BioEssays Vol. 1, No. 1 5

Cell Lineage Labels in the Early Amphibian Embryo Jonathan M. W. Slack

Summary

New methods of marking cells enable single clones to be followed during embryonic development. They can be usedfor the construction of fate maps and for the investigation of induction and determination.

Introduction

Experimental embryology depends to a large extent on the ability to identify the origins of cells or tissue regions both in normal development and in certain abnormal situations. For example, if a region which normally develops into structure A is transplanted to a new site in the embryo it may appear to form structure B instead. This might mean that the developmental pathway of the cells had been altered. On the other hand, the appearance might be deceptive -perhaps the graft cells have died or migrated away from the new site and structure B was actually formed by host cells already committed to this fate. This sort of uncertainty means that such experiments can only be performed satisfactorily if there is some method of distinguishing graft-derived from host- derived cells, which means that they must be marked with a label which is retained regardless of the developmental pathway followed.

Marking methods used in the past have included vital dyes, small carbon particles or the use of two related species whose cells differ in size or pigmentation. All of these have severe disadvantages which prevent discrimination at the level of individual cells. However, in the last few years this problem has been solved in a remarkably simple way by the introduction of a number of passive cell labels which are easy to administer, can be detected with great sensitivity, and allow single cell resolution. Passive cell labels become diluted out by growth and so find most application in develop- mental situations in which growth does not occur. For example, amphibian and other free living animal embryos show no net growth until the larva hatches

and begins to feed. Although there is plenty of cell division during embryonic development the cells become progres- sively smaller during this period. For mammalian and avian embryos which undergo extensive growth in early development, passive labels are much less satisfactory and genetic markers are to be preferred.

The New Labels

The first of these was the enzyme horse- radish peroxidase, which was intro- duced by Weisblat, Sawyer and Stentl for the study of cell lineage in the leech. This is an enzyme, molecular weight 40,000, and is therefore too large to pass from cell to cell through gap junctions. It is reasonably non-toxic and is not degraded for several days after injection into fertilized eggs or individual blasto- meres. It is detected in frozen sections by one of a variety of histochemical tests for peroxidase. In amphibian embryos endogenous peroxidase is absent until quite a late developmental stage and so does not confuse the results.

A label which is used in a similar way, by microinjection into the cells of interest, is dextran conjugated to fluorescein and lysine (FLDX).~ The dextran is of high molecular weight and unable to pass from cell to cell; the lysine enables fixation by aldehydes and the fluorescein enables visualization of the labelled cells in sections by fluores- cence microscopy. FLDx has certain advantages over HRP. It is less toxic, it can be visualized in paraffin sections which are easier to deal with than frozen sections, and because it is a fluorescent label it does not obscure the cytology of the labelled cells as tends to happen with HRP.

A rather different type of label is [1251]Bolton Hunter reagent (BHR).a This is a reagent used for protein iodin- ation which reacts with primary amino groups. It is lipophilic and so penetrates cells rapidly. The labelling reaction is irreversible and, because of the insta- bility of the reagent in aqueous solution, of short duration. The macromolecular

components of the tissue which are labelled are retained during fixation and histological processing and are visua- lized by autoradiography. BHR is suit- able for labelling isolated cells or ex- posed cell sheets; because of its short chemical half life it will not penetrate very far into a volume of tissue.

Lastly there is the fluorescent dye tetramethyl rhodamine isothiocyanate (TRITC).4 Normally, fluorescent dyes of low molecular weight are not retained by individual cells, indeed they are often used to investigate intercellular coupling. However, if isolated amphi- bian blastomeres are exposed to TRITC the dye becomes strongly absorbed by the yolk granules and other protein con- stituents and while these are retained the label persists and does not spread to adjacent cells. Like FLDx, TRITC labelling can be detected in paraffin sections by fluorescence microscopy.

This article deals with the application of these labels to the early amphibian embryo. Two developmental stages that will be referred to, the early gastrula and tailbud stage, are shown in Fig. 1. However, the techniques are equally applicable to any non-growing system and recent papers have described blasto- mere injections in the leech? starfishs and ascidian' embryos as well.

Fate Mapping and Clonal Analysis

A fate map reveals what will become of an embryo region during the course of normal development. It thus conveys information about where cells move to, and into what structures they differentiate.8 If an extended region of tissue is marked the fate map gives no information about the state of determin- ation at the time of labelling, but if a single cell is marked it can do this too. This is because a cell whose progeny contribute to structures A and B cannot itself have been determined with regard to this decision at the time of labelling. So the latest time at which a single cell can populate both A and B is a terminus post quem, or lower bound, for the

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Animal pole

Vent margi

zon

Vegetal pole

Fig. 1. Embryos of a urodele amphibian. (a) early gastrula stage, (b) tailbud stage.

estimated time of developmental commitment .

An extensive investigation of fates of single cells has been carried out by Jacobson and Hiro~eO-~~ on Xenopus laevis using horseradish peroxidase. At the 2-1~11 stage a single labelled blasto- mere labels a meridional half of the larva, confirming that the first cleavage separates future left and right halves, the only crossover being of cells contri- buting to the ventral third of the retina. The later studies concentrated on the central nervous system. At the 4-cell stage all cells contribute to the CNS, at

the 16-cell stage 10 blastomeres do so, and at the 64-cell stage, 38 contribute to the CNS and surrounding structures. The lack of clonal restriction at these stages shows that developmental com- mitment to principal body regions has not yet occurred.

The most surprising result from these early studies was that there was quite a lot of mixing of labelled with unlabelled cells at the border of each clone (see Fig. 2). The fate map for an amphibian is therefore not quite precise because a given clone will show a different distribution in different individual em- bryos. For example, the Mauthner cell, which is a giant neuron arising in the rhombencephalon, is sometimes labelled by injecting blastomere D222 of the 32-cell embryo and sometimes not. By the 512-cell stage there appears to be some restriction of clones to each of seven regions within the CNS. This means that the descendants of a blasto- mere appear to mingle freely within a region but not to cross its boundary. Whether this is a real restriction or arises because of the small size of clones marked at this stage is not really clear. If it is real then it means that the first regional specifications within the pros- pective nervous system arise before neural induction occurs, which would be a surprise to most embryologists.

Since clones spread out and mix with unlabelled cells it is not possible to mark a coherent region at later stages by injection of an early blastomere. To do

this an alternative procedure was intro- duced by Smith and Slack13 in which a fertilized egg was injected with HRP to create a uniformly labelled embryo. Then at the appropriate stage, early gastrula in this case, grafts were per- formed from labelled donors to un- labelled hosts in such a way that there was an exact replacement of tissue with its labelled equivalent (an orthotopic graft). An example is shown in Fig. 3. Using this method the fate maps of dorsal and ventral marginal zones were deduced and used to establish the nature of the inductions which occurred when dorsal and ventral tissues were juxta- posed (heterotopic grafts), as described below.

A rather different sort of fate mapping experiment was conducted by Smith and Ma1a~inski.l~ They treated early gastrulae of Xenopus and axolotl with [1251]B~lt~n Hunter reagent under con- ditions in which only the outer cell layer became labelled. They were then able to find which regions of the late embryo derived from the gastrula surface. In Xenopus it is the outer epidermis and the luminal surfaces of archenteron and neural tube, as expected from the orthodox understanding of gastrulation. In the axolotl, however, there was quite a pepper-and-salt mixture, showing that there must be some interchange of surface and deep cells, a result which suggests that passive inheritance of the original egg membrane does not guaran- tee an external position at later stages.

Fig. 2. Part of the gut of a Xenopus larva which received an injection of FLDx into a vegetal blastomere at the 44-cell stage. (a) Phase contrast, (b) DAPI fluorescence shows cell nuclei, (c) fluorescein$uorescence shows cells derived from the injected blastomere. ( x 264.)

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Studies on Induction

The availability of the passive cell labels makes possible much more dis- criminating studies on induction than have been possible before. Gimlich and Gerhart2 have performed an experiment showing clearly that the first signalling centre is made up of blastomeres from the dorsal side of the vegetal cell octet at the 64-cell stage (DV cells). It is known that fertilized eggs irradiated with ultraviolet light before first cleavage are unable to form axial struct~res.~5,16 Gimlich and Gerhart have shown that this ability can be restored by trans- planting one to three DV cells. Further- more, by labelling the transplanted cells with FLDx they could show that their progeny do not themselves enter the axial structures but remain in the yolk mass at later stages. The rescue effect must therefore be due to some inductive signal emitted by the DV cells.

The existence of a mesodermal in- ducing signal from the vegetal pole region was already known on the basis of experiments in which the marginal zone (prospective mesoderm) of axolotl blastulae was removed and the animal and vegetal regions joined together." There was, however, some uncertainty about whether the animal pole region normally contributed cells to the meso- derm and if so whether the induction was actually a permissive rather than an instructive effect. Recently, Slack, Dale and Smith'* have shown with FLDx labelled grafts that mesodermal induc- tion is a reality. The animal pole region does not normally contribute many cells to the mesoderm but does form large masses of mesodermal tissues when cultured in combination with vegetal tissue.

Smith and Slack13 had previously carried out a series of grafts at the stage of early gastrula in which tissue from dorsal and ventral marginal zone was interchanged. The dorsal to ventral graft is the famous organizer graft of Spemann and Mang01d.l~ It yields double dorsal embryos in which the host axis is known as the primary axis and the axis associated with the graft as the secondary axis. The question was: which parts of the secondary axis are derived from the graft and which from the host? The answer with HRP-labelled grafts is very clear: the notochord is graft-derived and the somites as well as the neural tube are host-derived. Smith and Slack argued that two inductions are manifested in this experiment: a dorsalization which causes the host ventral mesoderm to form somites, and

a neuralization which causes the host ectoderm to form neural tube. Dorsali- zation is also apparent in the graft of ventral to dorsal marginal zone. But here it is the graft whose fate is changed, so it seems that whichever way dorsal and ventral tissues are juxtaposed the dorsal tissue retains its commitment but the ventral tissue becomes dorsalized.

Gimlich and CookeZo have confirmed the reality of neural induction by the use of ' lineage-labelled ' hosts. They injected 32-cell embryos with FLDx in two cells which they could show never normally contribute to the neural tube but did contribute to ventral epidermis. Then an organizer graft was performed at early gastrula stage and the neural tubes of the resulting secondary embryos were shown to be heavily labelled.

Three inductive interactions can therefore be distinguished in early amphibian development : mesoderm in- duction in the blastula, dorsalization in the early gastrula and neural induc- tion in the late gastrula.

Determination of Single Cells The various types of developmental commitment are often assumed to be properties of single cells rather than tissues, not depending on any particular extracellular components or three- dimensional cellular arrangement. How- ever, the principal evidence for this has until recently depended on results from a single system : the inner cell mass of the mouse blastocyst whose cells become determined to form primitive ectoderm or endoderm and retain this determina- tion after injection into another blasto- cyst.21 Studies on other systems have often suggested that tissue subjected to disaggregation and reaggregation behaves as though its commitment is more labile than intact

The passive cell labels have now made it possible to investigate this question in the relatively well understood amphibian embryo. In a recent paper by Heasman et u I . ~ the vegetal pole cells from Xenopus were isolated, labelled by exposure to TRITC, and inserted into the blastocoel of control 2mbryos. Progeny from the injected cell were found to populate derivatives of all three germ layers at early and mid- blastula stage, but by late gastrula they were restricted to the endoderm. The same cells were also fate mapped by injection of HRP and the normal fate was shown to be restricted to endoderm by mid-blastula stage.

This experiment supports the idea that determination is indeed a single cell

23

Fig. 3. Transverse section through a Xenopus tailbudstage which received agraft of HRP labelled animal pole tissue cw' an early gastrula. The section is stained for peroxidase and activity is present only in the epidermis. ( x 65.)

property but suggests also that certain functions can be expressed before commitment at the single cell level has become irreversible. For example, as described above, mesodermal induction is a property of the vegetal pole tissue at early stages.

The Future

The power of the passive cell labels is demonstrated by the fact that the principal conclusions of experimental embryology with regard to fate maps and induction, originally obtained over 50 years, have been confirmed in only a few months of work. In addition, quite new types of experiment have become possible concerning determination in single cells, and clonal domains visual- ized by restriction of cell movement. It seems likely that in the next few years our knowledge of early amphibian development will be greatly deepened by these techniques. However, the ability to follow the Iineuge of early embryo cells must highlight our inability to determine the intrinsic character of these cells except by rather indirect arguments. We glibly speak of 'mesoderm' and non- embryologists sometimes are fooled into thinking we know what we mean. In reality there is no way of identifying a cell as mesodermal before terminal differentiation takes place, and it may be that there is actually no such thing. The

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real distinctions between cell types in the early embryo may cut across the names we give them and we will not be able to understand early development until we have found out what the real cell types are and what they do.

REFERENCES 1 WEISBLAT, D. A., SAWYER, R. T. & STENT, G. S. (1978). Cell lineage analysis by intracellular injection of an enzyme. Science, Wash. 202, 1295-1298. 2 GIMLICH, R. L. & GERHART, J. C. (1984). Axis formation in Xenopus laevis. Deol Biol. (In the Press.) 3 KATZ, M. J., LASEK, R. J., OSDOBY, P., WHITTAKER, J. R. & CAPLAN, A. I. (1982). Bolton-Hunter reagent as a vital stain for developing systems. Devl Biol. 90, 419429. 4 HEASMAN, J., WYLIE, C. C., HAUSEN, P. & SMITH, J. C. (1984). Fates and states of determination of single vegetal pole blasto- meres of Xenopus laevis. Cell. (In the Press.) 5 STENT, G. S., WEISBLAT, D. A., BLAIR, S. S. & ZACKSON, S. L. (1982). Cell lineage in the development of the leech nervous system. In Neuronal Development (ed. N. C. Spitzer), pp. 1-44. Plenum: New York. 6 KOMINAMI, T. (1983). Establishment of embryonic axes in larvae of the starfish, Asterinapectinifera. J. Embryol. exp. Morph.

7 NISHIDA, H. & SATOH, N. (1983). Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. I. Up to the eight-cell stage. Devl Biol. 99,

75, 87-100.

382-394.

8 SLACK, J. M. W. (1983). From Egg to Embryo: Determinative Events in Early Development. Cambridge University Press, Cambridge. 9 JACOBSON, M. & HIROSE, G. (1978). Origin of the retina from both sides of the embryonic brain: a contribution to the problem of crossing at the optic chiasma. Science, Wash. 202, 637-639. 10 HIROSE, G. & JACOBSON, M. (1979). Clonal organization of the central nervous system of the frog. Devl Biol. 71, 191-202. 11 JACOBSON, M. & HIROSE, G. (1981). Clonal organization of the central nervous system of the frog. 11. Clones stemming from individual blastomeres of the 32- and 64-cell stages. J. Neurosci. 1, 271-284. 1 2 JACOBSON, M. (1983). Clonal organiza- tion of the central nervous system of the frog. 111. Clones stemming from individual blastomeres of the 128-, 256-, and 512-cell stages. J . Neurosci. 3, 1019-1038. 1 3 SMITH, J. C. & SLACK, J. M. W. (1983). Dorsalization and neural induction : proper- ties of the organizer in Xenopus laevis. J . Embryol. exp. Morph. 78, 299-317. 1 4 SMITH, J. C. & MALACINSKI, G. M. (1983). The origin of the mesoderm in an anuran, Xenopus laevis, and a urodele, Ambystoma mexicanum. Devl Biol. 98, 250-254. 1 5 MALACINSKI, G. M., BENFORD, H. 8t CHUNG, H. M. (1975). Association of an UV-irradiation-sensitive cytoplasmic local- ization with the future dorsal side of the amphibian egg. J. exp. Zool. 191, 97-1 10. 1 6 SCHARF, S. R. & GERHART, J. C. (1980). Determination of the dorso-ventral axis in eggs of Xenopus Iuevis: complete rescue of UV-impaired eggs by oblique orientation

before first cleavage. Devl Biol. 79, 181-198. 1 7 NIEUWKOOP, P. D. (1973). The 'organi- zation centre' of the amphibian embryo, its origin, spatial organization, and morpho- genetic action. Adu. Morphogen. 10, 1-39. 1 8 SLACK, J. M. W., DALE, L. & SMITH, J . C. (1984). Analysis ofembryonicinduction using cell lineage markers. Phil. Trans. R. SOC. B (In the Press.) 1 9 SPEMANN, H. & MANGOLD, H. (1924). Uber Induktion von embryonenanlagen durch Implantation artfremder Organisa- toren. Arch. microsk. Anat. EntwMech. 100,

20 GIMLICH, R. L. & Coon, J. (1983). Cell lineage and the induction of second nervous systems in amphibian development. Nature, Lond. 306,471473. 21 GARDNER, R. L. & ROSSANT, J. (1979). Investigation of the fate of 4.5-day post coitum mouse inner cell mass cells by blastocyst injection. J. Embryol. exp. Morph.

22 FORMAN, D. & SLACK, J. M. W. (1980). Determination and cellular commitment in the embryonic amphibian mesoderm. Nature, Lond. 286, 492494. 23 KIENY, M., PAUTOU, M. P. & CHEVAL- LIER, A. (1981). On the stability of the myogenic cell line in avian limb bud develop- ment. Archs Anat. microsc. Morph. exp. 70,

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JONATHAN M . W . SLACK is with the Imperial Cancer Research Fund, London.

Uteroferrin: A Protein in Search of a Function R. Michael Roberts and Fuller W. Bazer

Summary

Uteroferrin, a purple-colored, iron-con- taining acid phosphatase, with many of the properties of a lysosomal hydrolase, transports iron from the mother to the conceptus in pregnant pigs. Uteroferrin, however, is but one member of what may be a broad class of iron-containing phosphatases with unusual spectral prop- erties which result from a novel type of di-iron active site. The biologicalfunction

Introduction

Murray et al. demonstrated the presence of a basic glycoprotein with a purple coloration in the uterine secretions of pigs in 1972. This protein is now generally known as uteroferrin. Its production requires progesterone, and large quantities can be isolated from pigs of high progesterone status. In 1974, Schlosnagle et al. showed that uteroferrin had acid phosphatase activity

with a basic PI and an acid pH optimum. More recently, it has been recognized that several other acid phosphatases from a diverse array of sources share many of the characteristics of uteroferrin and the spleen enzyme, suggesting that a broad class of iron- containing acid phosphatases might exist.3 Here we describe the properties of these enzymes and speculate about their function.

of uteroferrin is unknown. We argue here and contained iron.' In both these that the in vivo function of uteroferrin, respects it resembled a phosphoprotein and Pink Uteroferrin

- .

despite its undoubted ability to act as a potent acid phosphatase, is that of a transplacental iron transporter.

phosphatase which h a d been described in extracts of beef s ~ l e e n . ~ Like utero- ferrin, the spleen enzyme is also purple,

Uteroferrin and the beef spleen enzyme have a purple color which originates from a broad absorption band centered