Axial Elongation in the Mouse and its Retardation in ...dev.biologists.org/content/develop/10/1/73.full.pdf · Lp/Lp embryos from other lines show abnormal flexure of the back, but

Axial Elongation in the Mouse and its Retardationin Homozygous Looptail Mice

by L. JEAN SMITH1 and KATHRYN F. STEIN2

From the Clapp Laboratory, Mount Holyoke College

WITH ONE PLATE

INTRODUCTION

M I C E carrying the gene Looptail were first described by Strong & Hollander(1949), who named the mutation from the looped or kinked appearance of thetail in heterozygotes. Animals homozygous for Lp, as the gene was designated,have neural folds closed only as far posteriorly as the myelencephalon, and dieshortly before or at birth. Stein & Rudin (1953) traced the abnormality of thenervous system back to the time of closure and found that it resulted from thefailure of the neural folds to fuse rather than from a secondary breakdown ofthe roof of the neural tube, and that histological differentiation of the varioustissues of the embryo was comparatively normal. Various skeletal abnormalitieswere reported by Stein & Mackensen (1957), who considered them secondaryeffects of the abnormality in the neural tube.

This paper is concerned with the finding that, at the time closure of the neuraltube begins, the notochord, somites, and gut, as well as the neural tissue, showabnormalities in growth possibly traceable to a failure of the primitive streak toshorten normally. It presents a striking illustration of the manifold effects ofa disturbance in the delicately balanced relationships between the processesinvolved in the elongation of the normal embryo.

MATERIALS AND METHODS

With the exception of a few older embryos, the mice used in this study resultedfrom matings within lines inbred brother by sister for 20 to 25 generations.Embryos from 14 to 19 days' gestation (by Griineberg's (1943) criteria) wereobserved by gross inspection only, and are from six different lines all derivedfrom the same heterogeneous population. Embryos 9 to \2\ days of age camefrom timed matings within only one of these, line 55. Line 55 females werechecked for vaginal plugs in the morning, and 1 a.m. of the day a plug was

1 Present address: Albert Einstein College of Medicine, Department of Anatomy, New York,N.Y., U.S.A.

2 Address: Clapp Laboratory, Mount Holyoke College, South Hadley, Mass., U.S.A.[J. Embryol. exp. Morph. Vol. 10, Part 1, pp. 73-87 March 1962]

74 L. J. SMITH AND K. F. STEIN

found was arbitrarily assigned as the time of fertilization. Since physiologicalage may vary by as much as a day between different strains of mice, estimatesof somite numbers or stage of development also are given. Embryos to be sec-tioned were fixed in Carnoy's fluid, cut at either 7 or 10 /x and stained withAzure B.

Two methods of estimating length were used in this study. For structuresconfined to the posterior part of the body, the number of sections in whichthese structures appeared was determined in embryos cut in true cross-sections.This number was recorded relative to a fixed point, the tail-tip. Because of thecurvature of the trunk, however, it is impossible to get only cross-sectionsthrough an entire embryo. Therefore, to compare the relative lengths of spinalcords, for example, in two embryos, scale reconstructions were made on graphpaper. Relatively equal magnification in both dimensions of the reconstructionwas secured by the following method. Projection drawings at X 100 magnifica-tion were made of every 15th section of embryos cut at 7 [x. Dorsal-ventraldistances on the graph were plotted from direct measurement of structures inthe midline of each projected section. Points for successive sections were plotted10 mm. apart on the graph and those for like structures were then connected bylines. This gave, for structures in the midline, longitudinal outlines from whichdirect measurements could be made (Text-fig. 1). It should be noted that placingthe points 10 rather than 10-5 mm. apart resulted in a magnification in length ofthe embryo of only 95 rather than 100 times. As a result the ratio of length todepth in the magnified outlines of embryos sectioned at 7 /x is slightly lessthan 1.

RESULTS

Embryos at 141 to 19i days'1 gestation

The phenotype of the 69 looptailed homozygotes obtained from six differentlines (Table 1) differs little from that previously described by Strong & Hollander

TABLE 1

Numbers of embryos of different genotypes at 14$-19j days of gestation frommatings between heterozygous Looptails (Lp/+) of six inbred lines

Line

81644556671

TOTALALL LINES

Number of embryos

LpILp

17213

10126

69

Lpl+39523

172832

171

+ /+243557

1421

106

Total

8010811345459

346

Per centLpjLp

212027292210

20

AXIAL ELONGATION IN LOOPTAIL MICE 75

(1949) and Stein & Rudin (1953). All but one or two were markedly smallerthan their normal littermates but the decrease in size was not proportional inall parts of the body. The trunk seems relatively short for the size of the headand the nervous system looks too large for the body. Superficially, the trunk ofthe Lp/Lp embryo appears compressed along the anterior-posterior axis. Thepicture of six sibs from line 8 (Plate, fig. A) illustrates this disproportionatedecrease in size. It also shows the frequently observed 'crooks' in the back. Thehernia described as characteristic for the homozygotes by Strong & Hollanderhas disappeared from all our lines except line 8, in which not only intestinebut liver is found outside the body-wall. This does not show clearly in the figurebut was present in all Lp/Lp homozygotes of this line.

TABLE 2

Length in mm. of umbilical cords of embryos of different genotypes at 19-20 days'gestation

(Each horizontal line contains embryos from one litter)

Line

8

1644

55

$ X <J

Lp/ + Lp/ +

Lp/+ + / +Lp/+ Lp/ +

+ /+ Lp/ +

Lp/+ Lp/ +

Total number of embryos

Average cord length in mm.

Average cord length: cf.pseudencephalic class

Length of individual umbilical cords in mm.

LpILp

2

67-56,4

5,75,5

8, 7, 1096,7, 8

16

6-4

6-4

Lpl +

9, 10, 10, 109,9, 119, 11, 12, 129-5

9-5,* 12, 109, 10, 9, 9, 91012, 11, 11, 5*6,* 6* 11, 10,119,98,* 97,* 9, 101214, 1410, 12, 12, 12

44

10

10-5

+ /+12

1210, 111012117-5*13, 15, 9*1211,9*117,* 9, 11,9, 101415, 15, 14, 1612

26

11-4

121

* Pseudencephalic.

The abnormal concave flexure in the back, seen especially well in the Lp/Lpembryo on the upper left (Plate, fig. A), suggests that embryos in this line do notcomplete the rotation around their long axis which normally produces the changefrom the S-shape characteristic of rodent neural-plate embryos, to the C-shapefound in all mammalian embryos. Perhaps for this reason the somatopleure inthe posterior trunk region is unable to fuse ventrally and herniation results.


Lp/Lp embryos from other lines show abnormal flexure of the back, but rotationis more nearly complete and the most obvious result is therefore a shorteningof the right side relative to the left rather than a hernia. This was noted previouslyby Stein & Rudin (1953).

The neural tissue in all these embryos, from the posterior border of themetencephalon back into the extremely shortened tail, consists of a flat platewith a deep median groove. In the brain region of some specimens, flaps ofmore posterior levels of tissue are apparently pushed forward so that theyoverlie the diencephalon and, in some cases, the cerebral hemispheres, whichare collapsed.

Skin is absent over all portions of the nervous system. The age at whichectoderm over the closed brain region is lost was not determined, but presu-mably its loss is due to rupture by the expanding brain, since it is still presentat 10| days. The failure of closure of the neural folds of course accounts for theabsence of skin over the rest of the nervous system.

Another interesting difference characteristic of the older abnormal foetuses istheir shorter umbilical cords (Table 2). The average length for 16 individualsat 19-20 days of gestation was 6-4 mm. (range 2-10). Comparatively, cords of44 heterozygous Looptails averaged 10 mm. (range 5-14), those of 26 straight-tailed 11-4 mm. (range 7-16). Included in the latter two groups were 11 pseud-encephalic specimens which also had shorter cords. Elimination of these, on thebasis of evidence that this condition is genetically distinct from that producedby the Looptail gene, gives 10-5 mm. (range 9-14) as the average cord lengthfor heterozygotes and 12*1 mm. (range 9-16) for normal straight-tailed foetuses.Variation is too great within these two groups for us to conclude that any signi-ficant difference exists between heterozygotes and normals, but the abnormalhomozygotes would appear to have definitely shorter cords. This conclusion issupported by the lack of any overlap within individual litters.

Embryos at 9 to 121 days of gestation

Twenty-four litters 9 to \2\ days of age from matings of line 55 Looptailparents (Lp/+) were examined (Table 3). Of the 177 embryos obtained, 43 hadopen hindbrains and spinal cords; 17 of the other 134 were abnormally smalland had failed to change from the S-shape characteristic of embryos of 13somites or younger (Table 3—'S-shape'). All 134, however, had closed neuraltubes. The 43 abnormal embryos were considered to be Lp/Lp homozygotes onthe basis of the observed abnormality, and their number agrees very well withthe theoretically expected 44-4. Evidence that the 17 retarded embryos, all ofwhich were at less than 10| days' gestation, were not of the Lp/Lp genotype wasobtained when matings of Lp/+ X + / + parents of the same line produced6 similarly small embryos out of a total of 32. Therefore they are excluded fromany further data. Of the 117 normals, only those 11 days or older could beclassified as heterozygotes (Lp/+) or true normals ( + / + ) . The genotypes of


the 9-10|-day embryos called normal in the following descriptions may thus beeither Lp/+ or + / + .

TABLE 3

Numbers of embryos of different phenotypes at 9-12j days of gestation frommatings between heterozygous Looptails (Lp/-{-)from line 55

Age

12*11*10*10i9 |9*9i9

TOTALS

No. oflitters

13512741

24

Numbers of embryos

LpjLp

24833

1472

43

Lpl+or

+ 1+

818195

182621

2

117

Closed neuraltube; embryo

S-shape

——

12932

17

Total

102227

9234931

6

177

Embryos from the one 12^-day-old litter were examined only grossly. Theabnormal homozygotes in this litter were phenotypically similar to the olderLp/Lp embryos except that the neural tube was less obviously degenerate,although a haemorrhagic area was present in the flap of metencephalon. Similarhaemorrhages were seen in three of the twelve 10^-11 ̂ -day-old Lp/Lp embryosin the eight litters examined at these ages. No evidence of pycnosis could befound in these areas, but earlier observations by Stein, Lievre, & Smoller (1960)of an apparent reduction in cell density in portions of the mantle layer of the hind-brain and cord were confirmed. These areas of 'sparse, loosely arranged cells'could be traced back to 10| days and may be the first indication of the sub-sequent degeneration which the open neural tube undergoes. However, Stein,Lievre, & Smoller (1960) observed this abnormal appearance of the neuraltissue only at 10 to 13 days of gestation. Examination of comparable sectionsof the brains of normal and abnormal 11 ̂ -day-old embryos suggested that,in spite of these areas of decreased cell density, tract formation in the Lp/Lpembryos is essentially normal, since almost identical patterns of darker andlighter staining cells were observed in both.

The 10|-day-old abnormal embryos offered evidence that the open neuralcanal, at least in the hindbrain region, may not be due to an inherent inability ofthe neural fold to fuse but to mechanical difficulties which may cause them torip apart. Figures D and E of the Plate are of successive sections of an Lp/Lpembryo in which the cells of the stretched roof of the metencephalon have pulledapart, apparently under tension. In the normal embryos even the membranouscovering of the myelencephalon shows no such signs of stretching.

A ventral ectodermal ridge of the tail as described by Griineberg (1956) was


observed in both the normal and the abnormal 10|-day-old embryos. Since out-growth of the posterior portion of the body of the abnormals is disturbed priorto the time of appearance of the ridge, it is believed that a defect in this structurecould not be a primary cause of the failure of tail-growth in these animals.

Even at 9£ days, or shortly after the mid-gut has closed, the Lp/Lp homo-zygotes show grossly all the anomalies which characterize them at later stagesexcept that their brain vesicles are not yet collapsed and folded. Neural foldsare open and lie spread out over their backs, producing an appearance of excessneural tissue. Their bodies are relatively shorter than normal, at least from thelevel of the otocysts backwards, and although anterior somites appear normal insize, posterior somites are often very small and/or irregular in shape, making an

TABLE 4

Lengths of gut and nervous system in LpjLp and normal embryos(Measurements in mm. from level of posterior limit of right otocyst to posterior limit of tissue

measured, made from reconstruction drawings magnified x 100)

Exp. no. of$ parent

M15C20449

16163163163

Somite number ofnormal embryo

12-1318-19

2222-25

»>

>>

Nervous system

LpjLp

14020022-527-830-5340350

Normal

26030030036038-544047-0

Gut

LpILp

13019818-324-524028025-5

Normal

23-526-526029-831338-8380

accurate count impossible. For example, in a litter in which 18-25 pairs ofsomites could be counted in normal animals, one Lp/Lp homozygote appearedto have 22; two others 10. The heads of all three were about the same size andstage of development as those of normal littermates. When the two with 10somites were sectioned, they were found to have closed guts, a condition whichis usually not present before the 18-19-somite stage. Whether these somiticirregularities are due to reduced growth rates, to originally smaller somites, orto destruction of some of the cells, cannot be decided on the basis of presentevidence. Extensive areas of pycnosis were, however, found in somites of someof the 9£-day-old Lp/Lp embryos. All the abnormal embryos of this age showa typical bend in the region of the forelimbs which might produce the pycnosisby compression or conversely might be the result of cell loss. Slightly youngerabnormal embryos appear to have smaller somites and little or no evidence ofpycnosis.

Although the total amount of neural tissue at 9\ days may be similar in normaland abnormal embryos, the neural anlage in the region of the floor plate isconsiderably shorter in the latter. This difference can be noted in the scaled(x 100) reconstruction outlines of a typical pair of littermates (Text-fig. 1) andalso in measurements made on a series of such outlines (Table 4).


The gut and notochord are also shorter in Lp/Lp homozygotes. This is obviousfrom the reconstruction outlines (Text-fig. 1) and from measurements for thegut (Table 4). Neither notochord nor gut in the abnormal embryo show anycurves or bends not found in the floor plate of the neural 'tube'. That both

TEXT-FIG. 1. Projection drawings (median sagittal) constructed to scale from cross-sections of embryo9£ days of age. (Measurement in Table 4 obtained from similar reconstructions at x 100 magnification.)

these structures, and particularly the gut, are relatively more shortened thanthe neural floor plate is indicated by their wider separation from it in the scalereconstruction of the abnormal compared with the normal (Text-fig. 1). Furtherevidence, but less convincing because of the paucity of data and greater chancesof error, is also furnished by the measurements. Averaged data for the fivesets of embryos with 22-25 somites (Table 4) show that while the neural floorplate of the Lp/Lp embryo is 23 per cent, shorter than that of a normal embryo,the gut is 27 per cent, shorter. Also worthy of note in Text-fig. 1 are the greaterdiameter and extent of the thickened portion of the notochord in the abnormalembryo relative to the normal.

Lp/Lp embryos seem to be more closely fastened to the yolk sac than theirnormal littermates and this, together with the shortness of the embryos, mayaccount for their apparent difficulties in changing from the S to the C shape.Whether there is an actual difference in length of the allantois which may later

5584.10


be reflected in the difference (previously noted) in umbilical cord length wasnot determined.

Many of the differences reported above were traced to even earlier stages ofdevelopment, i.e. in embryos of 9|-9 days' gestation (16-8 somites). Text-fig. 2

Primative streak -—=*-—y E^r Notochord

Hind gut - ^ ^ / \ fi\ Foregut

Neural f loorplate

lOOu

10mm

TEXT-FIG. 2. Projection outlines (median sagittal) of an abnormal embryo Lp/Lp (A) and a normallittermate (B) at beginning of 9th day of gestation. (Dotted lines represent region of separation between

numbered somites.)

shows projection outlines of embryos from the youngest of the litters examined.The normal had 8 somites and a neural tube just beginning to close; the ab-normal, 9 somites and no evidence of neural-tube closure. A comparison of theseprojection outlines with photographs of embryos of similar stage (Plate, figs. B,C) indicates that the outlines reliably reflect differences in form. Well shown inthese reconstructions is the difference in length, the 9-somite Lp/Lp embryobeing much shorter especially in the region of the somites than the 8-somitenormal. Even at this age, therefore, there is a difference in somite size betweennormal and abnormal embryos. In addition, greater thickness and length ofthe primitive streak and of the enlarged portion of the posterior notochordcharacterize the abnormal. The primitive streak was considered as extendingfrom the posterior limit of the embryo to a point where neural tube and noto-chord seemed distinct structures by virtue of the smooth outline of the ventral


surface of the neural plate and the regular arrangement of its cells. Photographsof the 50th, 40th, and 30th sections from the tail-tip of two slightly olderembryos clearly show the differences in diameter of the notochord, and themore anterior point of separation of neural tube and notochord from primitivestreak in the Lp/Lp (Plate; compare figs. G-I with J-L). They also show theslightly larger tail gut of the abnormal embryos. The same differences charac-terized all the pairs examined.

Text-fig. 3 gives in graphic form, for all available embryos, the number ofsections from the tail-tip to (1) the point where the umbilical artery joins the

110

100

90

B0

70

60

50

40

30

20

10

0Somite No. 9-10,8 10-11,9-11Age (in days) | g |Litter No. 503 862Embryo No. 2,1 2,1,5,3

9-11,11-12 13,12-14 1314-16 13,15-17 11,16 18

7031,7,5,8

4915,6,3,8,7

7056,5,7,1

5615,8,7,4,2

4892,1,3,7

14,17-19 12-13,19 22

405 2 1634,1,5,2 R 3,2,4

TEXT-FIG. 3. Graph of primitive-streak lengths and of relative distances from tail-tip of indicatedstructures in abnormal (Lp(diag.)Lp) embryos and normal littermates 9-9£ days of gestation. (Streaklength, abnormal H i , normal a ; area in which notochord forms part of gut roof in abnormal embryo•̂ — • , in normal < — > ; point of entrance of umbilical artery into dorsal aorta in abnormal • ,

in normal x.)

dorsal aorta, (2) the anterior end of the primitive streak, and (3) and (4) themost posterior and anterior points between which the gut roof has not yet fusedunder the notochord. These embryos are arranged in order of average physiolo-gical litter age of normal embryos, but this arrangement is somewhat arbitrary.Relative position within the group timed as 9£ days of age was determined onthe basis of somite numbers of normal embryos. This order corresponds fairlywell (litter 705 excepted) with increasing distance between the tail-tip and pointof entrance of the umbilical artery into the dorsal aorta of the normal embryowith increasing age. In spite of the variability in physiological age, even withinlitters, and that resulting from differences in the angle of the cross-sectional cuts,several striking differences between normals and abnormals are apparent. Inthe normals the primitive streak becomes progressively shorter with increasing


age. In the abnormals the pattern is less regular and the streak, in each of theseembryos, is longer than in any of their normal littermates. Average lengths arecompared in Table 5.

TABLE 5

Primitive-streak lengths in LpjLp and normal embryos

Age in days

9

No. of embryos

LpjLp

283

Normal

4143

Average no. of sections in P.S.

LpjLp

6044-5±5-3

32

Normal

42-524-8±l-8

15-3

Average length in /x of P.S.

LpjLp

450334240

Normal

319186114

The length of the abnormal embryo, measured from the point of exit of theumbilical artery posteriorly, also fails to increase in the regular fashion of thenormal embryo. There seems to be an inverse relationship between the lengthof the streak and the length of the embryo from this point, even though theprimitive streak of the Lp/Lp does shorten somewhat during the period of timerepresented. This relationship seems to hold also for the normal embryo inlitter 491, which has a longer than average streak for its age and a shorter trunk.The time of completion of the gut roof (see Plate, fig. F for incomplete gut roof)by fusion of the gut endoderm under the notochord was also markedly differentin normal and abnormal embryos (Text-fig. 3). Not only was this fusion completein many of the older abnormal embryos at a time when it was still incomplete inall but the oldest of the normal embryos: it was also farther advanced in theyounger abnormal embryos than in their normal littermates.

DISCUSSION

The early mesodermal cells of the mouse segregate out of, or are proliferatedfrom, the median ventral surface of the more posterior portion of the embryonicepiblast; the head process comes from the anterior end of this same region. Thisregion of epiblast is, of course, the primitive streak. Normally, the primitivestreak shortens during the course of development. By the time the mouse embryohas assumed a C-shape, at the 13-14-somite stage, it is only about three-fifthsas long as at the time of the beginning of neural-tube closure and is furthershortened by the time of mid-gut closure. According to Spratt (1947) and Vakaet(1960), decrease in length of the primitive streak in the chick involves bothregression of the anterior portion and a 'shortening' at the posterior end. Inthis study it was found that the substitution of two Lp genes for their normalalleles in the mouse causes a delay in shortening and perhaps in the regressionof the primitive streak during and following the period of neural-tube closure.

Axial elongation of notochord and associated somites and neural tissue isalso less than normal in Lp/Lp embryos. This could be a direct consequence ofan abnormality of the Lp/Lp primitive streak which retarded conversion of its


cells into definitive notochord cells. Increased bulk of the notochord and thesmallness and irregularity of the somites of Lp/Lp embryos might also result ifsome presumptive paraxial mesoderm cells were organized to become noto-chord cells instead. This would be in line with the experiments of Spratt (1957)-When he forced the 'notochord organizing center' in cultured pieces of chickembryos to deviate so that it organized presumptive somite and lateral platecells into notochord, the diameter of the resulting notochord was approximatelytwice as great as in the normal non-deviated part. Notochord cells, whetherderived from paraxial mesoderm or not, might also be abnormal and thisabnormality might then delay the reorganization which normally produces achange in width of the differentiating notochord (10-12 cells wide at its juncturewith the primitive streak; 1-3 cells wide in the region of the somites). Sinceit seems reasonable to suppose that it is this reorganization which normallyaccounts for part of early notochord elongation, such a delay in reorganizationwould explain the increased length of the bulky portion of the notochord ofLp/Lp embryos and the decreased length of the total notochord.

The abnormal reaction of the gut-roof endoderm as expressed in its precociousfusion under the notochord in the abnormal embryos would seem to supportthe idea of abnormality in the notochord cells. Since the roof of the mid-gut isitself believed to originate from the head process (Snell, 1941, p. 24), its preco-cious fusion may be another and direct expression of abnormality in Lp/Lp headprocess or notochord cells. The failure of elongation of the notochord wouldaccount for the shortness of the 'uncovered' portion.

The implied view of normal notochord and somite differentiation in the mousecontained in the above attempt to account for some of the abnormalities in thedevelopment of Lp/Lp embryos is based on Spratt's (1955) efforts to explainnormal axial formation in the chick from results of his carbon-marking, vital-staining, and isolation experiments.

4 The results of many types of experiments seem to show that the upper (epiblastic)portion of the node (prospective posterior spinal cord and tail bud epidermis) alongwith at least a part of the surrounding posterior portion of the neural plate "slipsover" (i.e. moves posteriorly relative to) the ventral and originally more posteriorchorda and somite forming cells in the mesoblast. At least some of the chorda andsomite center cells remain in close association with the node epiblast and regress withit, leaving behind (anteriorly) the differentiated notochord and somites' (Spratt, 1955,p. 160).

It remains to be demonstrated by similar experiments how appropriate thisview will be in explaining axial organization in the mouse. It also remains to bedemonstrated, as Spratt has pointed out, what the relationship is between theelongation and regression of neural plate and endoderm, and the regression ofthe node. A discussion of causal relationships between the shortness of the neuralplate in the abnormal embryo and the shortness of the notochord becomestherefore even more speculative. It is possible that regression of the ' organizing


center' is responsible not only for the major and initial increase in length of thenotochord in early embryos but also for the elongation of the posterior neuralplate, perhaps by orientation of its cells. If this is the case, failure of notochordelongation in Lp/Lp embryos would then account for the failure of the neuralplate to elongate normally.

On the other hand, reorganization and elongation of the differentiating neuralplate may move the node with its postulated notochord-organizing centreposteriorly. It might then be postulated that 'intrinsic' failure of elongation ofthe neural plate in Lp/Lp is the primary site of abnormality and that shortnessof notochord and irregularities of somites are secondary. The shortness ofneural tube and notochord might produce a mechanical pressure resulting inshortening and fusion of the somites. Holtfreter's (1945) experiments, in whichgrowing Amblystoma larvae were prevented from elongating by encasement inagar, did result in embryos with a bulkier than normal notochord, mesodermoccupying less space relative to other tissues than normal, and tail somites whichlacked an orderly segmental pattern.

Many experiments in amphibians seem to indicate that the presence of anotochord is necessary for the normal elongation of the embryo. However,Kitchin (1949), who removed definitive notochords from Amblystoma neurulaeat the stage of the fully formed plate, found that stretching of the embryos im-mediately after the operation was normal, though shortening was obvious bythe time the tail buds were fully formed. This he interpreted as indicating thatthe elongation in the early period was 'the result of activity which is intrinsicto the differentiation of these individual structural rudiments and it is not at allaffected by the removal of the notochord' (p. 403). The somites in these amphi-bians segmented normally even though they fused in the mid-line. Spratt (1955)has also observed neural-plate elongation in posterior pieces from which thenotochord centre has been removed and which have, as a consequence, no noto-chord. The somites appear to be unaffected. Rothfels (1954) reported that amarked shortening of the notochord, produced in chicks in vitro by the additionof an amino-acid analogue to the culture medium, was not accompanied byextensive somite fusion. Occasional neural-tube abnormalities, including both'zigzag' and open neural tubes, occurred, however, in some shortened embryos,typically in explants treated with /8-2-thienylalamine, and the majority of theseembryos showed somite blocks, i.e. fusion of two or three adjacent somites.

In any case, the primary defect in the Lp/Lp embryos cannot be easilyexplained in terms of either an increased or decreased number of notochord orneural-tube cells. Although the diameter of the posterior region of the noto-chord and the amount of neural tissue in individual cross-sections of Lp/Lpembryos is greater than in normal embryos, the body-length is clearly shorterand the total number of cells in both normal and abnormal embryos may besimilar.

Whatever the original cause of the failure of the neural plate to elongate, its


failure to close appears to result from the failure of elongation. An examinationof the photographed sections shows an apparently normal floor plate and basalportions elevated in normal fashion. The alar portions of the plate seem toreceive no support from the relatively much too small somites and thus to beunable to fold upward and fuse. In the head region, the brain may close, notbecause of any difference in adhesive properties of the neural-plate cells in thisregion, but because the disproportion between neural and mesenchymal cellsmay not be as great, or because lengthening in this region is clearly not due tonotochord stretching.

No other mouse mutation produces just this array of effects. In his descrip-tion of Brachyury which results in a kinky and shortened tail in heterozygotes(77+), Griineberg (1958, p. 428) says that 'the growing end of the notochordtends to be larger than normal and is often very bulky'. He further states thatthe notochord is slow in separating from the overlying neural plate or tube, thatit divides frequently into two parts, the more dorsal of which eventually mergeswith the neural tissue while the ventral portion becomes the definitive notochord.This ventral portion is slow in separating from the gut roof, which is thereforeretarded in fusing beneath it. Although in most respects this description isdifferent from that for Lp/Lp embryos, Griineberg illustrates 9 | T/+ embryoswhich appear to have neural plates somewhat larger than those of the normalembryos, and he states that the tubes close later. He believes that an abnormalityof the notochord which is part of a more general primitive-streak abnormalityaccounts for the defects of these mice.

Splotch is another mutation which like Looptail results in an open neuralplate in homozygotes. In all Sp/Sp embryos the plate is open in the hind-limbregion and in 56 per cent, of the cases in the region of the hind brain. Auerbach(1954, p. 310) reported that the rachischitic area remains proportional to thelength of the embryo but ' the degree of neural overgrowth shows a relativeincrease when compared with the growth of normal tissues in the same region'.These embryos, however, also show a reduction in size or absence of spinalganglia and their derivatives and it is suggested that difficulty in closing resultsfrom the incomplete separation of neural crest from neural tube rather thanfrom overgrowth of neural tissue. Overgrowth is not demonstrable prior to thetime of normal neural-fold closure (Gluecksohn-Waelsch, 1955). In Lp/Lpembryos the size of the ganglia appears normal but no attempt to count theirnumber has been made.

Another example of a mutation in mice in which the neural folds occasionallyfail to close is Fused (Theiler & Gluecksohn-Waelsch, 1956). In homozygotesfor this mutation, overgrowth of neural tissue was observed but was usuallymanifest either as a greater thickening of the walls of the closed neural tube oras multiple closed neural tubes. No relative reduction in size of other tissues inthe open region of the tube was reported and no defect in notochord was ob-served in Fused embryos.


SUMMARY

1. Mice homozygous for the mutation Looptail have shorter bodies thannormal homozygous or heterozygous littermates. Shortening is relatively greaterin the posterior portion of the embryo.

2. The regression or the shortening of the primitive streak is retarded but theseparation of notochord from gut occurs prematurely in the abnormal embryos.

3. Umbilical cords are shorter in Lp/Lp mice than in normal embryos or inheterozygotes.

4. The possible significance of these facts in relation to the open neural foldsin the homozygous Lp/Lp mice is discussed.

RESUME

U allongement axial du corps de la Souris et son retard chez lesSouris homozygotes Looptail

1. Les souris homozygotes pour la mutation Looptail ont un corps plus courtque chez les souris normales homo- ou heterozygotes de la meme portee.

2. La regression ou le raccourcissement de la ligne primitive est retardee,mais la separation de la notochorde et de l'intestin a lieu prematurement chezles embryons anormaux.

3. Les cordons ombilicaux sont plus courts chez les souris Lp/Lp que chezles normales ou les heterozygotes.

4. La discussion porte sur une relation possible entre ces faits et la persistancede l'ouverture des plis neuraux chez les souris homozygotes Lp/Lp.

ACKNOWLEDGEMENTS

The authors are indebted to Sandra Durick for photomicrographs and toRosemary Lee and Susanne Foster for technical assistance.

This investigation was supported by a research grant, B-684, from theNational Institute of Neurological Diseases and Blindness of the NationalInstitutes of Health, Public Health Service.

REFERENCES

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GLUECKSOHN-WAELSCH, S. (1955). Genetic factors and the development of the nervous system. InBiochemistry of the Developing Nervous System, ed. H. Waelsch, pp. 375-96. New York:Academic Press.

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SNELL, G. D. (1941). The early embryology of the mouse. In Biology of the Laboratory Mouse, ed.G. D. Snell, pp. 1-54. Philadelphia: The Blakiston Co.

SPRATT, N. T. (1947). Regression and shortening of the primitive streak in the explanted chickblastoderm. / . exp. Zool. 104, 69-99.(1955). Analysis of the organizer center in the early chick embryo. I. Localization of prospective

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/ . Embryol. exp. Morph. 8, 6-19.

EXPLANATION OF PLATE

FIG. A. Six 19-day embryo sibs of line 8. From left to right one normal ( + / + ) , 4 abnormal (Lp/Lp),and one Lp/ + . In abnormal embryos note short body posterior to forelimbs, crick in back, x 1-7.

FIGS. B, C. Abnormal embryo (Lp/Lp) and normal 9-day littermate. x 17.FIGS. D, E. Successive cross sections of lOJ-day Lp/Lp embryo 499-3. x 100.FIG. F. Cross-section of normal 9J-day embryo 491—7. Notochord cells form part of roof of gut.

X533.FIGS. G, H, I. Sections 50, 40, 30 from tip of tail of normal 9i-day embryo 491-7. x 100.FIGS. J, K, L. Sections 50, 40, 30 from tip of tail of abnormal 9i-day littermate 491-6. x 100.

(Manuscript received 14: vii: 61)

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