The Embryology of Tubularia larynx (Allm.)By

Esther Lowe, M.Sc,

A s s i s t a n t L e c t u r e r i n Z o o l o g y , U n i v e r s i t y o f S h e f f i e l d .

W i t h P l a t e s 3 1 a n d 3 2 a n d 4 T e x t - f i g u r e s .

C O N T E N T S .

1 . I N T R O D U C T I O N . . . . . . . . . 5 9 9

2 . O B I G I N O F T H E G E R M - C E L L S . . . . . . . 6 0 0

3 . G R O W T H O F T H E O O C Y T E . . . . . . . 6 0 4

4 . M A T U R A T I O N . . . . . . . . . 6 0 S

5 . C L E A V A G E A N D F O R M A T I O N O F T H E B L A S T U L A . . . 6 0 8

6 . F O R M A T I O N O F T H E P L A N U L A . . . . . . 6 1 3

E c t o d e r m 6 1 3E n d o d e r m . 6 1 3

7 . D I F F E R E N T I A T I O N W I T H I N T H E E C T O D E R M . . . . 6 1 4

8 . F O R M A T I O N O P T H E A B O R A L T E N T A C L E S . . . . . . 6 1 5

9 . F O R M A T I O N O F T H E A C T I N U L A . . . . . . 6 1 6

1 0 . S U M M A R Y 6 2 4

1 1 . L I T E R A T U R E . . . . . . . . . 6 2 5

1 2 . E X P L A N A T I O N O F P I . A T H S . . . . . . . 6 2 5

1. INTRODUCTION.

THE early development of T u b u l a r i a m e s e m b r y a n -t h e m u m is well known from the work of Brauer (3, 1891).The development of the American species, T. c r o c e a , up tothe stage of the germ-layer formation has been worked out byAllen (1,1900) and Hargitt (9, 1909). The bare outline of thedevelopment of the British species, T. l a r y n x is also knownfrom the work of Alhnan (2, 1871) ; and the cytology of theearly stages of this species and of T. be l l i s has been workedout by Perez (12, 1912). There is not, however, as far as canbe determined, a complete account of the development of anyBritish species.

The present investigation of the embryology of T. l a r y n x

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was undertaken at the suggestion of Mrs. Bisbee, M.Sc. (Euth C.Bamber), Lecturer in Zoology at the University of Liverpool.My thanks are due to her for much valuable advice and helpfulcriticism throughout the course of the work, as well as for theuse of an excellent set of sections prepared by Mr. H. 0. Chad-wick, late Curator of the Marine Biological Station at PortErin. The work was carried out in the Zoological Departmentof the University of Liverpool during the period October 1923to June 1924.

The slides made by Mr. Chadwick were prepared from materialcollected at Port Erin. My own material was collected at PortErin and at Hilbre Island. The investigations have been carriedout with the aid of stained serial sections of the gonophores, andof. whole mounts of embryos obtained by splitting open thegonophores under water. The material prepared by Mr. Chad-wick was fixed in Bouin's fluid ; some of the sections werestained with Ehrlich's haematoxylin, others with Brazilin.Some of my own sections have been made from material fixedin Bouin's fluid and stained with Ehrlieh's haematoxylin,others from material fixed in Plemming's chromo osmic mixture(without acetic) and stained with Haidenhein's iron haema-toxylin.

2. ORIGIN OF THE GERM-CELLS.

The question of the place of origin of the primitive germ-cellsis one which has given rise to much discussion among workerson the Tubulariidae. It is well known that in many of theHydromedusae the germ-cells arise in the tissues of the hydroiclgeneration, either in the ectoderm or endoderm and latermigrate to their final position in the gonophores, where theyattain maturity. Weismann (15, 1883), who was responsiblefor the discovery of this phenomenon in many species, did notobserve any such process in T u b u l a r i a , and several morerecent workers have failed to find any trace of migration.Thallwitz (13) believed that the primitive germ-cells arose inthe ectoderm of the spadix. Tichomiroff (14, 1887) finds themin the endoderm of the distal part of the gonophore bud.

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Gotte (6, 1907), working on T. i n e s e m b r y a n t h e m u m ,agrees with Thallwitz in believing that they originate in theirfinal position in the ectoderm of the spadix ; and more recentlyPerez (12, 1912), working with T. be l l i s , also agrees withGotte and Thallwitz. Brauer (3, 1891), working with T.m e s e m b r y a n t h e m u m , discovered certain small roundedamoeboid cells which originated in the ectoderm of the gono-phore stalk, and believed them to be the sexual elements.Jickeli (10, 1883) also held this view. Brauer finds these cellsmigrating from the ectoderm through the mesogloea into theendoderm and from the endoderm into the ectoderm of thespadix. He gives figures showing the germ-cells in the ecto-derm and in the endoderm, and one figure showing germ-cellsin the mesogloea.

After a careful examination of many sections of the develop-ing gonophores of T. l a r y n x , I have little hesitation inaccepting Brauer's view with regard to the actual origin of thegerm-cells ; but the stages in migration which have been seenare not in accordance with his account for T. mesem-b r y a n t h e m u m .

The primordial germ-cells in T. l a r y n x appear to originatein the ectoderm at the base of the young gonophore bud (fig. 3,gc1, PI. 31). They arise in this position by the gradual modifica-tion of certain of the interstitial cells. This modification beginswhen the gonophore bud is nothing more than a small hollowoutgrowth from the blastostyle. The ectoderm of this gono-phore bud is one cell thick at the distal end ; but at theproximal end, which is destined to become the gonophorestalk, the ectoderm is several cells thick, due to the rapidproliferation of the small unmodified interstitial cells whichlie between the bases of the columnar cells in this position.1

1 In some cases this proliferation of interstitial cells seems to take placeeven earlier, in the ectoderm of the blastostyle itself, at a point where agonophore bud is about to form. This, however, is not certain, for nemato-oysts are being formed from the interstitial cells in considerable numbers inthis position, and therefore it is difficult to decide whether the proliferationseen at this stage is nematocyst formation or germ-cell formation.

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The gradual modification of these interstitial cells into germ-cells can be traced. Those which are destined to becomegerm-cells grow slightly larger than the other cells of theectoderm, and their cytoplasm becomes dense and granular.In sections stained with Ehrlich's haematoxylin these cellstake a very much deeper stain than the other cells of the ecto-derm, and are therefore easily distinguished from them(fig. 1, gc1, and fig. 3, gc1, gc2, PI. 31). These cells are found inboth sexes and are very similar at this stage. Their develop-ment has not been traced past this stage in the male, but inthe female gonophores numbers of these primordial germ-cellstravel in the ectoderm to the tip of the gonophore bud. Manysections of the young gonophores show these deeply stainingcells scattered singly at irregular intervals along the ectoderm,between the place of origin and the tip of the gonophore(figs. 1 and 2, gc3, PI. 31). They are more or less oval in shapeand can be clearly distinguished from the ordinary ectodermcells both by their shape and by their characteristic staining pro-perties. That they have travelled to this position and havenot arisen in s i t u is clear from the fact that no intermediatestages in their development are ever found except in theectoderm of the gonophore stalk. Having reached the tip ofthe gonophore bud, these cells pass singly from the ectodermthrough the mesogloea and take up a position between themesogloea and endoderm, where they form a dark roundedmass which is the bell-rudiment (' Glockenkern ' of Germanauthors x) (figs. 1 and 2, glk., PI. 31). The migration from theectoderm into the endoderm seems to take place in a circulararea surrounding the tip of the gonophore more frequently thanat the tip itself, and consequently the best examples of thismigration are found in longitudinal sections of the young gono-phore which are not quite median. Brauer does not describethis kind of migration for T. m e s e m b r y a n t h e m u m ; butone of his figures shows a germ-cell in the ectoderm at the tip

1 The development of the ' Glockenkern' is not being discussed in thispaper, but it is fully realized that the above description of its origin is notin harmony with the accounts given by either Gotte or Varenne.

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of the gonophore. With the aid of figures, he describes themigration of the germ-cells from their place of origin in theectoderm of the gonophore stalk, through the mesogloea in thisregion into the endoderm ; from which position, after variousstages of growth, they travel forward through the endodermof the gonophore bud to their final resting-place in the bell-rudiment. In T. l a r y n x no suggestion of a migration ofgerm-cells through the mesogloea has been seen, except aroundthe tip of the gonophore bud, and the young germ-cells arenever present in the endoderm of the gonophore stalk. Cer-tainly there are sometimes a few small rounded cells in thisposition, but they never exhibit the deeply staining propertiesof the cells which occur in the ectoderm and later in the bell-rudiment. The kind of migration described above, of whichall the stages have been seen quite clearly, only occurs in theyoung gonophore buds, and is not seen later than the stagewhen the split appears in the bell-rudiment. It follows, there-fore, in view of the enormous numbers of germ-cells which arepresent in the ectoderm of the spadix of a mature gonophore,that either the germ-cells which migrate into the bell-rudimentat this early stage undergo a tremendous amount of prolifera-tion, or that the great majority of the germ-cells migrate in adifferent way and join the bell-rudiment later in its development.

No evidence has been obtained of migration other than thatdescribed above. There are, however, certain cells which appearto be germ-cells, scattered in the ectoderm of the hydranth inthe region between the bases of the oral and aboral tentacles :but as they were never seen in any other position it is difficultto believe that they ever travel to the bell-rudiment. Thelarge cells in the endoderm of the gonophore stalk which Brauerdescribes as the later stages in the growth of the germ-cells, andwhich Perez believes to be gland-cells, are clearly visible inT. l a r y n x , and they certainly bear a very strong resemblanceto the young oogonia in the ectoderm of the spadix. The fact,however, that they are as large and well developed in the maleas in the female gonophores suggests that they are not germ-cells, for the cells of the male germinal mass are, at all stages

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of their development, very much smaller than these cells whichare found in the endoderm. Also, although many sections ofgonophores in all stages of development have been examined,these cells have never shown any signs of migrating towardsthe germinal mass. Their typical position is that shown infigs. 1 and 3, PI. 31, and it seems highly improbable that themigration of such large numbers of germ-cells could escapenotice altogether.

With regard to the alternate possibility that the germinalmass is formed by the repeated division of the bell-rudimentcells, Hargitt (9), Perez (12), and Allen (1) all describe mitoticdivisions of the germ-mass cells. In T. 1 a r y n x these cells arealways so closely packed that it is difficult to see cell divisions ;also the mitotic figures are very small and indistinct; but thatcell-division is taking place is clear from the numbers of thesecells which are ingested later by the developing ovum in groupsof two, three, or four (fig. 8, dc, PI. 31) showing incompletedivision of the cytoplasm.

3. GROWTH OF THE OOCYTE.

Only a very few of the many oogonia which cover the spadixof a mature gonophore are ever destined to grow to maturity :the others merely serve as food for the few whose developmentcontinues. Some workers say that the cells which are destinedto become eggs can be distinguished from the others beforethere is any difference in size. Brauer (3) believed that thegerm-cells were differentiated into two categories during migra-tion. Gronberg (7,1898), working with T. c o r o n a t a , foundthat the difference was first noticeable in the cells on thespadix; those which were destined to become ova having morechromatin in the nucleus than their neighbours. Hargitt(9, 1909) also found that in T. c rocea there is a differencein the nuclei of the oogonia even before growth begins. Hefound that in some cases the chromatin was scattered in grainsalong a network, and in others a nuclear spireme was formed.He says that the latter begin to grow at once and become theova. Hargitt believes, however, that the better nourishment

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of the cells which lie nearest the spadix is the original cause ofthis differentiation. Doflein (5,1896), in his examination of thedevelopment of the egg of T. l a r y n x , finds that all theoogonia are similar in structure, and concludes, also, that itis simply a question of nutrition as to which of them shallbecome the eggs. Perez (12) also supports this interpretationfrom his work on T. i n d i v i s a and b e l l i s , and from thepresent investigations it appears to be true also for T. l a r y n x .In this species the first visible difference appears to be one ofsize, and the egg usually arises from one of the cells which lie

' next to the spadix at the base of the germinal mass. All theoogonia in this region are much larger than those at the distalend of the spadix.

As a rule only one ovum is matured at a time. The growthof a second oocyte is delayed until the first embryo has reachedan advanced stage of segmentation.

The oocyte which is destined to become the egg increasesenormously in size, and soon loses its regular shape and becomesamoeboid. Before much growth has taken place the chromatinin the nucleus becomes arranged in the form of loops Avhoseends converge at one point (fig. 4, PI. 31). Also during thisperiod of growth the cytoplasm which has hitherto shown agranulated appearance becomes vacuolated. Gronberg (7)suggested that this vacuolation of the cytoplasm was mainlyresponsible for the increase in size of the oocyte. Perez (12)also supports this view for T. i n d i v i s a and b e l l i s . Har-gitt (9), on the other hand, for T . c r o c e a , and Doflein forT. l a r y n x , are of the opinion that during the whole of thegrowth of the oocyte the neighbouring cells are being an-n e x e d . Hargitt has never found any ingested nuclei duringthe early stages, but believes that this is due to the rapiditywith which they are digested. Doflein, however, has actuallyobserved this annexation. He states that in the first placea number of oocytes fuse and all their nuclei disappear exceptone, which is the nucleus of the dominating cell. The mass thusformed goes on annexing its neighbours and increasing in size.He believes that the cytoplasm is always a n n e x e d and never

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d i g e s t e d . Labbe (11, 1899) also finds this phenomenon inT. m e s e m b r y a n t h e m u m , i n d i v i s a , and c o r o n a t a ,but says that the oocytes undergo degeneration before theyfuse. During the present investigation many instances havebeen seen of the process of annexation of oocytes whichDoflein describes. There is no doubt that in this species theincorporation of the neighbouring cells takes place during thewhole of the growth period. It has not been possible to deter-mine with any degree of certainty whether the growing oocyteo r i g i n a t e s by the fusion of several neighbouring oocytes ornot, as the larger oocytes are always somewhat irregular inshape and have very indefinite edges, and some of the caseswhich appear to be fusion may be merely cells which areadhering together due to pressure. However, it is certain thatthe neighbouring oocytes are absorbed by the growing cell ata very early stage (figs. 5 and 6, PI. 31).

The cytoplasm of the oocytes which are absorbed during thisearly period seems to be added to the cytoplasm of the growingcell rather than digested. The nuclei, however, are digestedalmost immediately. No change is noticeable in the distribu-tion of the chromatin, but the nuclei gradually become fainterin outline and soon disappear completely. The nuclei of thecells which are absorbed later behave differently.

As the oocyte increases in size it wraps itself around thespadix, which it covers completely when full grown. Aftera considerable amount of growth has taken place, and thecytoplasm has assumed its vacuolated appearance, numbers ofvery conspicuous deeply staining bodies appear in the cyto-plasm. They appear as small vesicles each lying in a vacuoleof the cytoplasm and each containing several small rounddeeply staining bodies. These were named ' pseudozellen ' bythe early workers, who did not investigate their origin butconcluded that they were bodies in which reserves of food werestored by the oocyte. It is now known that these ' pseudo-cells ' are merely the degeneration stages of the nuclei of theoocytes which are ingested during the later stages of growth.During this later period in the growth of the oocyte, the nuclei

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of these ingested cells are not digested immediately, but remainin the cytoplasm for a considerable length of time. The processof ingestion at this stage takes place chiefly in the groovesbetween the numerous blunt pseudopodial outgrowths whichare formed by the oocyte, and which push in among theundeveloped germ-cells massed around the spadix and develop-ing oocyte. • The ingested germ-cells are clearly visible eachlying in a vacuole of the cytoplasm, and progressive stages intheir degeneration can be seen. The cytoplasm is the firstpart of the cell to disappear, and the process now seems to beone of digestion rather than of annexation. The cells whichare ingested at this late stage are usually the smaller germ-cellswith a very small amount of dense cytoplasm. Usually in thenewly ingested cells there is a definite trace of degeneratingcytoplasm (figs. 7 and 8, PI. 31), which leads one to supposethat the process now is not one of mere annexation. The nuclei,before ingestion, contain a linin network on which are scatterednumbers of small deeply staining granules of chromatin. Alarge nucleolus is also visible. After the cell has been ingestedthe chromatin seems to become massed together into one ortwo larger rounded bodies which later break up into six or sevensmaller masses. The nucleolus persists through all thesechanges. Occasionally the pseudocells are seen adheringtogether in groups of three or four, suggesting that the cellswere in the process of division when ingested. The ingestednuclei stain deeply at first, but as degeneration advances theirstaining properties decrease. Finally the nuclear membranebreaks down and the contained granules are set free and areabsorbed by the cytoplasm of the oocyte. It is a curious factthat in many cases these degenerating nuclei become aggre-gated at the side of the oocyte which faces the gonophore wall,1

1 This phenomenon is particularly interesting in view of the fact thatthis pole is always the more active one during development. Here thepolar bodies are given off and here segmentation begins, and in irregularembryos this pole is always characterized by the more rapid cell division.In view of these facts one is tempted to speculate as to whether the con-centration of the pseudocells in this region may not be connected in someway with the greater activity of this part of the cell.

NO. 280 S S

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and the very outermost nuclei seem to be those in whichdegeneration is farthest advanced. Many of these nuclei,however, do not break down at this stage, but retain theirdeeply staining properties and persist until a very much laterstage, numbers of them being found in the endoderm of theactinula at the time of its liberation.

4. MATUEATION.

By the time that the oocyte is fully grown it has ingested mostof the cells which formed a layer separating it from the cavityof the gonophore. It now loses its amoeboid form, becomesseparated from the spadix, and lies free in the gonophore cavity.If the oocyte is a small one and does not completely fill thiscavity, its shape is more or less oval, with a concavity on theside which faces the spadix (fig. 10, PI. 31). But very often thegonophore cavity is too small for the oocyte, and it thenbecomes much deformed by pressure on the spadix and some-times by pressure of another embryo.

A very definite polarity is evident at this stage and persiststhroughout the later development. The nucleus is clearlyvisible and lies near the surface at the side of the egg whichfaces the gonophore wall, that is, on the convex side and rathernearer one end. Two maturation divisions now occur, and ateach a very small polar body is cut off. These two polar bodiesremain attached to the surface of the egg for some time afterfertilization has taken place (fig. 10, PI. 31).

5. CLEAVAGE AND FORMATION OF THE BLASTULA.

The early stages in the cleavage of the fertilized egg are veryirregular and are subject to a considerable amount of variation.In the most common cases, however, the zygotic nucleusdivides a number of times, and the daughter nuclei can beseen distributed in the cytoplasm, long before there is anytrace of the formation of cell walls (fig. 12, PI. 31). Throughoutthe whole of the early segmentation of the egg, cell-wall forma-tion tends to lag behind the nuclear divisions (figs. 14 and 17,PI. 31).

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After fertilization the zygotic nucleus lies near the peripheryof the cell on its convex side. When the first nuclear divisiontakes place the spindle lies with its axis in a tangential position.The asters are very large and the chromosomes, which are inthe form of short rods, are clearly visible in sections stainedwith Ehrlich's haematoxylin.

In all the cases examined the axis of the spindle was parallelwith the long axis of the egg. When the second division takesplace the axes of the spindles are again tangentially placed.Several more nuclear divisions occur until there are a number ofnuclei lying free in the cytoplasm at one end of the egg. Cellwalls now begin to form cutting off the blastomeres (fig. 14,PI. 31). The size of these first blastomeres varies tremendously,and appears to depend on the numbers of nuclear divisionswhich have taken place before cell-wall formation begins(figs. 14, 16, PI. 31 ; fig. 18, PI. 32). Brauer (3) found that inT. m e s e m b r y a n t h e m u m there are two distinct methodsof segmentation. In the most common method the egg segmentsquite regularly, dividing first into two approximately equalblastomeres, then into four, and so on until a perfectly regularhollow blastula is formed with a large blastocoele. In the otherless common method he found that the nuclear divisions tookplace first, then cell walls arose and cut off the blastomeres,beginning from one end. This method also resulted in a per-fectly regular blastula. Hargitt (9), Allen (1), and Perez (12)all find that both these methods of segmentation occur, butthat instead of representing two distinct and well-markedtypes, they are merely the ends of a series, all stages of whichcan be found. This is true also for T. l a r y n x . In thisspecies the regular condition seems to be the least common.The degree of irregularity appears to be connected with thelength of time which elapses between the beginning of nucleardivision and the beginning of cell-wall formation. There seemsto be a very strong tendency for cell-wall formation to lagbehind the nuclear divisions, and this is most noticeable in theembryos whose segmentation is very irregular. In these casesa considerable number of nuclei are seen lying free in the cyto-

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plasm of the unsegmented part of the egg, and the segmentedpart consists of a large number of small blastorneres (figs. 14,16, PI. 31). In the few cases of regular segmentation whichhave been seen, this multinucleate condition does not occur,since a cell wall is formed immediately between the two daughternuclei produced by each mitotic division (fig. 11, PI. 32). Inthe irregular type of segmentation, in which these first daughtercells are not cut off immediately by the formation of cell walls,the daughter nuclei seem to congregate at one end of theembryo ; and if cell-wall formation is delayed for a considerabletime the nuclei become very numerous, and there is very littlespace between them (fig. 13, PI. 32). Consequently, when thecell walls form, the first blastomeres cut off are very small(iig. 16, PI. 31). This, however, is an extreme case, and betweenit and the perfectly regular type there are many other inter-vening stages in which the irregularity is less pronounced(figs. 12,14, PI. 31). A very common case is that in which cell-wall formation begins when only three or four nuclear divisionshave taken place and the daughter nuclei are lying round theperiphery of the convex surface of the egg (fig. 12. PI. 81).The first-formed blastomeres are then much larger than in thecases of extremely irregular segmentation; and when the egghas become completely divided the blastomeres which are cutoff last are not much larger than those which were first formed(fig. 17, PI. 31 ; fig. 18, PI. 32). This is not so in the cases ofextreme irregularity. In these, when the whole egg is divided,there is a tremendous difference in the sizes of the early formedand late-formed blastomeres, the latter being very much largerthan the former (fig. 16, PI. 31 ; fig. 23, PI. 32). The sluggish-ness which characterizes these late-formed blastomeres at thisstage seems to be retained throughout the later developmentof the embryo, and modifies it to a considerable extent.

The blastula produced as a result of these divisions is nota regular one such as Brauer finds in T. m e s e m b r y a n t h e -m u m. Pig. 20, PI. 31, shows the nearest approach to a regularblastula that has been seen during the present investigation.The blastocoele here is merely an irregular space caused by the

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moving apart of the blastomeres, which are irregular in shapeand uneven in size. In many cases, particularly in those wherethe segmentation is very irregular, the blastula stage seems tobe profoundly modified, and cleavage results in the forma-tion of a solid mass of cells with practically no trace of ablastocoele.

Judging by the accounts of the later cleavage stages given byother workers, the different species of T u b u l a r i a seem tovary considerably in their behaviour during late segmentation.Brauer (3) finds that in T. m e s e m b r y a n t h e m u m aregular blastula is formed in all cases. G. T. Hargitt (9) alsodescribes a blastula in T. c r o c e a, but finds that the segmenta-tion cavity is much reduced in some cases. It is certainly muchreduced in all his figures. Conn (4), on the other hand, workingwith T. c r i s t a t a , C. W. Hargitt (8) with T. m e s e m -b r y a n t h e m u m , and Allen (1) with T. c r o c e a, wereunable to find any trace of a segmentation cavity. However,all are agreed that whether a blastula is formed or not, thelater development of the egg is characterized by the formationof a solid mass of undifferentiated cells. This mass of cellshas given rise to a good deal of controversy. Conn, Allen, andC. W. Hargitt, who have failed to find any trace of a segmenta-tion cavity, think that this mass of cells marks the end ofsegmentation, and is therefore a true rnorula. Brauer (3) andG. T. Hargitt (9), on the other hand, find that a blastula isformed which marks the end of segmentation, and concludetherefore that the solid mass of cells which follows the blastulastage is the result of germ-layer formation. G. T. Hargitt,however, although he believes that a true morula is not formedin T . c r o c e a , finds that there is a strong tendency to reduc-tion of the segmentation cavity, and suggests that in some casesit may be entirely lacking. He says there seems to be a ten-dency towards abbreviation in this stage of the development,and suggests that cleavage and germ-layer formation may notbe sharply separated from one another. According to thepresent investigations this interpretation seems to hold goodalso for T. l a r y n x . In this species, as in T. crocea ' , all

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stages in the reduction of the blastocoele are seen. In somecases it seems to be entirely lacking (fig. 15, PL 31).

In the cases where a blastula is formed the solid mass ofcells arises later by a process of budding from the walls, and theblastocoele is thus filled by a mass of cells which forms the rudi-ment of the endoderm. This budding process is almost cer-tainly multipolar (fig. 21, PL 32). Pig. 20, PL 81, shows thebeginning of this process. There are, however, in addition,numerous cases which might be interpreted as unipolar buddingover a large area. The area frequently extends along a wholeside, or completely around one end. In these cases the un-divided portion of the embryo is almost invariably occupied bythe large inert blastomeres which are the late-formed productsof irregular segmentation; and judging by their sluggishbehaviour throughout the whole of the early development onewould not expect them to bud in cells at this stage, even thoughmultipoJar budding were the rule in regular embryos. Brauerdescribes multipolar budding in T. m e s e m b r y a n t h e m u m .He illustrates this process by several figures which MacBridethinks might equally well represent unipolar budding. Theywould, however, lend themselves more readily to the aboveinterpretation.

However, this process of budding, whether unipolar or multi-polar, results in the filling up of the blastocoele and the forma-tion of a solid mass of cells. If this is derived from a fairlyregular blastula by rnultipolar budding, the cells are approxi-mately equal in size (fig. 19, PL 31) ; but if the ' blastula ' fromwhich it was derived was very irregular, and in consequenceof this the area of budding was limited, then the resulting massis composed of very unequal elements (fig. 23, PL 32). Ineither case there is now a rapid proliferation resulting in theproduction of a mass of small undifferentiated cells. Theirregularity which characterizes many of the earlier embryosis not always obliterated during this proliferation.

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6. FORMATION OF THE PLANULA.

The planula stage arises by the cutting off of a definite ecto-derm around the hitherto undifferentiated embryo (fig. 25,PL 32).

E c t o d e r m . — I n most embryos the ectoderm is now formedby simple delamination, a layer of oblong cells being cut offaround the periphery. These continue to divide after they havebeen cut off, but the spindles are always formed in a tangentialplane, and, in consequence, the layer remains one cell thick.When this process is completed the ectoderm consists of smallcubical cells approximately equal in size, whose cytoplasm ismuch denser and more deeply staining than that of the endo-derm cells. The ingested nuclei, or ' pseudocells ' as they havebeen termed, usually remain in the endoderm. The process ofdelamination in normal embryos begins on the convex side,and gradually creeps round to the concave side. In the veryirregular embryos this process of simple delamination seemsto be somewhat modified by the presence of the large inertcells in which the division is considerably delayed. In someembryos this results in slight epibole, caused by the smallercells from the more rapidly dividing area encroaching uponthe large inert cells. There is no evidence that this processever takes place to any great extent. Certainly if it does itis extremely slow ; cases have been seen where the ectodermis well established and is forming interstitial cells in one halfof the embryo, while the other half consists of the large cellswithout any trace of ectoderm (fig. 24, PI. 32).

Endoderm.—During the formation of the ectodermchanges also take place in the endoderm. Numerous small cellsappear among the larger cells. Their cytoplasm is denser thanthat of the large cells, and in general appearance they resemblethe ectoderm. Usually they appear, from their position, tohave been budded off from the larger cells of the endoderm;but in a few very irregular embryos they are concentrated justbelow the ectoderm, at the rapidly dividing pole of the embryo,and look as though they may have been budded off from this

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layer. No mitotic figures have been observed, however, and itis therefore impossible to determine the origin of this mass.These small deeply staining elements do not remain long in theendoderm. They have usually disappeared completely by thetime the mesogloea is formed.

7. DIFFERENTIATION WITHIN THE ECTODERM.

Before the process of differentiation sets in the embryo ismore or less hemispherical in shape, and has its flattened orconcave surface turned towards the spadix and often appliedclosely against it. The embryo consists of a solid mass ofendoderm surrounded by a single layer of undifferentiatedectoderm cells (fig. 25, PL 32). Differentiation now begins inthe ectoderm. Those cells which occupy the area in the middleof the convex surface of the embryo do not multiply, butgradually take on a more columnar shape, the elongation talcingplace in the direction perpendicular to the surface. This'areais comparatively large; in longitudinal section involving25-30 cells. The area of ectoderm surrounding this centralportion, and extending down almost to the edge of the convexsurface, also becomes much thickened (fig. 26, PL 32). Here theelongation of the cells is followed by further division, wherebynumbers of small cells are cut off inwards from the originalsimple ectoderm. By continued division a mass of interstitialcells arises. Very few columnar cells are formed in this region.A similar formation of interstitial cells also takes place overmost of the concave surface of the embryo (fig. 26, PL 32).In some cases a few undifferentiated cells are left in the centreof this area ; and in all cases there is an undifferentiated regionaround the edge of the embryo, which separates the area ofinterstitial cells of the convex surface from that of the concavesurface. From this region the aboral tentacles grow out ata later stage.

While this differentiation is taking place in the ectoderma series of irregular spaces appear in the endoderm. This is thefirst indication of the formation of a coelenteron and is probablycaused by shrinkage of some of the endoderm cells. These

TUBULAEIA LARYNX 615

irregular spaces become larger as development proceeds andcoalesce to form a single large space in the middle of the massof endoderm. The endoderm cells become more regularlyarranged after the coelenteron is formed, but they show nosigns of differentiation. The pseudocells are still clearly visibleas deeply staining opaque bodies containing several chromatinmasses.

8. FORMATION OF THE ABORAL TENTACLES.

The general shape of the embryo now tends to become moreflattened. Prom the circular undifferentiated area of ectoderm,which lies round the edge of the embryo between the two areasof active proliferation, a ring of small outgrowths appears.These outpushings are the young aboral tentacles. In longitu-dinal section they appear as little evaginations of the simpleunmodified ectoderm each filled with a solid mass of endodermwhich is carried with the ectoderm as it grows out. Thecoelenteron is never carried into these tentacles, and the endo-derm which fills them at this early stage shows the irregularformation characteristic of the ordinary endoderm of the bodyof the embryo. As the tentacles continue to grow out thesimple cubical ectoderm cells at their apices divide rapidly.The ectoderm of the tentacles is never modified during theembryonic period, but consists, at all stages of the development,of a single layer of undifferentiated cubical cells. The cells,at the apex of the core of endoderm are dividing rapidly,cutting off on their proximal side new endoderm cells whichhave a very characteristic appearance, being oblong in shapewith the long axis placed across the tentacle (fig. 27, PL 32).In section these cells are seen in either a single or a double rowdown the length of the tentacle, with their nuclei arranged oneabove the other and usually near the middle line. These nucleistain very deeply, especially those of the newly formed cells atthe apex of the tentacle. The proximal ends of the tentaclesstill contain the irregular endoderm derived from the body ofthe embryo.

6 1 6 ESTHER LOWE

9. FORMATION OF THE ACTINULA.

While the aboral tentacles are being formed further develop-ments are taking place in the body of the embryo. First amongthese is the formation of the mesogloea between the ectodermand endoderm. This first appears as a very fine line almostindistinguishable in places. It becomes much thicker, however,as development proceeds. Further changes also take place inthe ectoderm. The columnar cells in the middle of the convexsurface, which are destined to become the fixation cells of theActinula, elongate until their length is about six times theirbreadth. This elongation does not take place regularly overthe whole surface, but seems to be delayed in the case of thecells in the middle of the area. This delay in growth is accom-panied by a slight evagination of ectoderm which carries outwith it a solid core of endoderm (fig. 27, PI. 32). By the timethe Actinula is fully grown the cells in this central region havebecome very much longer, and are indistinguishable from theelongated cells of the surrounding area.

This peculiar evagination of the cells of the fixation area isa constant feature in the development of the embryo ofT. l a r y n x , but its meaning is not easy to understand.Certainly the thinning out of the ectoderm has no connexionwith the formation of a mouth, for this breaks through muchlater at the opposite pole. In none of the records of previousworkers on the different species of Tubularia is there anymention of the peculiar aboral evagination described above forT. l a r y n x . The stages Brauer (3) figures and describes arenot old enough to show this phenomenon, even if it occurs inT. m e s e m b r y a n t h e m u m . Allen's (1) description of thelater stages of T. c rocea is very brief, and she makes noreference to any evagination in that species. None of theworkers on other species of T u b u l a r i a have carried theirinvestigations past the planula stage.

There seems to have been some uncertainty as to the orienta-tion of the embryo, at least in T. m e s e m b r y a n t h e m u m .Brauer did not trace the development of this species past the

TUBULARIA LARYNX 617

stage where the aboral tentacles begin to grow out, and there-fore did not discuss in detail the formation of either the fixationcells or the mouth. Prom his figures, however, and from a fewbrief references to the subject in his descriptions, he wasevidently very uncertain as to the pole at which the mouthwould eventually break through. In the figure of his lateststage he shows the elongated fixation cells in the middle of theconcave surface in that part of the ectoderm which faces the

TEXT-FIG. 1.

Diagram of T. m e s e m b r y a n t h e m u m (after Brauer, fig. 35).o., spot whore mouth will be formed ; ab.t., aboral tentacle ; drz.,fixation cells ; sp., spadix ; cod., coelenteron.

spadix ; and at the opposite side of the embryo, in the middleof the convex surface, there is a place where the ectoderm isthinner than elsewhere, and this he labels as the point wherethe mouth will eventually break through (Text-fig. 1). Mac-Bride, in summarizing Brauer's work on T. m e s e m b r y a n -t h e m u m in his 'Textbook of Embryology', definitelydescribes this point in the middle of the convex surface asthe place where the mouth breaks through. Brauer himself,however, says that he is not sure at which pole of the embryothe mouth is formed ; and he gives two other figures of earlierstages in which the fixation cells appear at the pole of theembryo which faces the gonophore wall (Text-figs. 2 and 3).

618 ESTHER LOWE

This latter condition is exactly comparable to that found inT. l a r y n x . The fixation cells are found in the same positionby Allen (1) in T. c r o c e a , and it seems probable that theorientation is the same in the three species and that some con-fusion has arisen in interpreting the later stages of T. m e s e m -b r y a n t h e m u m . In T. l a r y n x the definite polaritywhich characterizes the embryo throughout the whole of itsdevelopment makes confusion at this stage impossible.

TEXT-FIG. 2.

Diagram of T. mesembryanthemum (after Brauer, fig. 29).sp., spadix ; drz., fixation cells ; g.w., gonophore wall.

While the evagination is being formed in the middle of thearea of elongated fixation cells, further changes take place inthe rest of the ectoderm of the body. In the regions whereinterstitial cells were formed rapidly during the early differen-tiation of the ectoderm (fig. 26, PI. 32), cnidoblasts are nowformed in large numbers by the gradual modification of the in-terstitial cells (figs. 27 and 28, PI. 32). The cytoplasm of thesecells becomes denser, passes with the nucleus to one end of thecell, and forms an opaque deeply staining mass. The rest of thecell appears to be empty, save for the stinging thread which canoften be seen as a small deeply staining cylindrical body (fig. 27,cnb., PL 32). While this modification is taking place the cell

TUBULARIA LARYNX 619

grows, so that the fully developed cnidoblast is usually abouttwice as large as the original interstitial cell. These cnidoblastsare not orientated in any definite direction in the ectoderm,but are scattered irregularly through the thickness of this layer.They appear in enormous numbers and are usually more abun-dant on the concave surface than on the convex.

In the middle of the concave surface there are usually severalundifferentiated ectoderm cells which do not take part in the

TEXT-FIO. 3.

Diagram of T. m e s e m b r y a n t h e m u m (after Brauer, fig. 31).TO., mouth; g.w., gonophore wall; end., endoderm ; t., aboraltentacle.

interstitial cell-formation. This small area, which at firstconsists of only a very few cells, extends rapidly during thetime when the aboral tentacles are growing out. As a resultof this the hitherto concave part of the embryo, which facesthe spadix, becomes slightly convex in the middle. The areaswhere the cnidoblasts occur now appear, in longitudinal section,as four large patches, two on the convex or aboral and two onthe concave or oral surface (fig. 28, PI. 32).

The endoderm has so far differentiated very little. Thecoelenteron has now extended considerably and reaches to thebases of the aboral tentacles (fig. 28, PI. 32). By this time theevagination of ectoderm at the aboral pole of the embryo is

620 ESTHER LOWE

becoming obliterated by the continued elongation of thegland-cells in the middle of this region. The greater part ofthe endoderm now lies on the future aboral side of the coelen-teron. The pseudocells are still present in large numbers,particularly in the aboral mass of endoderm.

Across the base of each of the aboral tentacles a layer ofmesogloea now begins to form, cutting off the endoderm of thetentacles from the coelenteron (fig. 28, PL 32 ; Text-fig. 4, A).There is at this stage no endodermal lining of the coelenteronover the newly formed mesogloea. This layer of mesogloeastretches across the base of the tentacle and is continuous withthe earlier formed mesogloea of the embryo. It does not appearsuddenly across the whole of the base of the tentacle, but seemsto be somewhat uneven in its development, and is often foundin some sections of a tentacle and not in others. This irregu-larity in its appearance is still found in the advanced actinula.After this layer of mesogloea has been formed the endodermof the body grows across on its inner side separating it from thecoelenteron.

The embryo now undergoes a great modification in shape.The area of undifferentiated ectoderm in the middle of theoral surface continues to extend and becomes evaginated withinthe circle of the aboral tentacles, carrying the layer of endodermwith it (fig. 29, PL 32). This area of undifferentiated ectoderm,when developed to the fullest extent, often occupies abouttwenty cells in longitudinal section. The long axis of theembryo now lies between the oral and aboral surfaces. Thenarrowing which now takes place is initiated just above (i.e.on the aboral side of) the bases of the aboral tentacles, in thelower part of the region occupied by the cnidoblasts (figs. 28 and29, PL 32). Over most of this area of cnidoblasts the columnarcells are very widely separated ; but in the lower part a regionappears, completely encircling the embryo, in which thereare a number of columnar cells adjoining one another. Usuallyabout eight cells are seen in longitudinal section (fig. 29, PL 32).The ectoderm of this area is only about half the thickness ofthe ectoderm in the cnidoblast region above it. This circular

TUBULAEIA LARYNX 621

area becomes pushed in to form a groove which separates theaboral region of the embryo from the oral region (fig. 29, PL 32).The mass of endoderm at the aboral pole becomes much com-pressed by the narrowing of the embryo, and is pushed downso that it almost fills the aboral part of the coelenteron (fig. 29,PI. 32).

TEXT-FIG, 4.

Diagrams showing the development of the endodermal rim and theassociated layers of mesogloea.

While the groove is being formed in the ectoderm furtherdevelopments are taking place in the endoderm at the basesof the tentacles, and in the mesogloea. The layer of endoderm,which has grown across the base of the tentacles separating thelayer of mesogloea from the coelenteron, now proliferates, andthe individual cells enlarge until a circular ridge of tissue is

622 ESTHER LOWE

formed which gradually encroaches on the coelenteron (Text-fig. 4, C). The cells composing this mass of endoderm are largeand irregular in shape, with small nuclei and very little cyto-plasm, and, as a rule, are devoid of pseudocells. Before thedevelopment of this mass of cells has proceeded far a layerof small cubical cells grows over it on the inner side from thegeneral endoderm lining the coelenteron (Text-fig. 4, C, end2).

Pseudocells are usually found in this layer of cubical cells(fig. 29, PI. 32). Soon after the appearance of this layer anotherlayer of mesogloea is formed separating it from the mass oflarger cells which form the rim (Text-fig. 4, D). Thus the endo-derm of the rim is separated from the endoderm lining thecoelenteron by this new layer, and from the endoderm of theaboral tentacles by the mesogloea which was previously formedacross the bases of the tentacles (fig. 29, PI. 32 ; and Text-fig. 4, D).

By this time the evagination which appeared at the aboralpole has been completely obliterated. All the fixation cellshave increased enormously in length and have become muchattenuated. In the middle of each cell there is a clearly definedoval nucleus and many of these cells appear to be divided upinto threads at their outer ends (fig. 29, PI. 32). While thesechanges are taking place a further advance is seen in thedevelopment of the oral region. The evagination continuesand is due in part to the division of the patch of cubical undif-ferentiated cells which occupy the middle of this area, and inpart to the narrowing of the embryo as a result of the forma-tion of the circular groove. That part of the ectoderm whichcontains the cnidoblasts on the oral surface undergoes nofurther change ; but plays a passive part, and is merely carrieddownwards by the outpushing of the central region.

Before the evagination of the oral surface takes place theendoderm at this pole consists of a thin layer of cells irregularlyarranged ; but as soon as the area of undifferentiated ectodermcells in the middle of the oral surface begins to extend, theectoderm of this region becomes altered also. The central cellsof the oral endoderm divide rapidly to form a mass of very

TUBULAEIA LARYNX 628

small cells just internal to the layer of undifferentiated ectoderm,and as this patch of ectoderm extends, the newly formed patchof endoderm extends also. The rest of the endoderm of theoral region undergoes considerable modification. The cells areplaced with their long axes perpendicular to the surface of thelayer, and as development proceeds certain groups of thesecells elongate more than others, giving to the internal surfacea peculiar folded appearance. This peculiar formation beginsbefore the oral end of the embryo is evaginated, and becomesa very conspicuous feature in the fully developed actinula(fig. 29, PL 32).

When the evagination of the oral surface has been completedand the developing actinula has attained its full length, theoral tentacles are formed in a ring around the lower end, bythe evagination of areas of the simple ectoderm. The out-growths are usually five in number, and at first are formed ofectoderm only. They are bent slightly towards the oral poleand are first hollow, but each one soon becomes filled with asolid plug of endoderm which is pushed into it from the bodyof the embryo. These oral tentacles always remain quiteshort, the ectoderm which covers each consisting of abouttwelve or fourteen cells in longitudinal section. The endodermof the oral tentacle is now cut off from that lining the coelen-teron by a layer of mesogloea which grows across the base ofthe tentacle. Both ectoderm and endoderm retain their simplecharacter.

The mouth of the actinula is also formed at the same time asthe oral tentacles. At the lower extremity of the embryo, inthe middle of this ring of tentacles, a thin place forms in thewall of the embryo. The cells of both ectoderm and endodermgradually move back from the centre of this region, andeventually the wall is completely broken through. The mouth,which is thus formed, does not enlarge while the actinula is inthe gonophore : it never develops into anything more thana simple break in the body-wall.

While the mouth and oral tentacles are being formed, muchof the endoderm which has hitherto formed a solid mass in the

NO. 280 T t

624 ESTHER LOWE

aboral part of the embryo is pushed down into the oral part.Some of the lower cells of the mass appear to be degenerating,and there are many pseudocells. As soon as the mouth breaksthrough, part of this mass of endoderm is extruded through itinto the cavity of the gonophore.

The embryo is now a fully developed actinula and is readyto be liberated.

10. SUMMARY.

The primitive germ-cells in this species originate in the ecto-derm of the stalk of the gonophore bud and migrate from thatposition along the ectoderm to the tip of the bud where theypass through the mesogloea into the bell-rudiment. Duringthe growth of the oocyte the surrounding cells are at firstannexed, but later the process is one of digestion. Fertilizationhas not been observed.

The early cleavage stages show a good deal of variation rang-ing from a perfectly regular type which results in the formationof a regular, hollow blastula to an extremely irregular type in

. which a blastocoele is never formed. The blastula is followed bya stage in which the embryo consists of a solid mass of cells.This stage arises by a process of modified multipolar buddingfrom the walls of the blastula. The ectoderm is now formed bya process of delamination. The coelenteron appears and themesogloea is formed between ectoderm and endoderm. Theembryo is now saucer-shaped and the solid aboral tentacles arenow formed in a ring around its edge. A layer of mesogloeaappears across the base of each tentacle. A solid circular rimof endoderm is formed at the bases of the tentacles, and thisagain is separated from the endoderm lining the coelenteron byanother layer of mesogloea. Meanwhile a gradual differentia-tion has taken place in the ectoderm, giving rise to nematocystsand fixation cells.

The embryo now becomes greatly elongated so that its longaxis lies between the oral and aboral poles. Five short oraltentacles are now formed, and the mouth breaks through in themiddle of the circle. Having reached this stage the embryo isreadv to be set free.

TUBULARIA LARYNX 625

LITERATURE.

1. Allen, Carrie M. (1900).—" A Contribution to the Development ofParypha crocea ", ' Biol. Bull.', vol. 1.

2. Allman, G. J. (1871).—' A monograph of the Gymnoblastic or Tubu-larian Hydroids.' London.

3. Brauer, A. (1891).—" tJber die Entstehung der Geschlechtsprodukteund die Bntwicklung von Tubularia mesembryanthemum Allm.",' Zeitschr. f. w. Zool.', torn. 52.

4. Conn, H. W. (1882).—"Development of Tubularia cristata ", 'Zool.Anz.', Jahrg. 5, pp. 483-4.

5. Doflein, F. J. Th. (1896).—" Die Eibildung bei Tubularia ", ' Zeitschr.f. w. Zool.', torn. 62.

6. Gotte, A. (1907).—" Vergleichende Entwicklungsgeschichte der.Geschlechtsindividuen der Hydropolypen ", ibid., torn. 87.

7. Gronberg, G. (1898).—" Beitrage zur Kenntniss der Gattung Tubu-laria ", ' Zool. Jahrb.', Abth. i. Anat., Bd. 11.

8. Hargitt, C. W. (1904).—" Notes on some Hydromedusae from theBay of Naples ", ' Mittheil. Zool. Stat. Neapel', Bd. 16.

9. Hargitt, G. T. (1909).—" Maturation, Fertilization, and Cleavage ofPennaria tiarella and Tubularia crocea ", ' Science ', vol. 29.

10. Jickeli, C. F. (1883).—" Der Bau der Hydropolypen ", ' Morphol.Jahrb.', torn. 8.

11. Labb6, A. (1889).—" L'ovogenese dans les genres Myriothela et Tubu-laria ", ' Arch. Zool. Expdr. et Gen.' (3), torn. 7.

12. Perez, C. (1912).—' Observations sur l'ovogenese et la segmentationdes Tubulaires.'

13. Thallwitz, J. (1885).—" t)ber die Entwicklung der mannlichen Keini-zellen bei den Hydroiden ", ' Jen. Zeitschr. f. Naturw.', Bd. 18.

14. Tichomiroff, A. (1887).—" On the development of the Hydroids "(in Russian), ' Nachr. d. K. Ges. d. Liebh. d. Naturw., Anthrop. u.Ethnogr.' Moskau.

15. Weismann, A. (1883).—' Die Entstehung der Sexualzellen bei denHydromedusen.' Jena.

EXPLANATION OP PLATES.PLATE 31.

Fig. 1.—Longitudinal section of young gonophore showing origin ofgerm-cells and formation of the bell-rudiment or ' Glockenkern'. gc1, germ-cells at place of origin; gl., gland-cells; gc3, germ-cells in ectoderm ; glh..bell-rudiment (' Glockenkern').

Fig. 2.—Longitudinal section of young gonophore (same as fig. 1) showingwandering of germ-cells, gc3, germ-cells travelling to bell-rudiment;go1, germ-cells at place of origin ; glk., bell-rudiment.

T t 2

626 ESTHER LOWE

Fig. 3.—Part of longitudinal section of young gonophore showing originof germ-cells. gcl, very early stage of germ-cells ; <jc2, later stage ; gl.,gland-cell in endoderm ; ect., ectoderm of gonophore bud ; ect.sl., ectodermof stalk ; n., nucleus of ectoderm cell.

Fig. 4.—Early stage in growth of oocyte showing chromatin of nucleusin loops.

Figs. 5 and 6.—Two sections of same growing oocyte showing ingestionof neighbouring cells, g-oc., growing oocyte; oc, one of surroundingoocytes; i.n., nucleus of ingested cell: sp., spadix ; g.w., gonophore wall;ic, cell in process of ingestion.

Fig. 7.—Late stage of growing oocyte showing ingestion of the smallercells and formation of pseudocells. oog., oogonia; ic, cell in process ofingestion showing cytoplasm ; ps., pseudocells ; sp., spadix.

Fig. 8.—Section of growing oocyte showing pseudocells in groups,rfc, group of pseudocells; ic., ingested cell showing degeneration of cyto-plasm.

Fig. 9.—Section of ovum showing concentration of pseudocells at theside of the egg facing the gonophore wall, einb., another embryo ; sp.,spadix ; ps., pseudocells ; ch., remains of broken-down pseudocell.

Fig. 10.—Section of egg after fertilization has taken place showing apolar body. The other polar body is visible in another section. Theegg contains four nuclei, n., nucleus; p.b., polar body; p.s., g.w., as above.

Fig. 11.—Section of regular embryo just later than the four-cell stage.c.w., cell wall.

Fig. 12.—Section of embryo whose irregularity is not very pronounced.Six other nuclei are shown in other sections, sp., mitotic figure; n.,nucleus ; cw., cell wall.

Fig. 13.—Longitudinal section of embryo showing an extreme degreeof irregularity. Other nuclei are shown in other sections. Cell-wall forma-tion is just beginning.

Kg. 14.—Longitudinal section of irregular embryo. Cell-wall formationis considerably delayed. An examination of other sections shows numerousnuclei in the unsegmented portion, inf., mitotic figure; nu., nucleus;cw., gw., ps., ii., as above.

Fig. 15.—Longitudinal section of embryo without any trace of blasto-coele.

Fig. 16.—Longitudinal section of very irregular embryo showing verysmall blastomeres (sb.). bow., cell wall forming to cut off a large blastomere.

Fig. 17.—Longitudinal section of late stage of slightly irregular embryo.The cell division seems to be less active on the side facing the spadix.Budding is taking place from a limited area on the.opposite side, blc,blastocoele ; cb., late blastomere.

TUBULARIA LARYNX 627

PLATE 32.

Fig. 18.—Longitudinal section of late stage of slightly irregular enibryo.The blastocoele here is merely an irregular space.

Fig. 19.—Longitudinal section of late segmentation stage showing solidmass of undifferentiated cells.

Fig. 20.—Longitudinal section of regular blastula showing the verybeginning of multipolar budding, mpb., cells budded off from blastulawall; We, blastocoele.

Fig. 21.—Longitudinal section of another regular embryo showing multi-polar budding from the walls of the blastula.

Fig. 22.—Longitudinal section of late segmentation stage showingreduced blastocoele.

Fig. 23.—Section of very irregular embryo showing large blastomereson the side facing the spadix. The actively dividing part is forming ecto-derm, lb., large blastomeres; ect., ectoderm.

Fig. 24.—Section of another very irregular embryo. Interstitial cell-formation has begun in the upper part of the embryo ; the lower consistsof the large blastomeres. Slight epibole is shown on one side, lb., largeblastomere ; ect.cp., ectoderm encroaching over the large cells.

Fig. 25.—Longitudinal section of planula stage, ect., ectoderm ; end.,endoderm ; sc, small deeply staining cells.

Fig. 26.—Longitudinal section of stage later than planula showingdifferentiation within the ectoderm and formation of the coelenteron.fc, fixation cell; inc., interstitial cells; at., region from which aboraltentacles grow out; we, undifferentiated cells on future oral surface.

Fig. 27.—Longitudinal section of embryo showing aboral evagination andstructure of aboral tentacle, abt., aboral tentacle ; fc, fixation cells ; nbev.,aboral evagination.

Fig. 28.—Longitudinal section of later stage showing the formation of thefirst layer of mesogloea across the base of the tentacle. Differentiation ofthe oral endoderm has begun, fe., oral endoderm showing folded appear-ance ; mes., first-formed layer of mesogloea; cnb., cnidoblasts; c.c,columnar cells of circular groove.

Fig. 29.—Longitudinal section of actinula showing oral tentacles, ot.,endodermal rim ; end., the aboral mass of endoderm is being pushed down-wards towards the mouth ; fe., folded endoderm of oral region; mes1,first layer of mesogloea ; mes2, second layer of mesogloea ; e.g., circulargroovs.

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