterian strategy of early development. Thus, these fossils imply that lobe formation is an ancient evolutionary device, and that the general strategy of precocious blastomere specification still used in most bilaterian groups was extant at least 40 million years before the Cambrian.

Supporting Online Material

www.sciencemag.org/cgi/content/full/312/5780/1644/DC1

Fig. S1

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