INDUCED C LEFT LIP AND FACE SHAPE IN MICE

Harry Rajchgot Human Genetic 5 Sector

M. Sc. Department of Biology Harry Rajchgot Human Genetic s Sector, M. Sc. Department of Biolog y

ABSTRACT

6- AMINO:-.l'ICOTINAMIDE- INDUC ED C LEFT LIP AND

EMBR YONIC FACE SHAPE IN MIC E

Studies of 6-aminonicotinamide-induced median cleft lip in the C 5ïBL/ 6

strain of mice indicated that the rate of differentiation of the nasal placode

is not a factor in the pathogene sis of this defect. After treatment, neithe r

greater divergence of the incipient nasal placodes nor a wider embryonic

face shape was seen, and all embryonic head measurements were reduced.

The latte r observation sugge sts a drop in mitotic rate. Reduction in early

nasal placode thickness and an increase in variance of its width, together

with an apparent retardation in neural cre st ce 11 mig ration by t reatme nt,

suggest a threshold model in which the interaction of· these factors pro­

duce critically small nasal placodes, resulting in abnormal mergence and

consequent median cIe ft lip.

Selection for two lines of mice, one responding to ~-aminonicotinamide

t reatment specifically with median cleft lip, the othe r with late raI c le ft lip,

now in their seventh generation, is presently successful for the forn,er,

but not for the latter. Comparisons of morphological stage at the time of

treatn,ent and embryonic face shape sho·..... ed no diHerences between the t",.. o

lines at the present time. 6-AMINONICOTINAMIDE-INDUCED C LEFT LIP AND

EMBRYONIC FACE SHAPE IN MICE

by

Harry Rajchgot

A THESIS

Subrnitted to The Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Deg ree of Maste r of Sc ience

Human Genetic s Sector Department of Biology McGill University Montreal July, 1971

@) Harr1 Raj cbgot 1971 TABLE OF CONTENTS Page 1. lnt roduction l

Il. Lite rature review 2 A. C left lip 2 B. Neural cre st mig ration 6 C. 6-Aminonicotinamide 30

Ill. Expe rime nta l h ypothe si s 33

IV. Materials and Methods 37 A. Maintenance 37 B. Expe riments 37

1. Early embryology of the lip 37 a. AnimaIs 37 b. Experimental design 38

2. Se lection expe riment 47 a. AnimaIs 47 b. Backg round of the expe rimental procedure 47 c. Present status of the experiment 50

V. Results 52 A. Pathogenesis of induced median cleft lip 52

1. lnduced median cleft lip 52 2. Early development of the nasal placode 53 3. Rate of development of the nasal placode in treated and untreated embryos 54

a. :--:asal placode stage and somite numbe r 55 b. Ch ronological age and nasal placode stage 55 c. Ch ronological age and somite numbe r (.3

4. Effects of (,-aminonicotinamide on the early nasal placode (,(. 5. Effects of ~,-animonicotinamide on the face at the oblon1! and crescent sta1!es of the nasal 'P lac ode ïe t.. Effects of t.-an1Înonicotinan-1ide on neural l-rest cclI migration il -11- Page B. Selection expe riment 81 1. Method of selection 81 2. lnduced c left lip frequency 81 3. Spontaneous cleft Hp frequency 96 4. Morphological stage at the time of treatment 98 S. Embryos examined on Day 10, 20 hours 99

a. Morphological stage 99 b. Face shape 104

VI. Discussion 105

A. Effect of 6-aminonicotinamide on the C S7B L face 105 B. Success of selection n8

VII. Summary 125

VIII. Ack now ledge me nt s 127

IX. Appendices A-E 128

X. Bib 1iog raph y 139 1. INTRODUCTION

The specific embryological mec.hanisms which are involved

10 the production of cleft lip are unknown. Embrvonic face shape ha s bee n implicated as one pos sible factor influencing predisposition to this malformation, as it has been found that certain strains which differ in face shape also differ in both their spontaneous c1eft lip frequencie s and in their induced cleft lip re sponse to various te ratogens. The se te ratogens lead to different types of c1eft lip depending on the time of administration during gestation. One way a teratogen may produce a cleft lip is by influencing face shape.

The present thesis studies normal embryonic face shape and its relation to c1eft lip predisposition as well as the effects of a te ratogen on face shape. -2-

II. LITERATURE REVIF:W

A. C left lip

Cleft lip, in mice, results from the abnormal development of the

fronto- nasal p roce sse s. ln the ea rly embryonic lHe of the mouse

(Patten, 1911; Trasler, 1968), the nasal placodes are originally h'.'o

flaltened oval l..ulges lying in antcro-inferior positions on either side

of the midline of the head. As growth proceeds, the mesodermal

tissue of the placodes proliferates to a greater degree near the cir­

cumference than at the center of the oval. This results in a deprC's­

sion which will later form the external nare or nostrils. This de­

pression, the nasal pit, divides the nasal placodc into lateral and

medial clements. At ils posterior limit, the nasal pit b<.:comcE

extended as a caudally-directe:d invagination. This invagination liés

superior to the area ...... here the surface epithelia al the posterior

ends of thcse lateral and medial processcs are continuolls ,vith one

another. This epithelium infC'rior ta the invagination of the: naEéd

pit is known as the isthmus. The epithelia of the: lateral and mediéd

procC'sses which line the invaginatio:1 of the: nasal pit bccome opposéd

inCeriorly to form a double layer of epithelium lying in the antero­

postt:rior pl<~n('. This double layer is the ndsdl fin, and lies im- -3-

The invagination of the nasal pit later forms the nasal passage, leading from the external to the internaI nare. The internaI nare will later open into the nasopharynx superior to the secondary or ha rd pa late.

At this stage the inte rnal pit or primary choana invaginate s until it is separated from the invagination of the nasal pit by a double layer of ectode rm, the oronasal membrane, one laye r being continuous with the ectode rmal lining of the nasal pit, the othe r continuous with the ectode rmal lining of the inte rnal pit. The nasal fin now disappears as the two epithelia which make it up separate. As development

proceeds, the poste rior portions of the late ral and medial proce s se s

come to oppose one another in a postero-anterior wave. Their

epithelia come into contact with one another to form a double epithelium

separating their mesodermal cores.

Shortly after these surface epithelia lining the sides of the nasal

pit come in contact, they break again in an postero-anterior direction

and fusion of their mesodermal cores occurs. The mesodermal cores

of the medial and late raI proce s se s thus become continuous. Event­

ually, the opening of the nasal pit is quite small. and its posterior

in .... agination connects it to the internaI pit. By this time, the oronasal

rnen1brane separating the tv.:o pits has broken do".. -n, 50 that the two

nov.: form one continuous nasal passage. -4-

In some cases, the normal fusion of the lateral and medial nasal processes does not occur. This may be because they are too w idely divergent. As the embryo continuesto grow, the processes failto

fuse and the isthmus is torn. The consolidated isthmus normally

forms the upper lip and primary palate. Thus lack of fusion of various

degrees results in a lateral cleft of the lip and primary palate of

varying degrees of severity.

Reed (1933) suggested that lateral cleft lip in the mouse results

from insufficient pre ssure of the maxilla ry proce s s on the late raI

na sa 1 proce ss, re sulting in abnormal fusion of the late raI and medial

nasal processes with a resultant lateral cleft lip.

Stark (1955) proposed that a deficiency of mesodermal tissues in

eithe r of the se proce s se s may p revent normal pe net ration of meso­

de rm afte r apposition and fusion of thei r adjacent epithe lia. This

may lead to a lateral cleft lip.

Trasler (1968) has postulated that it is the degree of separation

of the late raI and medial proce s se s which dete rmine s '\... hethe r a

lateral cleft lip will result. There is quantitative cvidence (Trasler,

unpublished) that the frequency of spontaneous lateral cleft lip in

certain strains of mice can be correlated with the degrce of di .... er-

).!ence of their lateral and medial nasal processes (i. e. their embryonic -5- face shape).

The middle of the upper lip in mice becomes continuous by merging of the two medial nasal processes (Patten, 1971), and sub­

sequent overgrowth by the anterior portions of the maxillary processes. Smith and Monie (969) administered methyl salicylate, trypan blue, and 9- methyl PGA to rat embryos. Examination of

these embryos at various times after treatment revealed lateral and

medial clefts of the lip and primary palate as well as maxillary clefts.

The authors proposed that a me sench ymal defieiency as well as

failure of merging may result in indueed median cleft lip.

The mesoderm of the nasal processes, as of most of the head,

develops from neural crest eeUs, which have immigrated into this

region (McAlpine, 1955). One mutant gene, dancer (Oc), in mice

usually cause s a white blaze on the forehead, always produee s abnormal

development of the inner ear, and results in lateral eleft lip in

homozygotes. Theacoustic gang lion is a neural e re st de rivative

(Adelmann, 1925; Berke, 191:,5) and Deol and Lane (1966) have pos­

tulated tha t a neu ra 1 cre st defect lead s to abnormal deve lopment of

the inner ear. This allele also causes lateral cleft lip when homo­

zygous. The postulated neural cre st defect mentioned abovc may

result in the clcft lip defect as ,-';eU (Trasler, personal communication). ,. -0-

Measurement s of embryonic face shape of animaIs homozygous for this allele (Trasler, 1969) have shown that their medial and lateral processes diverge from one another more than do those of animaIs with the same genetic backgro'ùnd but of the normal genotype.

B. Neural Crest Migration

Along with studies on ge rm ceU mig ration from the ge rminal ridge, and the development of the blood system, inve stigation of the movement of ce Us de rived from the neural cre st have yielded insights into the properties of moving embryonic ceUs.

The neural cre st, a t ransitory embryonic st ructure in ve rte brate s, sparked interest soon after its discovery as a possible exception to the germ layer theory. Hs ectodermal origin and apparent mesodermal

(or, more correctly, ectomesodermal) fate was investigated by a large number of \,:orkers, at first to map its contributions to the anatomy of the g rowing embryo. Later, it was studied in order to

shed light on possible mechanisms involved in the interaction of

different embryonic tissues.

:--':eu raI cre st mig ration may be divided into seve raI stage s:

difCe rentiation fron, ectoderm and proliferation, initiation of

rnig :'ation, rnig ration, and sett ling and difCe rentiation into the final

phenotype. Ti is re .... iew will present evidence accurnulaterl to rlate

·... hich has elucidated sorne of the events and interactions involved ln crest has been thought to condense in an antero-posterior direction before or at the time of closure of the neural tube. 1ts structure is not, however, continuous at all axial levels. In the head region, conflicting reports have been produced by a numbe r of inve stigator s concerning the spatio-temporal sequence of condensation of the

neural cre st ceUs.

Adelmann (1925), working with the rat embryo and using

histological obse rvation alone, reported that neural cre st cells

we re fir st seen in the region of the mesencephalon and late r in that

of the rhombencephalon. Initially, the rhombencephalon subdivide s

into three 'pouches' known as proneu'omeres A, Band C. These sub­

divide again and finally result in seven rhombomeres (aiso known as

neuromeres), proneuromere A dividing into Al and 3, Al then

dividing into land 2, at which time B divides into 4 and 5, and C

into neuromeres 6 and 7.

According to Adelmann, neural crest in the cephalic region is

fi rst seen at the five- somite stage in the me sencephalon and

rhon.bome re ALBy the eight- somite stage, howeve r, the neural

cre st of the me sencephalon has been depleted by mig ration, but i s

still present in the region of rhombomere Al, proneuromere B, and

in the ante dor portion of the t runk neural tube. Gaps in ",\'hich no -j-

neural crest is formed occur at rhombomere 3 and proneuromere C.

Thus the neural crest of the trunk region appears to form before that of the most poste rior axial level of the head.

Ade Imann found no evidence for the formation of neural cre st in the forebrain region, although it had been found before in the guinea pig as far forward as the anterior neuropore (Celestino da

Costa, 1920, 1921, 1923, as cited by Adelmann, 1925).

Forebrain neural crest was studied in the amphibian Amblystoma by Baker and Graves (1939), who found by histological observation that the neural cre st formed in the neural fold s of the h ead prior to neural tube closure. lt extended forward to the anterior neuropore, and the cre st eeUs formed a plug in the roof of the neural tube once

closure had occurred. After migration had been completed, there was no evidence for retention of the neural crest in the forebrain

region.

Be rke (1965), ,-,,:orking with rabbit s, found that the ea rly neu raI

cre st of the head formed in rhombome re s 2 and 4 at the 10- 12 somite

stage, in conjunction .... ·ith neural tube closure, rather th an before

closure. As well, he observed what he termed optic neural crest at

the time of closure of the anterior neuropore (14-1( somites). This

ccli mass may have been derived from the forebrain, as was found

by Baker and Graves n93 Q I. He found no neural crest dt rhomboolere -10-

(; until the 16- somite stage. ln ag ree ment with Adelmann (1925), he found no neural cre st forming at rhombome re 3. Although he found a neural crest derivative at rhombomere 5, it was part of the extending facial-acoustic ganglion, and could have been derived from rhombome re 4.

The derivatives of this cephalic neural crest, as found by the workers mentioned above and many others, are listed below:

Rhombomere 2 ------trigeminal ganglion

Rhombomere 4 ------t.- facial-acoustic ganglion

Rhombomere 6 ..... glosso-pharyngeal-vagal ganglion

Thus the neural cre st of the c ranial region appears to fo rm in a rostro-caudal direction and to be a discontinuous structure. lt may also form before or in conjunction with c losure of the neural tube, which also occurs in a cephalo-caudal direction in the head.

The non-linearity of formation of neural cre st in the head and body reported for the rat by Adelmann (1925) was not found by

Kàllén (1953). He observed. in the mouse. that the neural crest of the

head ha s reached the a rte rior- most limit of the somite s be fore the

'spinal' neural crest forms. Kallén correlated the discontinuity of

the cephalic neura 1 cre st with the subdi vi sion of the proneurome re 5

into neu rome re s. and th·.! appea rance of neu raI cre st immediate 1y

aCter that oC the corresponding proneuromere. This suggests but dues -11-

not prove that there is a causal relationship between the two devel­ oprnental processes, and that there rnay also be a causal relation­ ship between the closure of the neural tube and the appearance of the neural cre st.

Landacre (1921), working with urodeles, found that the neural crest forrned in the head at the tirne of closure in three different patterns. One form of condensation was a wedge in the roof of the neural tube which included the overlying epidermal ectoderm; a second, two masses of ceUs between the dorsal portion of the neural tube and the exte rnal ectoderm; the third involved contact of the neural crest only with the external ectoderrn. These may, of course, have been different stages of the same process.

Det",.. iler (1937), working on Amblystorna, also found a wedge of the type de sc ribed by Landac re (1920. The same wa s found by

Di Virgilio!:.!.. ~ (1967) and Marin-Padilla (970) the former in chick embryos, the latter in the golden hamster.

Di Virgilio E..!.. ~ (1967), using histological methods, found no presumpti .... e neural crest v.. ·hich could be distinguished before the neural tube closed in tre head, and found no wedge forming in the prosencephalon (no neural crest forms in this region in birdst. The neural crest ".... edge separated into t"... ·o parts, one remaining in the o .... erl'fing ectode rn1, and anothe r as a wedgc ir the dorsal neural tube. -12-

This latter wedge itself divided into two, one part lying free between the ectoderm and neural tube, the other remaining in the neural tube. lt was this free portion of the neural crest which migrated. The other portions remained behind and did not migrate, although the lateral expansion of the ectoderm as growth proceeded, might carry this portion to areas which receive a neural crest con­ tribution in the form of melanoblasts. If there is a correlation between neural tube closure and neural cre st formation in the normal embryo, it is unlikely that the relationship is causal, unless the primary cause rests in the neural crest rather than the neural tube. Exencephalic embryos which are known to have abnormal neural tube closure ...... ould otherwise be expected to have defects in the

neural-crest-derived structures, such as the nasal placode of the

mouse, and this is not the case.

In the trunk region, Marin-Padilla (1970) found that the closure

of the neural tube was correlated with the formation of what he took

to be neural cre st ceUs. The neural tube starts closure in the region

of the fifth to seventh sonlite and then proceeds both anteriorly to the

cc rvica1 region and caudally towards the poste rior neuropore. The

appca rance of the sc cc Ils in two wave s, one di rectcd art e rior1y, the

other posteriorIy, conflicts with data gathered by Adelmann (1925), -13-

who states that the trunk neural crest imrnediately caudal to the head forn'1s in an antero-posterior direction. The evidence is not, hQ'\,t/evcr. in conflict with that of other investigators.

Thus the neural crest first forms in conjundion with neural tube closure, although the inte ractioils involved in gene rating it are unclear.

2. Neural Cre st Mig ration

Once the neural crest has condensed in the neural rolds or

dorsal neural tube region, the cells begin to migrate out of the crest

into the body. This migration occurs in an antero-posterior

direction, beginning at the levcl ci tbe hindbrain. occurs slightly

later in the foreurain region, and thcn Croln progressively more

postcrior regions (Weston. 1970). Derivatives of the neural crest

include pigment ceUs, parts of the nervous system, skelctal and

connective tissues.

Mig ration in the t runk occu r s along two path"va ys. One group

of cells move lateral to the neural tube and travcl just bclow th(: surface

ectodertn and eventually become the mclaneJcytes of the skin and

hai r or fcathc r s. The othe r gr oup n'love vent rad towa rd the sornitc s.

U sing auto radiot.: raphic technique s, \1.· e ston (19{'3) t ran~plankd nCl~ral 3 tubes from dunor chick enluryos incubatc-rj '.• ;ith II-thymidine ir,to

un la h(' 11(' cl ho~ t s. li c f ound th a t the n1Ït. :-oc 11 vi ron n-:e nt ur the- -';0 rr-i tic -14-

mesoderm facilitated migration, whereas that of the intersomite mesoderm eithe r did not, or actually inhibited migration. The enhancement of intrasomitic move ment of the neu raI cre st ce Us corre late s with the positioning of the ganglia deri ved from the se ceUs.

P revious methods used to foUow migration of the neural cre st ceUs were either cytological or used staining of the neural tube.

The former was limited in that the ceUs of the neural crest were often indistinguishable from mesenchymal ceUs. The latter method did not permit the study of individual ceUs. At present, the use of autoradiographic techniques for the study of migration in

mammalian ceUs is limited, although it can be used successfully

in ce rtain cases as with pigment ceU migration. Following migration

in the mammal has had to rely on differences in enzymatic activity

between neural crest ceUs and mesenchymal cells. Using the

g reate r intensity of alkaline phosphatase activity shown by neural

crest cells compared with other ce Us of the rat embryo McAlpine (1955)

".. ·as able to trace the migration of neural crest cells in the head

and show that the fronto-nasal and maxillary processes were derived

from the neural cre st.

In the head of the chick (Johnston, personal communication) the

neural crest cells migrate bet\... ·een the surface ectodern1 and the -15-

mesenchyme which forms-the 'packing material' between the brain and the epidermis. Here they ap~ar to move either actively by amoeboid motion through the mucopolysaccharide matrix lying between the IT'lesenchyme and ectoderm or passively by secreting this mat rix or by a c ombination of the two.

Johnston (l966}, using radioactive labelling in the chick, was able to follow precisely the paths of neural crest cells in the he ad by labelling very limited regions of the neural crest. It was

seen that a neural crest cell derived from a particular portion of the

neural cre st a rrived in a we ll-defined region and t rave lled over a definite pathway. The region of arrivaI coincided with that which had

been abnormal when corresponding portions of the neural crest

" .. e re removed in ablation experiment s (J ohnston, 1964). This showed

that neural crest cells, as had been found indirectly by extirpation

studie s, did not rnig rate randomly aite r leaving the neural crest

Working with a breed of chicken having t ransitory piebald

spotting, Schaible (19(,8) "vas able to show that diHerences in the

rate of migration of neural crest cells "... ·hich differentiate to form

pig ment ce lis did not e xist. Sc haiiJle could have inve stigated wh ethe r

these cells had only a limited time during which they could migrate,

as this would influence the final phenotype. It is possible that the -16-

cre st cells might be limited in the energy supply or regulatory control capacity nece s sary for mig ration. Thus, they might not reach the limits of the growing wing tip because they literally

'run out of fuel'. These birds have no pigment in their wing feathers at birth. lnward migration of melanocytes from surrounding regions re sults in pigmentation in previously non-pigmented a reas after the first molto

Schaible g rafted portion s of the late raI plate of the e mbryos of

Ancona (piebald spotted) and Brown Leghorn (norrnally pigmented) intracoelomically into White Leghorn (unpigmented) hosts. These grafts were taken from different positions along the surface of the embryo at the axial level of the wing, between the neural tube and the presumptive wing tip. The grafts were taken at different embryonic ages. The appearance of pigment after grafting was taken as an indication that mig rating cells had reached the levei of the g raft at the time of transplantation. Schaible shO'.... ·ed that the rate

of migration in the Ancona brced ".. ·as slower than that of the normal

breed. As ".. ·e 11, the forme r showed pI'ecocious diffe rentiation of

mclanocyte s in the head rcgion comparcd to the normals. He explained

this featurc (slow migration) as being due to this carly diffcrcntiation.

The carly partial diffcrentiation of these cells might have s,,';itched

thei r de .... c lopm<,ntal path 5 to sorne extent. They wou Id then be le s 5 -17-

efficient in migration.

Alternatively, and Schaible did not investigate this, these slowly migrating ceUs may be unable to differentiate normally on reaching the wingtip. According to Schaible, " ... retarded migration is the simplest mechanism since it is mere1y the deceleration of a normal process. Inhibition of differentiation in the white regions would require the addition of unknown factors." These "unknown factors" might, however, be operating and could be tested using the techniques devised by Raw1es (1947) and Mayer ( 1962), to be discussed 1ate r in this review. Whether it turns out that the re­ tardation is due to the migrating ce Us or their environment, the features of early differentiation and partial differentiation may be similar to those described by Mayer 0965, 1967) in the piebald mOUSe. In the pieba1d mouse, Maye r sho\',;ed that the deficiency 1ay in the reduced ability of me1anob1asts ta respond ta enviromenta1

stimuli. ln the case discussed here, this ability ta respond may be enhanced, leading to ea rly diffe rentiation and the conseque nt depression of motility. lt is equally possible that the cue to dif­

ferentiate may be greater than normal, leading ta the phenotype de sc ribed.

W(>ston 0'1t.3) inverted labelled chick neural tubes in unlabelh~d -18-

hosts from which corresponding segments of tube had been removed.

He found that the orientation of the neural tube controlled the di rection of mig ration of its as sociated neural cre st ce Us. The se ce 11s we re found to migrate in a normal configuration relative to the

neural tube, but 'upside-down' relative to the host embryo mesoderm.

We ston te rmed this imposition of polarity "contact guidance".

Inverting the neural tube without doing the same to its neural crest

ceU complement is unfortunately impossible. It is therefore not

possible to determine whether the neural crest ceUs themselves are

cont rolling their polarity of movement or whethe r this control doe s

indeed reside in the neural tube.

The que stion of whethe r the neural cre st ce 11s are predete rmined

as to their final location and phenotypic fate was studied by Weston

and Butler (19{,{,), who devised an experiment in which labelled neural

crest and tube from an 'old' embryo were transplanted into a

'young' hosto These were taken at one axial level such that some

emig ration had already occurred in the 'old' donor, but not in the

'young' hast. If it i sas sumed that the neural cre st ce II i s predete rmined

before it leaves the crest region, then it might be expected that

derivatives of the neural crest ..... ·ould be missing in these hosts some

time after g:-afting. This would be due to the depletion of sorne pre-

cursor cclls in the 'old' ).!raft(~d neural crest. :--;0 n1issing structures -19-

were found. The hypothesis stated above rests on the assumption that those ceUs which would form a particular structure migrated as a group at the same time. This migration of predetermined ceUs might be random, howeve r, and extend ove r the entire pe riod of migration from the neural crest region. ln order to test whether this 'was so, these workers performed the opposite experiment, transplanting

'young' labeUed crest to progressively ol:ler msts. If. on the one hand, ce U s le ft the cre st in random 0 rde r but we re predete rmined, then some of the ceUs leaving at aU stages would be found in more distal derivatives of the crest. On the other hand, if these cells

settled in any region they reached, filling a 'niche', they would prevent

ceUs which left later from entering it, and the older embryos would

have the se niche s at least pa rtially fiUed. It would the refore be

expected that fewer labelled cells would seUle in these distal

de rivatives as the age difference between donor and host increases.

The conclusion d ra ...... n from this re suIt would be that the cre st eeUs

settled in a definite order related to their order of dispersal from the

neural crest. Weston and Butler did indeed find that, as the in-

crement in age bet ...... een donor and host increased. there ..... as less

labelling in the more distal regions. This finding. coupled with that

of the previous transplantation experiment. convinced these -20-

researchers that the ceUs of the neural crest were not pre­ determined but in fact pluripotent, and settle and differentiate in re sponse to environmental cue s.

One would expect, however, that if 'niche-filling' ( the exclusion hypothesis) was indeed occurring, then the first cells to migrate from the crest should settle in the most proximal, rather than the most distal positions in the embryo. Otherwise, if th.e earliest cells fiU sites most distal, then cells migrating later would necessarily have to either sense beforehand that more distal sites were filled or reach them, stop, and turn a round. The aging of the medium th rough which the crest ceUs dispe rse with a concomitant drop in the ability to support mig ration could account for We ston and Butle r' s results.

Ra",.. les (1948), in her review on pigment cells, mentions seve raI experiments which would seem to disagree with Weston's contention that the neural crest is not predetermined. Several in- ve stigators ( Lcuenbe rge r, 1942; Twitty and Bodenstein, 1939), working

", .. ith interspecific and inter generic transplants of neural crest

matcrial fron"l one species or genus to host embryos of another, found

that when the graft was interspecific, the pigmentation pattern of the

dono r was pre se rved, whe reas in inte rgenc ric g raft s, the patte rn

,-\"as that of the host. This finding seems to indicate that there is -21-

some predetermination of the neural crest. ln other words, these cells are canalized to some degree as to developmental pattern, but thi 5 canalization is w..fficient to re spond to a li mi ted variation only, in the cellular environment. There thus appears to be some specificity re siding in both the cre st cell and in the tissue s th rough which it move s.

Along similar Unes, Chibon(1966, quotedbyWeston, 1970), found that transplants of cranial neural fold to trunk regions will not give rise to Rohon- Béard cells (the primary sensory neurons of the central nervous system in fish and amphibia). Thus, although neural cre st cells from one axial level may be pluripotent, their versatility may be limited to a range smaller than that of the neural crest mass as a whole. Also, they may have some degree of intrinsic dete rmination as to their final phenotype, this being modified to

va rious de gree 5 by the mic roenv ironments th rough which the y pas s.

The inductive role of the neural tube on the cre st tissue is an

issue 'which has not been investigated, except as to its role in the

o I"Ïentation of move ment of the cre st ce 115. Because of it 5 contiguit y

to the neural crest, it is very difficult to isolate the two. This

factor has prevented any study of their interaction.

The fillin!! of niches by early-migrating cells had originally been

postu1ated by \\ïllier and Ra ....-les i 19401. They found that ",,'hen young - 22-

Pigmented donor crest was implanted in an aIder unpigmented hast, the neural crest cells did not migrate into the skin. The skin had pre sumabl y been 'filled' by me lanoblasts which could not synthe size

pigment. These then excluded the new supply in the transplant. It

was assumed that the aging of the substrate for migration, as

opposed to the filling of available settling sites, is not the cause

of this inability ta mig rate. Thi s obse rvation is in ag reement

with Weston and Butler's (1966) finding.

That mig rating neural cre st cells can stop and then re sume

mig ration was shawn by Maye rand Reams (1962), who used g rafting

techniques similar ta those of Rawles (1947), and worked on a strain

of mice in which pigment cells were found in the muscles of the

hind leg. These cells could be of non-neural-crest origin or could

havI' arrived by a route other than the usual ectodermal one. In

arder ta test these possibilities, these workers grafted ectodern1

and mesoderm from the level of the mouse hindlimb at different

embryonic ages into the coeloms of chicks of an unpigmented line

(White Leghorn). They found that by Day 11 of gestation melanoblasts

had irnn1igra~cd ta the ectodermal grafts, but ",-ere not secn therc

previous ta this age. ~tesoderm grafted alone did not show the

presence of n1elanoblasts until Day 13 of gestation, although com­

bination n1esodcrn1-ectoderm !!rafts taken on Day Il sho"'-cd pi~rent -23-

formation in the mesoderm after culturing. Thus the melanoblasts

of the mesoderm must be initially resident in the e~toderm and only enter the mesoderm after a lag period of two days. This showed

that me lanoblast s which had pre sumably stopped rnig rating (on reaching

the skin) c Quld re-initiate this proce s s.

The lack of appearance of a te rminal phenotype in a neural- c re st­

de rived tissue might come about through several mechanisms. One

of these has already been discussed: the slowing of migration. There

could, howeve r. be othe r ways in which migration is controlled or

alte red.

The re is probably some mechanism which initiate s neural cre st

migration, although its nature is unknown. Cells which are travelling

may re.quire stimuli to make them continue to do so. Alte rnatively

they may require stimuli to slow or stop their motion. Once they

ente r re gions in which they wi 11 diffe rentiate, they may requi re

an environmental stimulus to undergo this change. Mutant neural

cre st ce Ils ".. ·hich do not diffe rentiate might be unable to re spond to

such a stimulus. The stimulus itself may be inadequate, the re suIt

of a mutant cellular environment. As well, some mutant cells may be

unable to survive in their final location. In this case, the mutation

may be acting either on the neural crest cells' ability to survive or

on the environment' s ability to maintain them. -24-

Both Lane and Deol (Deol, 1964 a, b, 1966, 1967, Deol and Lane, 1966;

Lane, 1966), have proposed that ce rtain mutants in mice, in which there occurred syndromes of abnormal pigmentation, inner ear defects, or megacolon (due to reduction of the myenteric ganglion) cou1d be the re sult of a neural cre st defect.

Mayer, working on a variety of mutants in mice, known to affect the melanocytes, as well as other crest-derived structures, investi­ gated a large numbe r of factors involved in neural cre st cell mig ratioOr localization, diffe rentiation, and survival.

W orking with mice bearing a mutant gene which cause s piebald spotting (s), and using the int racoe lomic grafting technique already

mentioned, Maye r (1965) showed that the mutation affected the ability of melanoblasts to differentiate. By making combination grafts of

skin and neural tube from sI sand +s I+ s embryos, h.e found that when

neural cre st ..... as normal but the skin mutant, normal pigmentation

occurred. When the neural cre st was mutant and the skin normal,

mèlanocytes, when found, were localized to the hair !ollicles. They

wc re ne .... e r found in thp. surrounding epide rmis. A cont rol g raft of

norn.al skin alone resulted in no pigmentation, while a second control

of normal skin with normal neural crest gave normal pigrn entation.

Th<> stimulus ta differentiate would thus he sub-threshold in the

nlutant strain, as con.parcd to the normal, in regions .... ·here pigment -25-

did not appear. He explains this as resulting from modifying or minor genes in the genome of the mutant animaIs.

Mayer obtained further evidence on which to base his idea by comparing the presence of pigment in various tissues of +5/+5 adults,

5/5 adults, and adults which were heterozygous (+5/5). Mayer found that the heterozygotes had fewer melanocytes in some regions of the

body than the normals. The mutants had no pigment in most regions which were pigmented in the normals and the hete rozygote s, and had

a reduction in one area, the ankle skin. The melanocyte population

of the membranous labyrinth of the inner ear was, however. the

same for aU three genotypes. This suggested that the mutant melano­

blasts were capable of differentiation in fewer tissue environments

than those of normal animaIs. This is presumably due to differences

in the st re ngt..h of the stimulus to diffe rentiate.

The interaction of the mutant (5/5) neural crest and its own

ectoderm was next investigated by Mayer (1967). Mutant melanobla!;ts

might be spread more thinly in the mutant skin than normal melano­

blasts. They might only be able to localize in the hair follicles, since

they had been found (as diffe rentiated me lanoe yte s) only in the se

positions in normal skin. To test these possibilities, ~1ayer did the

same type oi g raft s to chick coe loms as before. thi s time combininl,!

norn1al skin ..... ·ith normal neural crest and mutant skin with mutant -26-

crest. The neural tubes were taken at a time when no extrusion of neural cre st had yet occu rred, and the skin at a time at which there was no melanoblast immigr~tion. By injecting the pregnant females previous to transplantation with radioactive label, he could follow the mig ration of cells without requiring their differentiation. The mutant melanoblasts were found to be quite similar in extent of mig ration and in final patte rn in the skin to tho se of the normal embryos. This left no doubt that it was the ability to respond and/ or survive in the skin which diffe rentiated the mutant from the normal phenotype.

The cellular environment might have diffe rent capacities to support differentiation at different times and this was investigated by Maye r (1968), again using the s/ s mutant. He g rafted, as before, normal Day 8 neural tubes (from which no neural crest emigration had yet occurred) with mutant skin of increasing age taken from a future unpigmented area. He v,,'as able to show that although the re was a 510'.'" increase with age in the percentage of grafts containing pigmented hair, the re ".... as no pigmentation in the skin until Day 15 of gestation. After this, there was a rapid increase in the percentage of grafts containing melanocytes in the skin. As a control, to make ce rtain that the skin was indced not taken from a region •... ·hich wouici have bccomc pigmcnted, he g raftcd isolated mutant skin of inc reasing -27-

age. He found no pigment production in the se g rafts. Thus it would appear that the skin of this mutant has only a temporat:y inability to present a stimulus sufficient to cause differentiation.

There are a large number of mutant genes in mice which result in various degrees of spotting. This raises the question of whether the mechanisms of spotting are related in all of them. Some mutants act in the neural crest and others in the cellular environment. Mayer and Green (1968) looked at the mutants dominant - spotting (W) and steel (SI), which showed similar phenotypes: pigment defects, macrocytic anemia, and sterility in the homozygote, as weil as a tendency to develop gonadal tumours. By doing skin-neural tube mutant - norma 1 c ombination transplants as before, they found that while the W mutant gene affected the neural crest, as does the pie­ bald mutant, the SI mutants had normal neural crest with an abnormal ectoderm. The SI gene thus prevented the expression of the normal

neural crest phenotype. The SI skin thus also prevented the expres-

sion of the phenotype of it s o ...,'n normal neu raI crest. Sub sequently,

~layer (19ïO) also studied ho... '" these defects were acting; whethe r on

mi~ration itself, or on differentiation, as with the si s mutants.

~1aye rand (j reen (19t.-81 had been unable to obtain emb ryos of

unknown genotn>e because mating the homozygotes .... ·as impossible.

They had been forced to rely on the expected segration of alleles -28-

when mating heterozygotes to yield one-quarter of the homozygous mutant variety. Mayer (197), however, was able to distinguish the homozygotes of both the W and 51 alleles on Day 12 of gestation by their pale livers (due to their anemic condition). He used normal

Day 8 neural tubes and grafted these as before with skin of various ages taken from a1bino c/c, (which had unpigmented me1anocytes in normal numbe rs and distribution), W /W, and SI/51 embryos. When the albino skin was taken on Day 10 (when no melanoblasts were yet present), melanoblasts from the norrnal neural tube nligrated into it quite weIl. When they were taken on Day 12, most of the skin grafts did not acquire pigment. This hints at a 'site-filling' block to migration. When the skin genotype was W /W, taken between Day 12 and Day 17 of ge station, neural cre st migration did take place into it.

This suggested that if the exclusion hypothesis was correct, in that sites filled exc1uded later cells, then the W neural crest was de­ fective in its ability to migrate.

51/51 skin, on the othe r hand, not only prevented the appearance of pigment in itseU, but of mouse melanoblasts in the chick host coelomic lining as 'weIl. Thus the steel skin could be acting by blocking eithe r immig ration or diffe rentiation of me lanoblast s. Thi s black ".. 'as quite powerful, but did not completely prevent pigmentation in the hast. When pig ment wa s found, it wa 5 close ta the skin g raft s. -29-

If the action of the mutant gene was on differentiation, then one would expect ceUs migrating into the host to be able to express their normal phenotype at some distance from the skin graft. The appearance of pigmented ceUs, when pre sent, close to the graft, therefore indicates that the SI allele is preventing migration of these ceUs, although Mayer did not attempt to explain this block to cell movement.

It has not been shown, ha.v ever, that the neural crest ceUs which settle earliest prevent later immigration. This apparent

site-fiUing block to immigration may merely be the result of a change with age in the ability of the substrate to support migration.

It may also be the result of change s in the celi environment by neural cre st ce lis migrating th rough it, which then act s to inhibit late r

mig rating ce 11s.

Although it could be thought that any sudden morphogenetic

change must have a controlling stimulus, it is possible that this is

not so. Soon after the neural crest begins to proliferate, it begins

to migrate. It has been postulated (Johnston and Listgarten, 19(.9;

Weston, 19ïO) that the neural crest ceUs suddenly become contact

inhibited. Thus they ""'ould repel one another and beg in to migrate.

The neural tube, which is known to determine the polarity of mig­

ration, might be implicated in its initiation as weIl. Tr.us, if the -30-

neural cre st cells were initially moving slightly and at random, a stimulus to direct their movements in one direction could start migration. lt is also possible that the neural crest cells, as they proliferate and secrete their mucopolysaccharide rnatrix, might simp ly be squeezed out of the cre st region, and with the polarizing effect of the neural tube, move away from it (Johnston, personal communication) .

Neural crest cells may migrate out of the crest region until they a re aU depleted. On the othe r hand, the re may be a 'signal' which stops migration at a particular time. Although Di Virgilio

!:!. al. (1967) found what they considered to be retention of part of the neural cre st in the neural tube, this inte rp retation re sts on their definition of what constitutes the neural cre st. Mulnard (1955), using an enzyme- staining technique for alkaline phosphata se in the rat and mouse embryo, found what he considered to be complete depletion of the neural crest. Other workers (Baker and Graves,

1939), ag ree with this view. Thus the re doe s not seem to be a second sti mulus, following that to mig rate, to stop dispe r sion of cc 11 s fron1 the cre st re gion.

C. l.-Aminonicotinamide

The nicotina mide antagoni st (-aminonicotina mide was fir st uscd a 5 a te ratogen by ~lu rphy ~~. (1 ~5 ÎI in a c ompa rati ve stud y of the - 31- chick and rat embryo. .A large variety of defects we re produced in both specie s. Pre-treatment with nicotinamide immediately before application of the antimetabolic completely prevent its toxic and te ratogenic effects.

Dietrich ~~. (1958) found that b-aminonicotinamide was

conve rted to 6-amino- substituted analogue s of NAD and NADP in

vivo after its administration to mice. This conversion could be

carried out in vitro using pig brain DPNase, and 6-amino-NADP

sunthesized using DPN kinase. The 6-aminonicotinamide caused a

reduction in the activity of certain NAD-dependent mitochondrial

enzymes.!.!!. vivo. These workers postulated that the 6-amino­

nicotinamide exe rcised thi s inhibitory effect only afte r conve r sion to

6-amino-substituted analogues of NAD and NADP. Presumably, this

inhibition was caused by the attachment of these converted analogues

to these enzymes. They were then unable to function in hydrogen

and e lect ron t ransfe r reactions, thus blocking the normal functioning

of these enzymes.

The antagonism of nicotinamide by 6-aminonicotinamide in mice

could be reduced considerably by simultaneous adn1inistration of

nicotinan1ide, less 50 by nicotinic acid, and to a smalt extent by

tT',-ptophan lJohnson and ~1cColl 19551. These authors found that ..... hen

the dose of t.-arr'.inonicotinan1ide was 100 mg/k~. body ' ... eight d .... pn -32-

together with 25 mg of nicotinamide per kg. body weight, oxygen uptake i!!. vitroby tissues of treated animaIs was only 30% of the normal level. The addition of either lactate or NAD increased the rate of the oxidation, and when they were added together, brought it up to almost normal levels. It was suggested that these two sup­ plements were depleted in these tissues after treatment. Neither treatment in vivo with a 50 mg. /kg. dose of 6-aminonicotinamide nor its in vitro addition to Liver homogenate had any appreciable effect on oxygen uptake, however.

Coper and Neubert (1964) found that this teratogen did not inhibit the phosphorylation of NAD to NADP in vit ro but that 6-amino- NAD caused a 34-90~v inhibition of this transformation. They could not find

any inte de rence "vith the t ransphosphorylation reaction leading from

NADP to ATP.

lngalls !:.!. ~.(1964) reported that treatment of pregnant mice

with 6-a minonicotinamide (dosage not stated) without subsequent pro­

tection with nicotinamide resulted in chromosome fragmentation and

polyploidy. -33-

Ill. EXPERIMENTAL HYPOTHESIS

Treatment of C57BL/6 pregnant females with the teratogen

6-aminonicotinamide (6AN) on Day 9, 12 hours of ge station leads to median cle ft lip in their offspring (Trasle r, unpublished). The mechanism and locus of action of this teratogen in the production of median cleft lip is unknown. Trasler (968) has proposed that a wide divergence of the median nasal processes (i. e. a wide face shape) predisposes an embryo to respond with a median cleft lip when presented with an environmental insult at the appropriate time.

A possible way in which the te ratogen may produce a median cleft lip is by acting at the time of treatment on the neural crest of the head. The se form the me sode rmal core s of the nasal processes (McAlpine, 1955). The action of 6AN may be on the

normal migration of these cells, by preventing a full complement

of ce Ils from settling in the nasal proce s se s. Mucopol ysaccharide s

a re the medium within which neural cre st cells are thought to

migrate and it is known that 6AN depresses mucopolysaceharide

synthesis in the hearts of rat embryos of an equivalent age

(Ove rman and Beaudoin, 1971). AIso, treatment with salie ylate s,

" .. hich also produces a median cleft lip in C57BL/fJ embryos

(Trasler, 1965" when administcred during the sarne period -34-

depresses mucopolysaccharide synthesis in mesenchymal tissues

(Larsson ~ ~,1968). Thus, if the locus of action of bAN is on

neural crest cell migration, it may result in their arrivaI in the

presumptive nasal placode region later than in the normal embryo.

Since the head is growing during this time, the se ce Ils may not be

able to travel far enough ~.o permit their settling in normal

positions and may settle further apart than normal. In this way,

a wider face shape might result, and this lead to a median cleft

Hp. Alte rnative ly, insufficient neural cre st ce 115 might reach

their normal positions, and this insufficiency of tissue could

lead to the same defect. A combination of the two is also possible.

Anothe r pos sible mechanism is that involving neural cre st

cells which settle normally but whose cellular metabolism is

altered by the treatment. In this case 6A':' may cause a temporary

depression in their mitotic rate. This could lead to a la te r

insufficiency of tissue with a resultant median cleft Hp.

The effects of 6A~ on the rate of development and the type of

abnormal developn1ent of the {ace resulting from treatmcnt were

studied. The following hypotheses were investigated: (al the

teratogen acts by changing face shape Ibl the incipient nasal

placodes di .... cr!!e to a !!reater del!rec i.n the trcatcd err.br;·os than -35-

in the controls (c) 6AN results in reduced size of the initial placode, and (d) the final size rather than position of the nasal p lac ode is changed. AIso, the occur rence of mig ration of neural cre st cells into the nasal placode region, and the effects of tAN, if any, on this migration were studied. The results should indicate whether the neural crest-derived tissue is affected by the teratogen before,

during, or after settling has occurred. It should also show

whethe r the effect of treatment is a change in placodal position

and face shape, or in placode size.

Whether or not face shape is a factor resulting in

susceptibility to a particular type of cleft lip, i. e. lateral cleft lip

or median cleft lip, is being studied through a selection experiment.

If face shape is a factor in cleft lip predisposition, selection for

lateral cleft lip and median cleft lip in response to 6AN in two

separate selection tine s should ultimately produce two lines, each

ha ving a high induced re spon se of the appropriate type of c left lip.

Selection for lateral cleft tip should result in a line whose

embryos have a characteristic "narrow" face shape, that is, one

in ",.. hich the median processes diverge very little, and the

distance bctween the nasal pits is small relative to the width of the

head. Se lection for median c le ft lip should re suIt in a line whose

ernbryos have a characteristic '··... ·ide .. face shape, 'with the median -36- 1 processes widely divergent and a larger distance between the 1 nasal pHs relative to the width of the head.

Anothe r factor which may be invo1ved in specific cleft lip predisposition is the morphologica1 age of the embryo, or more specifically, the stage of deve10pment of the nasal placode at the time of treatment. Treatment of pregnant females of the C57BL/6 st rain on Day 9-1-, as already mentioned, re sults in median c1eft lip in their offspring. T reatment of the se animaIs on Day 10,

8 hours leads to latera1 c1eft lip offspring (Tras1er, unpublished).

Seleclion for either lateraI cleft lip or median c1eft Hp may thus re suIt in two se Iection line s whose embryos are at two different morpho1ogical stages at the time of treatment. The embryos of the Median line (median c left Hp re sponse) would be at a stage

similar to that of the Day 9i C57BL/ 6 embryos at the time of treatment (Day 9, 10 hours). Those of the Lateralline (Iateral

cleft lip re sponse) might be at an olde r stage c loser to that of the

Day 10, 8 hours C57BL/6 embryos. The hypothesis that

selection has been for either (a) embryonic face shape, or (b)

n1orphological age at the time of treatment was examined. -37-

IV. MA TERIALS AND METHODS

A. Maintenance

The mice were fed Purina Laboratory Chow and water ad

libitum. For the C57BL/6 animaIs used in the study of the early

embryology of the lip, the diet was supplemented once weekly with

lettuce and whole wheat bread soaked in milk. The laboratory

tempe rature was maintained at 710 Fahrenheit. The room Eghting

schedule was sixteen hours of light and eight hours of darkness,

the light s going off automatically at 11 p. m. and on at 7 a. m. Up ta

five mice of one sex were kept in each cage. Mating was carried

out by placing one male in each cage of female s two to three times

pe r week in the late evening, and the male s we re re moved when the

fe ma le s \'.:e re checked for vaginal plug s the next day.

B. Expe riments

The experiments had two aspects which will be dealt with

separately. One concerns the embryological study of early stages

of embryonic deve lopment of the lip, using the inbred st rain

C5ïBL/6, and the other selection for differing cleft lip responsc to

the te ratogen, t,- aminonic otinamide.

1. Early Embryology of the Lip

a. AnimaIs

~1ice used in the study of the early embryology of the -38-

induced median cleft Hp were of the C57BL/6 strain. The incidence of spontaneous lateral cleft lip in this strain is very rare. b. Expe rimental de sign

Females with a vaginal plug on the day after mating were weighed. This day was conside red to be Day 0 of ge station. and it was assumed that fe rtilization had occurred at 2 a. m. of the previous night (Snell.!:.! ~, 1940). Females were weighed again on Day 8 and we re conside red to be pregnant if they had a weight gain of at least two grams. Those which showed the required weight gain were randomly assigned to either the control group or the treatmenl group. Females in both groups were killed at six-hour intervals by cervical dislocation, beginning for the controls at the time at which treatment with 6-aminonicotinamide (6AN) would have been administered (Day 9, 12 hours) and ending at Day Il,

14 hours, and for the treated group from Day 9, 18 hours to

Day Il, 20 hours. The only interval which was not of six hours duration ...... as between bay 10, 0 hours and Day 10, 8 hours.

The ute ri we re re moved and fixed in Bouin' 5 solution for

se ..... e raI days followed by storage in 70'T~ alcohol, unless the e mb ryos ....';e re ta be examined fre sh, in which caSe the ute ri v;e re placed in Ringer-Locke saline solution. -39-

The treatment group we re injected intrape ritoneally with a full dose of bAN at Day 9, 12 hours followed by a full protective dose of nicotinamide (N) three hours late r at Day 9, 15 hour s. The full dose of bAN was 19 mg. /kg. of body weight on the day on which the vaginal plug was found. and that of N was 7.3 mg. /kg. The concentration of the bAN solution was 45 mg. /20 c. c. of sterile, di stilled wate r, and that of the N solution was 17 mg. /20 c. c.

Thp. incidence of induced c left lip was de te rmied by sacrificing treated females on either Day 12, 12 hours, Day 13,

12 hours, or Day 14, 12 hours, the majority at the ea rlie r time, when the presence of any median cleft Iip (MC L) and laterai cleft lip (LC L) was quite obvious.

Uteri which were fixed before examination were later dissected and the fixed embryos carefully removed. Embryos "·.. ere rated morphologically for stage of eye vesicle, ear vesicle and limb buds according to the Milaire rating system (Milaire, 1959), as well as somite number, the degree of closure of the neural tube in the head region, and the stage of the nasal placode s as defined by

Trasler (Trasler, 1968). 0:eural tube closure stages ean be seen in

FIGURE 1. .A. numerieal seale assigned to the various stages of

nasal placode development is deseribed in TABLE 1. -40- l FIGURE 1: Stages of neural tube closure in the head. Closure in the head begins at D and proceeds from there towards the stomodaeum and rostro-caudally towards the mesencephalon (arrows).

h

o. optic vesicle p. prosencephalon m. me sencephalon r. rhombencephalon e. otic vesicle D. point of origin of closure

STAGE 0: Neural tube completely open in head region

1: Closure at middle of prosencephalon

~ Neural tube completely closed over prosencephalon

3:. C losure to rniddle of me sencephalon

4: Closure to caudal end of mesencephalon

5: Closure over rhombencephalon incomplete

6: :'\eural tube complctcly closcd -41-

TABLE 1:

;'" Stages of nasal placode development.

Stage 0: No nasal placode development is visible

1: A slight flattening is seen in the nasal placode

region.

2. A slight bulge which is flattened is Seen.

3: A slight indentation has occurred into the bulge.

:'r:'': 4: Shallow oval

5: Oval

6: Deep oval

7: Early oblong

8: Oblong

9: Late oblong

10: Cre scent

11: Early comma

12: Comma

13: Late comma

14: Epithe liaI fusion

.'. The term "nasal placode" is used here loosely to refer to the area ",,'hich in later stages includes the lateral and medial nasal processes

and the nasal pit between them.

..... Stages 4 - 14 are those described by Trasler (l9t,Sl. -43-

dis secting mic roscope with sub- stage and over- stage lighting.

Calibration was carried out and the actual measurements calculated using a g rid micromete r on the mic roscope stage. The

ocular micrometer had a 10 mm. linear scale divided into 0.1 mm.

units. At the lower magnification used (2. 7x), one micrometer

unit equalled . 0370 mm. At the highe r magnification (5. 5x), one

micromete r unit equalled .0182 mm. Ear1y embryos were

measured at the higher magnification, later embryos at the lower

one. Seve raI embryos we re measured twice to check the accurac y

of the individual measurements, which was found to be very good.

Most measurements were identical in the two trials, and where

different, theywere within one ocular micrometer unit ofeach

other. An measurements were made viewing the heads only

th rough the eyepiece ca rrying the ocular mic romete r. The head s

were positioned in such a way that they appeared to be symmetrical­

ly placed, as much as this was possible, when viewed through the

eyepiece used in their measurement.

Some Day 9 embryos, both treated and control, \ ... ·ere

exanlined fresh. The methods used were similar to those already

de sc ribed for fixed embryos, except that the ute ri we re placcd in

Ringer- Locke saline solution and the embryos then dissccted out -44-

for obse rvation and manipulation. These embryos were examined for the criteria already discussed as weH as for the presence of a hea rtbeat.

The majority of fre sh treated and control embryos we re examined and then stained in an attempt to distinguish neural crest ce 11 mig ration. A large numbe r of dye s were te sted. Initially the dye brilliant cresyl blue, applied direct1y, appeared to give good

re suIt s. When it was app1ied to the limbs of Day 14, 12 hour embryos, however, the dye was taken up specifically in the inter­

digital necrotic zones, either by dying cells or macrophages. It

appeared, then, that the cells taking up the stain in the head were

probably dying cells, and not neural crest cells, as had been

supposed. An alternate staining technique was then tried which was

a modification of the Gomori alkaline phosphatase staining technique

for sectioned material (Pearce, 1961, p. 868). Whole embryos

\\;e re stained, and in 0 rde r to compensate for the large volume of

the embryo, the times for application of the various reagents and

the length of the rinsing periods between had to be g reatly inc reased.

\" e ry Ce'-\' re su It s wc re obtained due ta the long pe riod of time

required Cor this method ta give results, the difficulty of controlling

the water conditions during the long rinses, and the inability ta

distinguish control and treated embryos run in the sanîe vessel. -45-

Finally, a modification of a sirnultaneous alkaline

phosphatase technique (Boyer, 1961; Lawrence et al, 1960;

Knudtson and Evanger, 1962) for starch gels and leucocytes was

applied. Subst rate s te sted we re p-nitrophenyl phosphate, o<.-naphthyl sodium acid phosphate, and,8-naphthyl sodium acid

phosphate. Only the latter substrate gave results. A control ex-

periment was carried out using no substrate in the reaction

mixture. The procedure is shown below:

Simultaneous Alkaline Phosphatase Technique Modified for

Whole Embryos

(1) Prefixation

Fresh embryos are rapid1y dissected from the uterus,

their embryonic membranes removed, and fixed immediately

in cold 80~~ ethanol for 2 hour s.

(2) Incubation Mixture

Ve ronal buffe r (2~Ct), pH 9. 3 50ml.

Subst rate (~- naphthyl sodium acid

phosphate) 25mg.

Fast Blue RR salt 25mg.

~la~ne sium sulphate ('Omg.

The inc ubation n1Îxtu re rnu st be made up fre sh im­

mediately before USe and filtered. After prefixation, -46-

embryos are transferred to this mixture and stained at room temperature for 2 hours (until they are fairly darkly stained) in batches of 3-4 embryos in Coplin jars. The Fast

Blue RR salt spontaneously decomposes and produces a background staining, which cannot always be removed by de staining, so care should be taken to prevent ove rstaining.

This can be accomplished by changing the incubation at i -

1 hour intervals.

(3) De staining and Storage

After staining, emb ryos a re de stained in a mixture of methanol, distilled water, and acetic acid (in proportions 5:5:1) overnight and stored in the same mixture. After they were sufficic nt ly de stained, the embryos wc re examined for signs of neural cre st mig ration in the head and body re gions. -47-

2. Se Iection Experiment

a. AnimaIs

This experiment was begun in 1967 by Miss Lindsay Scott, who performed the initial crosses. Two FI groups were obtained, one from a reciprocai cross of DBA/lJ x CL/Fr animaIs, and another from a reciprocal C57BL/Fr x NS/Fr cross. These FI animaIs we re then c rossed reciprocally to produce a tetrahybrid

F2 generation. At this point the experiment was assumed by Mrs.

Carole-Ann Hamly. The F2 animaIs were mated randomly with non­

sibs, and litters of the first parity were raised.

b. Backg round to the Expe rimentai Proc.edure

The same matings were then made again and the pregnant

females were treated with a single dose of 6AN on Day 9, 10 hours,

follo .....:ed by a full protective dose of N on Day 9, 14 hours.

Pregnancy was determined by the sa me procedure as desc ribed in

Section lb. The se female s \"·e re sac rificed on Day 17 and the

embryos checked for MC Land LC L as weIl as any other gross mal­

formations. These included defects of the limbs, eyes, palate, brain,

and abdomen. The first untreated litters from [emales showing a

clcft lip response in their second treated HUer werc assigned to

cithc r th('l.ateral or ~tedian selection line on the basis of the type of -48-

cleft lip induced by treatment of their sibs in the second litte r. Un­ treated sibs of treated embryos giving a MCL response were

as signed to the Median line, while untreated sibs of embryos

showing an LC L response after treatment were assigned to the

Lateral line. The first litters of females showing no cleft lip

re sponse in their second litter were assigned to the Control line. A

first litter is defined as the first litter obtained from the chosen

cross, and so is not nece ssarily the first parity, as sorne females

became pregnant by their brothe rs before male and female sibs

were separated after weaning. Initially approximately 60 females

and 15 male s we re as signed to each selection line and 40 female s

and 10 male s to the Control line.

The selection procedure was then started on the F3 animaIs of

the two selection lines. The selected Iitters of the F3 animaIs were

considered to belong to the first generation of selection. In aU

cases, the generation number given will be that of the litter, not of

the treated females.

Randonl non- sib and non- first cousin mating was maintained

th roughout the expe riment (an exception to this rule occur red in F 3

and Fol; wherc a fcw sib and first cousin matings wcre pcrformedl.

Bcginning with the second generation of selection, no transfcrs of -49-

animaIs we re made from one selection line to the other.

Beginning in the third gene ration of selection, female s we re treated in their first recorded pregnancy, and allowed to give birth in separate cages over wire mesh. If their response was positive, they were remated to the same males as before, and their second litter raised to produce the next generation. A

positive reaction was defined as LCL in the Lateralline, MCL in

the Median line. A negative reaction was defined as LC L in the

Median line, and as MCL in the Lateralline. A non-reacting

female is one whose treated offspring showed neither type of cleft

lip, but showed other malformations. A double-reacting female

was one whose treated offspring showed both type s of c le ft lip.

Female s who se treated offspring gave no response to treatment,

i. e. had no visible malformations of any type were remated and

t reated again. The re sults of such a female' s liUer we re not in­

cluded in the results. since the lack of response could be due to

imprope r administration of the te ratogen.

Wi re me sh, th rough ".. :hich the t reated ne ...... borns d ropped.

was uscd to prcvent the females from eating or otherwise mutilating

thei r ncwborn oHsp ring.

A more rigorous selection procedure was introduced after -50-

generation 3, where only the offspring of positive-reacting females were used for the succeeding generations. The selection of generation itself was carried out on the basis of results ob- tained for gene ration 1.

In gene ration l, a number of female s we re ethe rized for easier handling at the time of treatment. The ether treatment was found to significantly reduce cleft lip frequency, and for this reason, ether was not used in subsequent gene rations.

The Control line in aIl gene rations was bred randomly, and selection was not based on treatment. A group of Control animaIs was treated in generation 4 in order to retest the ether effect and identify possible genetic drift.

A more complete description of methods used up to and in­ cluding generation 4 can be found in the thesis written by Carole­

Ann Hamly. The criteria for selection as well as selection theory are adequately reviewed by that author and are not being cove red here, as this is not the main emphasis of this thesis.

C. Present status of the experiment

Beginning with gene ration 5 and continuing until gene ration 7,

the expe riment ,-';as as sumed by the pre sent inve stigato r, afte r which

point the cxperiment was taken over by another v;orkcr. -51-

The dose s we re altered in gene ration 5, the embryos of this gene ration being treated with an inc reased dose of 6AN and N.

The majority of embryos were treated with either a li or 1 3/8 dosage level, and the latter dose was used in the later generations.

The ste rility of positive-reacting female s of the fifth

ge ne ration Late raI line re quired the use of offspring of fe male s

which we re eithe r non- reactors or unte sted in making up the next

generation of the Lateral line.

In generation 6, a number of untreated third litters of females

of known c1eft lip response were gathered on Day 9, 10 hours and

Day 10, 20 hours in the selection lines. These were fixed, and

morphological ratings were made on the basis of the same criteria

as in section 1. b. The Day 10, 20 hour embryos were measured for

face shape by the same procedure as de sc ribed in section 1. b., for

older embryos at the oblong and crescent stages of nasal placode

de ve lop me nt. -53-

re 50 rbed (16. 70/0) and none of the othe r 15 had MC L. No LC L wa 5 found. The combined resorption frequency was 20.0% and the total

MC L frequenc y was 14. 10/0.

On the basis of the se re sults, examination for deve 10pmental changes caused by the teratogen was carried out, with the object of finding change 5 which may cont ribute to the formation of a MC L.

2. Ear1y Development of the Nasal Placode

At Day 9, 18 hours, the majority of fixed untreated embryos obse rved had no externally visible nasal p1acode development when examined unde r the light mic roscope. The first signs of a placode appear to take the form of a flattening of the nasal p1acode region

(Stage 1). This stage may, howeve r. be abnormal, for although it is found in both the treated and control groups, it is only found in

retarded embryos of the latter. Embryos at this stage may be unde rgoing re sorption. A slight flattened bulge then develops in this area (Stage 2). This flattening of the bu1ge then develops into a

sh a 110,",' de ...... : ssion (Stage 3). As the lateral and medial portions of

this bulge grow, the indentation deepens and the placode reaches the

shallow oval stage of development (Stage 4). From this point on

(TABLE 1), development proceeds as described by Trasler 09bSl,

i. c. through stages nan1ed oval, oblong, crescent, comma, and

epithelial fusion. When the Cre sh Day 9, 18 hour cmbryo is -54-

examined with subillumination, an apparent thickening of the epi- thelium in the nasal placode region is seen at face stage O. When sectioned embryos of this age are stained for alkaline phosphatase activity, an intensely stained epithelium 1 to 3 or more cells in thickness is seen in this position (Trasler, unpublished). Pre- sumably, then, mig ration of neural cre st cells into the nasal placode area is in progress by this time. This aspect will be dis- cussed furthe r and more evidence pre sented in Section 6.

No obviously abnorrnal stages could be distinguished after treatment with 6AN.

3. Rate of Deve lopment of the Nasal Placode in T reated and

Untreated Embryos.

A total of 201 untreated and 191 6AN-treated embryos were examined at six-hour inte ryals afte r the time of t reatment, as de sc ribed in Materials and Methods, Section lb. Among the embryos

in these two groups, 42 obvious re sorptions (18. O~O) we re found in the

treated group out of a total of 233 recognizable implantations, and 48/249 re sorptions 09. 3(1"0) we re found in the c ont roI group... The resorption frequencies were not statistically different (~::{). 124;

P •. H-. Îl. The rate of development of the nasal placode was

determined in relation to somite number and to chronological age. -55-

a. Nasal Placode Stage and Somite Numbe r

The re sults of the comparison of nasal placode stage and somite numbe r of treated and untreated embryos examined are presented in FIGURE 2. Initially, there appears to be a lag in somite numbe r relative to nasal placode stage among treated embryos when compared with the controls. This divergence in de­ velopment of nasal placodes and somites within an embryo occurs at least up to stage 6 of nasal placode development, after which treated and controls tend to overlap. To determine whether this apparent lag may actually be due to an acceleration of nasal placode development rather than a slowing in somite development caused by the te ratogen, both rating s we re individually compared with chronological age of the embryos.

b. Chronological Age and Nasal Placode Stage

As can be seen in FIG URES 3 and 4, and in T AB LE 2, 6AN

causes a statistically significant retardation in the mean nasal

placode stage at and after Day 10, 8 hours of gestation. FIGURE 4

illustrates the changing distribution profile of nasal placode stage

with tin1e. At Day Il, 14 hour s the range of na saI placode stage s of

the t reated group spans almost the entire scale, and an apparent

bin10dal distribution occurs among the treated embryos at this time. -56-

FIGURE 2 ..Ali 10MIli .. A' IACII IIASAL PUCO" Il•• Ile CI7.. • ...'IOS u ~ COII'_ •• e-- -e 1l1A1I.I.AII• Il 1 IIAII.A..... 01 a.

M • • al ,, •:t , Z a. ,1 • ,1 ~ Il , •0 .. 16 /.... -~··f 1. , ,It Il '1-- ,,' 10 1 , • 1 a • • • 7 • • 10 11 12 ,. ,. .. AIAL PLACOD' STA.' -57-

FIGURE 3 CO.'MIIOII Of ..... MIAL fLACON nae. .. Of CI7I&16 .IIRYOI AI ...... , ••nAIIOIIAL A.II --- COIIftOI. 11 ..--. fllAn. C6AII. SlAII...... ,.. -1 11 f , , 11 • • •~ , • ,, -• , •0 • , u e.. 7 .. • ,f. .. \ e S , ," e -z \ • ,f----I' \ a .. .. \ , .. , , t ,A' .... '.IOUII.fIOflllfW ...... ,10 •• .... _ ... --1' • Ya .,...... " lIfM nit nIt "lM .,. '.C"A"" ...ae. CUY/ __ • -58-

FIGURE 4 CHANGE IN DISTlIIUT/ON OF NASAL PLACODE STAGE IN C571U IMIIYOS WITH 'IMI DCON'IOL • TlIATID 'IANI

011. 20H15 ~I - 1 • 1 011. 14H15 ~I • 1 • • - - ",dl -- 011, IHIS ~ ~I zC • n 2 - -- - - 011. 2HIS ~ ':, ...~ - • -o - ~I .1 ;':;-Sj] 0 n n o 1;;; C Dl0.14HIS -• ~I "! z ~ DlO.IHIS i ~ • ...cJ CI C"'"1 ...o ~ ~L 010. OHIS • C' Cl 0 • JI - JO -.. U o 20 IJ i 10 09. llHIS J ,.

09. 12HIS

ùo =r=y=rTTT'TTY=-:-=W4t'4"t'7t' NASAL PLACODI STAGI TAIII.E 2: COll1pllrison of HCllns of Trcatcd and Control Embryos at Various Chronologiea1 Ages.

CIIRONOl.

M;E Trt'atl'd Control Treated Control

x S.E. x S.E. t p(d.f.) x S.E. x S.E. t p(d.f.)

();1~' <),1"2 hl'. 0 0 16.05 0.46

(lI1y <),18 hr. 0.24 0.097 0.11 0.11 0.90 .4-.3(57) 18.61 0.58 18. 76 0.88 0.15 .9-.8(56)

(1l1~' 1 10,0 hr. 2.15 0.36 22.93 1. 63 \J1 'f ()./l V 10,8 Ill', 1. 20 0.20 3.88 0.30 7.51 .001 (11) 20.71 0.97 26. 75 0.56 5,40 .001(13)

(lll~' 10, Il. hl', 2. Il 0.21 3.61 0.31 3.96 .001 (39) 22.42 0.56 28.35 O. 73 6.42 .001(35)

(ln\, 10,20 hl'. 3.76 0.26 7.95 0.29 10.83 .001(84) 25.00 0.66 34.32 0.25 13.24 .001(80)

();lY Il,2 hr. f•• OO 0.33 24.57 1. 67

();1 \' Il.8 hl'. 6. 13 0.80 Il.50 1. 93 2.58 .02-.01(20) 29.86 1. 74 40.80 0.58 5.97 .05- .025(17) * 5.26 0.83 6.26 .001(51) 28.11 1.18 8.98 .001(52) Day Il,1!1 hl'. 11. 72 0.62 39.58 0.49 ** Il.33 0.40 0.53 .7-.6(28) 36.00 O. 70 4.21 .001( 30) (lll\' 11,20 hl'. 12.46 0.34 37.75 0.46

* Compllrisoll of ml'Illl of aIl treatcd cmbryos with mean of contro1s.

** Cll 11lpllrisOll Ill" "\l'Illl of ndvllncl'd mode of trentcd embryos with mean of controls. -60-

This distribution of nasal placode stage among treated ernbryos was tested for bimodality using Haldane' s method (Haldane, 1951).

This test is presented in TABLE 3a. The distribution was fa.lnd to be highly significantly bimodal (p=. 0049). The loss of bimodality on Day 11, 20 hours may indicate that the group of more retarded embryos are lost as resorptions, although a portion of this group may form the more retarded tail of the Day 11, 20 hour dist ribution, and pos sibly be malformed. Because the distribution of nasal placode stage s is bimodal on Day lI, 14 hour s. the means of the two groups are plotted separate1y in FIGURE 3. after arbitrarily dividing the two distributions between stages 8 and 9.

As can be Seen in TABLE 4, the variance of nasal placode stage of treated embryos is significantly larger than that of the controls

only on Day Il, 14 hours (p,. 005-.001). On Day 10, 14 hours

(p,. 005-. 001), the variance cf the trcated group is significant1y

smalle r than that of the c ont roIs. At othe r ch ronological age s at

"... hich comparisons could be made, the variances of the t",,,'o groups

did not diHer significant1y.

After removal of the group throught to be potential resorptions

front the treated en1bryos as a \ ... ·hole on Day 11, 14 hours, the

variance of the treated embryos is significantly smaller than that

of the controls. Since it was not possible to recognlze potential -61-

TABLE 3: Tests for Bimoda1ity(Ha1dane,1951) a.Nasa1 P1acode Stage:Sample at Day Il,14 hours grouped in

three partitions of equa1 range,five stages per partition.

24 22 20 016 >- d 2 =N 2 - 3( n 2 , =26 ~12 10 E S.E.2 - ~2N2 - 8.36 • 8 -o 4 3 o - 0/'2 c z - d2

0- 4 5-9 10 - 14 .. NaSC!1 Plaeode Stage

d2 -%-1/6 (z2_l) -2.815 X 1 2 3 zr: V2N 2 22 "x 3 10 26 d 2 1 1,..

-Correction for normolity

b.Somite Number:Samp1e at Day 11,14 hours grouped in six

partitions of equa1 range,five somites per partition.

10 10 8 ;8 ~ i6 1 E !4 3 i as above: o _' ____~ ~ __ ~ ~ 2[ " S.E.4 - 6.633 L!~~~I~~~.~~~!~~~I~~~l~~~ '" .. " ... -- ... -' ... -" ...... z'- 2.101 14-11 19-23 24-21 29-83 34-31 39-43 SOMITE NUMBER p =.0358

and X 1 2 3 4 5 6 'd.' -3;2 > 2S.E .• 3 8 10 2 10 2 "x ~ -_._- _._------16 d4 TABI~ 4: Comparison of Variances of Treated and Control Embryos of Various Chronological Ages.

CIiRONOI.OGICAI. Var!ance of Nasal Placode Stage Variance of Somite Number

AC:E TH'ated Control Treated Control 2 2 2 2 s ~ s d. f. F -L s d. f. s d. f. F -L Dlly 9,12 hour s 0 18 3.94 18

Illl)' 9, 18 hr. 0.39 40 0.22 17 1.75.25-.1 13.59 40 13.19 16 1.03 .50

Illly 10,0 hr. 1. 64 12 39. 78 14

Dlly 10,8 hr. 0.70 7 0.20 4 3.50 .25-.1 6.57 6 2.50 7 2.63 .25-.1 1 0\ IlJly 10,14 hr. 0.81 17 2.25 22 2. 78 '.025-.01 4.42 13 12.33 22 2. 79 .05-.025 1\) 1 Dlly 10,20 hr. 1. 69 2i. 5.01 60 2.96 .005-.001 9.52 21 3. 75 59 2.54 .005-.001

Dll)' Il,2 hl'. 0.86 7 19.62 6

Da)' Il,8 hr. 10.25 15 22.30 5 2.18 .25-.1 42.29 13 1. 70 4 24.88 .005-.001

*23.23 3i. 3.36 .005-.001 48.75 34 10.88 (.001 Dlly Il,14 hr. 6.92 17 4.48 18 ** 1. 88 11 3.68 .025-.01 6.33 12 1. 41 .25-.1

I~ly Il,20 hr. 2.69 23 5.15 23

* Cpmpl1rison pf vlIrll1ncl' of ail treated embryos with variance of contrcls.

** (~mpllriR0n of Vl1rlllnCe of treated embryos in advanced mode with variance of contro1s. -63-

resorptions at other chronological ages, their presence may be a confounding factor in the comparison of the variances of the two groups. As will be seen in the next section, this inconsistency does not occur in the comparison of variances of the somite number among treated and untreated embryos. Thus the nasal placode and the

somites seem to react differently to the 6AN.

c. Chronological Age and Somite Number

As can be seen in FIGURE 5 and in TABLE 2, mean somite

number among treated embryos is retarded in comparison with the cont roIs. It would appear, then, that 6AN is reta rding both somite and nasal placode development, but has a g reate r effect on the

somites, as seen in FIGURE 2. The change in the distribution of

embryos with various somite numbers with time is seen in FIGURE 6,

An inc rease in the range of somite number similar to that for

nasal placode stage is seen on Day Il, 14 hours. Application of

Haldane's method (TABLE 3b) shows that a bimodal distribution of

somite number exists at this time (p=. 0358). This bimodality

disappears by Day Il, 20 hours, suggesting again the 105s of the

more retarded group as resorptions. Here again, the mcans prc-

sented in FIGURE:; at Day 11, 14 hours arc calculated for the Iwo

unimodal distributions separately, the two groups being arbitrarily

divided between somite numbcrs 29 and 30. -64-

FIGURE 5 COMPAIIION Of MIAN SOMnl NO Of C57.LI. IM.. YOS A' DlffiIIN' GIS'AfiONAL AGES

42 --- CONflOL 40 .---. 'IIAUD C.AN' 3. t SfANDAID 11101

:32 1 • •:» z ,,y , 2 , , 1 , ,, \ POYlNYlAL :1 P10• A• U J IISOI" IONS 22

2

'II. .. ..,.. 1CIfIO "12 11#1 "1t4 INCIIAIING AGI CDAY/HOUI' -65-

FIGURE 6

Cil••• ' •••'''1' •• '''''' Of ""' ••••• CDa6

C COII'IOL • ' ••&n ...... '1 0 11.-. .1.111,,1

.'.11.'. l" •.

• • 1 •• ••••• • " ... 0 1

• ••• 1 • 1

a IL'.. nflo nO a

,.. • .. 00 0 n·:o- aD a OO '\.,,-•....d ...... " •••11- n TW 'lf,p.~ •••...... •••••••••... ·1 -66-

The variances of somite number of the treated and control groups differ significantly beginning at Day 10, 14 hours (TABLE 4), and the probability that the variances differ increase s consistently with time after treatment. When the group of retarded embryos on

Day lI, 14 hours is removed, the two groups are found not to differ significan.tly at this time (p,. 25-. 1). These results are in contrast with those obtained when comparing the variances of nasal placode stage (Section 3b).

4. Effects of 6-Aminonicotinamide on the Early Nasal Placode

Embryos, both control and treated, which had some externally visible early nasal placode development at shallow indentation and early oval stages were measured for head length, head width, distance between the medial limits of the ea rly placode s (distance between placodes), distance bet",:een their late raI limits (distance across placodes), and the width of the nasal placodes (placode size), as described in Materials and Methods (Section B. 1. b). A total of

21 embryos of the treated group and 17 of the control group wcre n1easurcd for thcse parameters, and these measurernents can be

secn in Appcndix A.

AU rneasurcrncnts wc re found to be highly significantly reduccd

ln the trcated group: hcad length (t=5. 2t,Î, 36 d. f. ;p(. 0005, I-tailedl,

head ..... ·idth (t=4. 03t., 3tJ d. f. P <.0005, I-tailcd), placode size -61-

(t=2.800, 34d. f. ; p,. 005-. 0005, I-tailed), distance between placodes

(t= 3. 227, 36d. f. ; p, . 005 -. 0005, I-tailed), and distance ac ros s the placodes (t=2. 728, 36d. f. ; p,. 005-. 0005, I-tailed).

Tests were made of the hypothesis that MeL results from either 1) a lateral shift in the positions of the early placodes, i. e. they become more divergent, or 2) a change in placode size, re lat ive to the head. The forme r predicts an inc rease in both the distance between the placodes and the distance across the placodes

relative to head size, while the latter predicts a smaller placode

size relative to head size. Head length and head width were taken in combination to be a measure of head size. Because the embryos were not a homogeneous group, i. e. they had a range of nasal

placode stages, and were not aU taken at the same chronological

age, variance due to regression was removed in comparison of

means. Since the mean head size s diffe red significantly, the com­

parison of placode parameters was carried out by obtaining means

adjusted for these differences and for regression of the placode

paran1eters on head size by a multiple covariance analysis. The

placode pa ramete r being compa red ' ... ·as taken as the dependent

va riable, while head length and head width we re both taken as

independent variable s.

The mean distance betwccn the placodes, after adjustmcnt, was -68-

found to be highly significantly smaller in the treated group than in the control group (F=8. 564, 1/34d. f.; p,. 01-. 005, and t=2. 927,34 d. f.; p,. 005-. 0005, I-tailed), the goodness of fit of the regression line being highly significant (F=21. 207, 2/34 d. f.; p(. 001). The cor rected mean of the distance ac ros s the placode s was also found to be highly significantly smalle r in the treated group than in the

control group (F=19. 703, 1/34 d. f. ; p(. 001, and t=4. 441, 34 d. f. ;

P (.0005, l-tailed), while the goodness of fit of the regression line

was again highly significant (F=185. 259, 2/34 d. f.; p(. 001). For

placode size, on the other hand, the adjusted mean of the treated

embryo s was neithe r significantly la rge r nor smalle r than that of

the control embryos (F=. 843, 1/32 d. f.; p, 15-.25, and t=3. 66,

32 d. f. ; p, .5-.45, I-tailed) .....-ith a highly significant goodness of

fit of the points to the regression line (F=17. 319, 2/32 d. f. ;p(. 001).

Thus it appears that the size of the initial placodes, i. e. the

size of the region populated by the neural crest ceU complement

which form the mesoderm of the nasal placodes, is unchanged.

AIso, the placodes are not more ...,;idely divergent with treatment.

The recluction in the distances between and across the placodes can

be i~terpreted in two ...';ars. Either the placodes have been shifted

cIo se r t 0 the n1 i cl li n e 0 f the he a d 0 r, bec au seo f the me th 0 cl 0 f -69-

measurement, these changes are a reflection of a reduction in the thickness of the nasal placode. Because the placodes are set at an oblique ang le re lati ve to line s d rawn joining eithe r thei r medial limits or their lateral limits. these measurements include in part the thickness of the placodes as weIl as the distances between them. The differences found between control and treated means,

63p ac ros s the plac ode s (adjusted mean of controls = 58.4% O. 59 micrometer units, and of treated = 54. 8:i: 0.53 micrometer units), and 67 tJ- between the placode s (adjusted mean of contraIs = 34. 3:b O. 94 micrometer units, and of treated = 30. 6:i: 0.85 micrometer units) encompass the diamete rs of only a few ceUs, and thus could easily be interpreted as a measure of a reduction in the number of celI layers of the nasal placodes, with treatment, relative to the general

reduction in head size.

Comparisons "'.. ere also made of residual variance of the three placode pa ramete r s with and without t reatment, afte r the re moval of va riance due to reg re s sion on head length and head width in a

O'mltiple regression analysis. The residual variances, for treated

and control, of distance bet""'een the placodes, were not significantly

different IF::l. 245,18/14 d. f. ;p, .5-.25). The residual variances

of both the distance ac ross the placode 1 F:::3, 58t, 18/14 d. f. ;p,. 01-

.005) and placode size t F:::3. r1 71, If_/14 d. f. ;p,. 01-. 0051 were highly -70-

significantly diffe rent, the t reated animaIs having a large r varianc e than did the c ont roI s. For the latte r, placode size, thi s inc rease in variance with treatment might produce a proportion of treated embryo 5 with a c ritically small placode. This aspect will be discussed at greater length in the Discussion (Section A).

5. Effects of 6-Aminonicotinamide on the Face at the Oblong and

Crescent Stages of the Nasal Placode

The hypothe sis that 6AN re sults in MC L by influencing the face th rough an affect on its width was tested. The faces of embryos

which we re at the late oblong and cre scent stage s of nasal placode

development in both the treated and control groups were measured as

detailed in Materials and Methods (Section B. 1. b.). All measure­

ments of the faces of treated embryos were smaller than those of

normals, and there was no difference in the ratio of head width

ac ross the placodes to distance between the anterior limits of the

nasal pits IAppendix B).

A total of g embryos in the treated group and 21 embryos of the

c ont roI group wc re mea sured fo r face shape. ~ot a 11 a spects of

face shape ".. -cre measurable in all embryos. The mean maximal

width of the head across the maxillary processes at the level of

Rathke's pouch was found to be significantly greater in the control -71-

than in the treated group (t=2. 094, 27 d. f. ;p,. 025-. 01, l-tailed).

Both the mean distance between the posterior limits (t=4. 593,

28 d. f. ;p<. 0005, I-tailed) and between the anterior limits (t=8. 537,

28 d. f. ;p<. 0005, l-tailed) of the nasal pits were found to be highly

significantly larger in the control than in the treated group. The width of the head ac ros s the nasal placode s was found to be highly

significantly greater in the control than in the treated group (t=

2.857, 27 d. f. ;p, .005-. 0005, l-tailed). Although the ratio of

head width ac ross the nasal placode region to distance between the

anterior limits of the nasal pits was greater in the treated than in

the control group (i. e. embryos of the treated group had a narrower

mean face shape than those of the control group), this diffe rence

was not significant (t=l. 282, 27 d. f. ;p, .15 - . l, I-tailed) and was in

. , the opposite direction to that expected. Comparison of the re sidual

variance of the distance between the anterior limits of the nasal

pits in the t".. ·o groups, after removing variance due to regression

on head width at the le ve 1 of the placode s, was found to be non-

significant (F=l. 046, 18/7 d. f. ;p, .75-.5). The teratogen 6A~ thus

results in a significantly smaller face, but does not alter face shape. -72-

é. The Effect of 6-Aminonicotinamide on Neural Crest CeU Migration

The possibility that 6AN administration on Day 9i of gestation affects neural crest cells migrating in the head to the nasal placode regions was studied using the histochemical method described in

Materials and Methods (Section B. 1. b). This hypothesis predicts that the extent of migration, i. e. the relative area of the head in which stained cells are found, will be less in treated than in control embryos at the same stage of development, here measured by somite nUll1.be r.

A total of 42 treated embryos and 69 control embryos were examined afte r the staining procedure was carried out. Migration was studied in control embryos at three-hour intervals of gestational age. beginning at Day 9, 12 hours. the time at which 6AN is admin­ iste red, and continuing until Day 10, 0 hours. Treated embryos \ .... e re examined at the same intervals but starting at Day 9, 15 hours, the time at which the protective dose of N is given. and continuing until Day 10, 0 hours.

What \\'ere assumed ta be selectively-stained ecUs were a very clark blue to black in colour. and werc ah,\'ays found as discrctc eeUs

,FIGCRF: -; and 8 and not as a sheet of eeUs. Backg round lHaining.

r.'sultin;-.: irom spontancous breakdown of the dyc, ·... ·as rcddish in

c()lour, ano iad('d to a li~ht pink aiter storage in the fixative for -73-

FIGURE 7: Magnified view of granules (g) seen in figure 8; surface view in vicinity of eye. Note also unstained cells (c), presumably not neural crest cells.

FIGL~E 8: 19-somite control ecbryo. Sote granules over mesencephalon. anterior ta eye. and in smaii number near base of maxillary process. -73-

FIGURE 7: Magnified view of granules (g) seen in figure 8; surface view in vicinity of eye. Note also unstained cells (c), presumably not neural crest ce 11 s.

~~S~~c~?~alc~. a~tcri~r tn c~c. a~d ~~ s~al! :1~:-:-.~t.·r ~I~'a'!"" ~ast: ,-': ::-.axillar~: ~!"CCt·ss. -74-

several weeks. In this section, the presumed neural crest cells seen after staining will be refered to as "granules".

In gene raI, when obvious artifacts we re seen, they we re found in aU members of a litter whieh had been stained in the same incubation bath. Artifaets were of the same colour and intensity as the granules, but were much larger and were often geometric in shape. They we re usually attached to the surface of the embryo, although they were sometimes found beneath it. Artifacts were found mainly among litters used to develop the technique. Granules tended to fade and finally disappear after the embryos had been stored for longer than a month. What were assumed to be dead embryos showed no specific granular stain and had almost no back­ ground staining. After removing embryos showing obvious artifacts, onlyabout 15 control and 10 treated embryos were available for comparison. The granules seen magnified in FIGURE 7 and unmag­ nified in FIGU RE 8 we re measured and their actual size calculated from the known magnification. Their diamete rs ranged from

1O-25~ , within the range of average eeU size. Embryos which had obvious a rtifact s we re ignored in de le rmining mig ration patte rn s.

In general, control embryos in which the neural tube had not yet begun to close in the head region (Stage 0, 7-10 somite 51. had onlya few granules at the crest of the neural folds at the level of the -75-

juncture of the presumptive prosencephalon and mesencephalon

(FIGURE 9 a). By Stage 3 of closure, (11-13 somites), more granules were seen over the mesencephalon and presumptive rhombencephalon and were found to range further from the dorsal midline of the head (FIGURE 9 b). Once closure was complete

(Stage 6, over 14 somites), these stained cells had dispersed ventrally to a greater extent than the previous stage (FIGURE 9 c) but were still found in the region of the mesencephalon and rhom­ bencephalon. When these embryos were examined in dorsal view, granules were found to be absent in a narrow band along the dorsal midline (FIGURE 9 dl. At 16-20 somites, the granules were now found further from the dorsal midline, surrounding the eye except ante riorly, at the base s of the vi sce raI arc he sand near the nasal placode regions (FIGURE 9 e). A wider band along the dorsal mid­ line was nov.: devoid of granules. At 21- 24 somite s, the granule 5

" .. ere seen in the nasal placode region, around the eye, within the

visceral arches, and throughout the rest of the head (FIGURE 9 f).

Granule s ""e re also seen dorsal tO, at the leve lof, and ventral to

the somites. posterior to the otic vesicles, often in the forelimb

buds. and over the heart. Granules were usually not present posterior

to the level of the heart. but some were seen in the membranes of

the yolk sac and the amnion next to the abdominal region. and are FIGURE 9: Apparent extent of neural crest cell migration (stippled area) in control and treated embryos of various morphological ages.

E, otic vesicle; H, heart; N, nasal placode region; 0, optic vesicle; V, visceral pouches. a. Control, neural tube stage 0, 7-10 somites. b. Control, neural tube stage 3, 11-13 somites. c. Control, neural tube stage 6, 14-16 somites. d. Control, neural tube stage 6, 14-16 somites; dorsal view of head. e. Control, 16-20 somites. f. Control, 21- 24 somites. g. 6AN- treated, 15-17 somites. h. 6AN- treated, 20-23 somites. -77-

a b

c d

e f

9 h

-79- probably migrating germ cells (B. V. Konyukhov, pe rsonal com­ munication). As we11, a few characteristica11y stained granules were seen around the posterior neuropore. Staining was relatively consistent in embryos in which artifacts were not apparent.

Several control experiments were run. One control experiment on untreated Day 9, 15 hour embryos was carried out with the reaction mixture lacking the substrate. A very light reddish back­ ground staining was seen, and no intense staining of granules occurred. ln another control experiment, oIder Day 10, 14 hour and Day 11, 12 hour embryos we re run in the complete reagent

mixture and examined for specifie staining. In Day 11~ embryos, the eye vesicles, the isthmus of the nasal placode, and the surface

of the heart were found to have the characteristic intense gt"anular

staining. Granules were aiso seen in the isthmus of the nasal

placode and over the surface of the head posterior to the eye. The

latter presumably are either precursors of the chondroblasts of the

skull or pigment ce Ils.

Examination of 6AN- treated embryos showed that the degree

of dispe rsion of the se granule s from the dorsal midline of the head

was less than that among control embryos of equal somite numbers.

T reated embryos of 15-17 somite s had granule dispe rsion (FIGURE

9 g) similar to that of 11-13 somite embryos de sc ribed above. T reated embryos of 20-23 somites had neural crest migration equivalent to that of 14-17 somite control embryos (FIGURE 9 hl.

It appear s that 6AN treatment re sults in a retardation of neural crest cell migration. The technique. however. is as yet imperfect. and it is yet to be shown histologically that the stained granules are indeed neural crest cells. at least in their positioning relative to the ectoderm. mesenchyme. and neural tube. There is good evidence that the granules seen are neural crest cells. since the change in the patte rn of thei r position s with age ag ree s with the re suIt 5 obtained by Johnston (1966) in the chick using a radioactive marker to trace the path s of the se ce Ils. -81-

B. Selection Experirnent l. Method of Selection

TABLE 5 lists the total number of females and males being tested in generation 7, as well as the breakdown of cleft lip

response. The numbers of animaIs and type of response in the

ancestry of these generation 7 animaIs is given as well.

2. Induced Cleft Lip Frequency

Selection in the Median line was for 6AN-induced MC Land

against LCL while in the Lateral line selection was for 6AN-induced

LCL and against MC L. Thus successful selection would be indicated

by a high frequency of MCL accompanied by a low LCL frequency for

the Median line and the opposite for the Late raI Hne.

The numbers and percentages of embryos showing a cleft lip

re sponse to 6AN are shown in T AB LE 6, and in FIGURES 10-14. For

reasons to be discussed below, the frequencies shown for the fifth

generation of selection are the results of the 1 3/8 6AN treatments

only.

ln the fifth gene ration of se lection, seve raI highe r dose s of 6A N

were te sted when it was found that the single dose used up to that

point "',a s p roduc ing ve ry low frequenc ie s of c left lip. Initially

a l~ dose was tried, which was then increased to l! dose. The

latte r ...... as te sted on only th ree female s, of which one re so rbed -82-

TABLE 5: Generation 7 C1eft Lip Response and Ancestry.

GENERATION LINE Total Positive Negative Non- Mixed Untested Reactors Reactors Reactors Reactors NO. 99 99 dt! 99

L 22 7* 1 1 6 4 6 5 1 1 8 o 7 M 35 8** 10 5 o o 8 4 2 1 15 o

L 10 4*** 2 2 o o 5 3 o o 3 o 6 M 10 7 9 6 o o 1 1 o o o o

L 6 3 o o o o 1 1 o o 5 2 5 M 8 5 3 3 o. o o o o o 5 2

L 3 3 3 3 o o o o o o o o 4 M 8 4 8 4 o o o o o o o o

dt! assigned to different classes if they gave severa1 different reactions.

*~--3 non-reaction,l negative reactions;l d--1 non-reaction,2 negative

reactions;l ~--l non-reaction,l mixed reaction;l ~--non-reactions only;

1 ~--positive reaction only;and 1 ~--negative reactiûn ûnly--tûtal 7 QQ.

**2 r't'--l non-reaction,2 positive reactions;l tf--1 positive reaction,2 mixed

reactions;2~j'--negative reactions only;and 3 ~--positive reactions only--

total 8 :~.

***1 ~--positive reaction only;2 r:",J--non-reaction only;and 1 ~--l positive

reaction,l non-reaction--total 4 r~' TAnu: 6 : tnduccd Cleft Lip Rcsponse to 6-AN up to the Sevcnth Generation of Selection (F 10).

C;EN~:RATION Ovcrall CL Lateral Une Median Une

NO. Frcquency Total CL LCL MCL Total CL LCL MCL

N CL 'ïo N CL 7.. CL ï.. CL 7.. N CL i. CL 'X CL 'X

0 255 94 36.9 131 42 32.1 42 32.1 0 0 124 52 41. 9 6 4.8 46 37.1 (F 3)

1 395 78 19.8 188 39 20.7 Il 5.8 28 14.9 207 39 18.8 8 3.9 31 15.0 & ~ 2 144 43 29.9 62 14 22.6 0 0 14 22.6 82 29 35.4 6 7.3 23 28.0 1

3 123 4 3.2 47 0 0 0 0 0 0 76 4 5.3 0 0 4 5.3

4 599 71 ll.9 312 26 8.3 Il 3.5 15 4.8 287 45 15.7 21 7.3 24 8.4

5* 76 16 41. 6 45 10 22.2 8 17.8 2 4.4 31 6 19.4 0 0 6 19.4

6 211 84 39.8 127 38 29.9 6 4.7 32 25.2 84 46 54.8 2 2.4 44 52.4

7 173 43 24.9 89 14 15.7 3 3.6 11 13.3 84 29 34.5 2 2.4 27 32.1

* R~sults of trcatmcnt with 1 3/8 dose only. -84-

FIGURE 10

.NDUCID CL alPON11 .N LAnRAL MD MIDIAIII UNII ...... &eL ' ...... 11_ 50 • ---. MCL ...... --. ~L •••••, 1.­ """'-IICL •.•.. 1.- .­ tA CL • .~ , ~ , ~ 10 , " ,, "" , "~ 10 ".

1 • 7 -85-

FIGURE 11 ... 1 .NDUCID MCl '.IOUINCY .. --. l ..... Il. __ •••, •• Il.

40 Cl" ... II 1 .. 1 .... 1 .. 20 , .. .. 1 " ...... u ,1 1 10 , 1 1 1 1 \ ,.------,/ \,:...... ',' U U 1 2 1 4 5 • 7 • __r •• '_, ...... c •••• -86-

FIGURE 12

60 INDUCID LCL ••IGUINCY

0--0 L.'.r.1 Il•• 50 • ---.....1 •• 11_

... 40 CL

20

10 , 7",. , ...... , ...... 1 2 a 4 5 • 7 .... ,.. 1_. .1 ••I.e .1.. -87-

FIGURE 13

60 INDUCID CU'T LIP -lATIIAl LlNI-

50 ·~lCl a----Q MCl

40 ~ Cl 30

Jf.2U ,1\ , \ , '\ 20 i , \ \ " ~" 1 \ , " ! " " "a lU 10. i , 1 1 , '. 1 2 a .. 5 6 7 ••.• r...... f ••I.c' ... -88-

FIGURE 14 601 • i INDUCID CLI.' LI' -MIDIAN LINI-

50~ .---. LCL ~MCL

40 Cft CL 30

i 201 1

10 7~ ... __ e" ~~ .------.----" " ~.', 1.. '....' '..... _~------\4 "1,' "'li_- - -- 1 23456 7 •••• r.tl... .1 •• I.ct ••• -89-

completely and another died. Finally an intermediate 1 3/8 dose was tried.

A total of fifteen HUers were treated with the l~ dose and fourteen with the 1 3/8 dose. The results of these treatments can be seen in TABLES 7a and 8. Since the response of the two lines to the two doses might differ, the interaction and/or independence of dose, selection line, and type of c1eft lip response in embryo frequencie s was tested (Lewis, 1962), with a X2.analysis of a three-dimensional contingency table. A summary of this statistica1 method as well as ,,2. va lue s obtained can be found in TABLE 7b.

It wa s found that the re was a significant difference in total c1eft lip frequency between the two strains (p,. 02-. 01). As well, there was a highly significant first order interaction (. 001) pl between line and type of cleft lip response. The Median line had significantly more

MC L than the Lateral line and the Lateral line had significantly more

LCL than the Median line. The results of different doses were inde­ pendent (p,. 25-.1) of both line and type of response. No other inter- actions were found, either Cirst or second-order.

When the results of the two doses were compared, it was found

that only the total cleft lip frequency produced was significantly

1 altered (X = 4. ï22;p,. 05-. 01) with a cleft lip frequency of 10. 4'7'~ in

the Median line against 15.6'7''0 for the Lateral line. Therc was a -90-

TABLE 7a Frequencies of C1eft Lip amang Embryos:Results of Different

6-AN/N Dosages in the Fifth Generation of Selection.

DOSAGE Total CL LCL MCL

6-AN/N LlNE N CL % CL '% CL %

L 45 10 22.2 8 17.8 2 4.5 1 3/8 M 31 6 19.4 0 0 6 19.4

L 77 9 11.7 7 9.1 2 2.6 1 1/4 M 36 1 2.6 0 0 1 2.6

TABLE 7b Method of Ana1ysis of Data in TABLE 7a. Using Three- 2 Dimensiona1 Contingency Table for (Lewis, 1962).

L Line= n = 19 •• 1 .. Co1umns(C) M Line= n = 7 6AI(IN11 ~ 10 •• 2 3 1 /8 0 6 16 n.jk 1 1/4 dose=nl. . = 10 0 7 Rows(R) 1 3/8 = 16 n··k 19 7 dose=n2 ••

LCL=n. • = 15 l Layers(L) MCL=n. 2• = Il N = 26 Ho: Pijk = Pi •• P.j.P •• k

2 Total::C 2(Theoretical) = (o-e) where e = 3.25 e =24.827 (rcl-l d.f. = 4 d.f.)

Independence X 2 :7'2 R (Theoretical) = 1.385 (r-l=1 d.f.) '2 Xc (Theoretical) = 5.539 * (c-l=l d.f.) (Theoretical) = 0.615 (L-l=1 d.f.) rJL where e = 13 -91-

TABLE 7b : ( Continued ). 2 InteractionX : Remove one dimension at a time

First - order interaction : 2 !:X = 0.615 (r-1 X c-1 = 1 d.f.) RC ~2RL=0.615 (r-1 X L-1 = 1 d.f.) 2 _ *** 'J; CL-12.462 (c-1 X L-1 = 1 d.f.)

where e = 6.5

Second - order interaction

=3.596 (r-1 X c-1 X L-1 = 1 d.f.)

* .02)p).01 '

** p < .001 -92-

TABLE 8: Frequencies of Litters Showing a C1eft Lip Response.

Resu1ts of Different Dose Leve1s in Generation 5.

DOSAGE LlNE Total CL LCL MCL Mixed CL *

6-AN/N N CL % CL 'X. CL '70 CL 'X.

L 7 5 62.5 3 42.9 1 14.3 1 14.3

1 3/8 M 7 4 57.1 0 0 4 57.1 0 0

L 12 7 58.3 5 41. 7 2 16.7 0 0

1 1/4 M 3 1 33.3 0 0 1 33.3 0 0

* Inc1udes 1itters in which at 1east one MCL and one LCL were

found. -93-

nonsignificant inc rease with the 1 3/8 dose in total MC L frequenc y

(X2.=3. 801;p,. 1-. 05) in the two lines combined, and in MCL frequency in the Median line alone (x""= 3. 2809;p,. 1-. 05). No significant difference (X2.= 1. 6598;p,. 2-. 1) was fcnnd in the percentage of litters responding to the two dosages (TABLE 8).

Comparisons of litter size (TABLE 9) among animaIs given the two dose levels showed that litter size in the Median line was higher than that of the Lateralline for the lower dose. A smaller litter size was obtained in the Median line than in the Lateral line with the higher dose although the difference was not significant (t=. 845,

12 d. f. ;p,. 5-.4). There was a significantly lower litter size for the higher dose than for the lower in the Median line (t(1-tailed) = 2.489,

8. d. f. ;p,. 025-. 01), while there was no change in liUer size in the

Late raI line using the two dose levels.

For the reasons given above, i. e. the trend to give a different cleft lip frequency in the Median line which did not occur in the

Lateral line, and the differential change in litter size, the results obtained at the h ..·o dose levels we re seg regated. Only those obtained using the 1 3/8 dose are included in TABLE 6 and in FIGURES 10-14.

The induced MC L re sponse in the Median line has consistently

tended to be higher than that in the Lateral hne (FIGURE 11) up to

the current generation. This is not so for the induced Le L frequency -94-

TABLE 9: Litter Size, Generations 5 to 7 (1 3/8 6-AN/N).*

GENERATION Lateral Line Median Line

NO. Litter Litter

Embryos Litters Size Embryos Litters Size

1 3/8 45 7 6.4 33 7 4. 7 * 5 1 1/4 77 12 6.4 26 3 8.7

6 127 23 5.5 84 15 5.6

7 90 15 6.0 89 18 4.9

* Except in generation 5,where resu1ts are shown for both dose 1eve1s. -95-

in the two lines (FIGURE 12). A large drop in LCL response in the

Late raI line was seen between gene rations 5 and 7. This may have occurred because it was not possible to breed from positive­

reacting Lateral line females in generation 5 due to their infertility.

It was therefore necessary to use the offspring of non-reacting

females and of females which had not been tested.

Although LC L incidence was significantly highe r in the Late raI

line than in the Median line (17. 8% vs. 0%. p=.0114) in generation 5

of se lection (1 3/8 dose only). this diffe rence was lost in the two

succeeding generations. The higher LCL frequency in the Lateral

line in generation 5 is in contrast with the significantly lower

(X2.=5. 04;p •. 05-.02) LCL frequency found in the Lateralline relative

to that in the Median line in generation 4 (3. 50/" vs. 7. 30/0). A trend

is evident for MC L response to be greater in the Median line than in

the Lateralline (FIGURE ll) in generation 5 (19.4% vs. 4. 4%;p •. 1-.05)

and afte rwards in gene ration 6 (52. 4~o vs. 25. 2%;p < . 001) and in

generation 7 (32. l~o vs. 13. 3%;p<. 001) this difference became highly

significant.

Within the Late raI line (FIGURE 13) the LC L frequency was non­

significantly higher than the MC L frequency (LC L=17. 8'1'0, MC L=4. 4(]'~;

p •. 1-,05) in generation 5, while in generation & LCL frequency was

significant1y lower (LCL=4. 7'7"ç, ~iCL=25. 2 11'v;p<. 001) and no -96-

significant difference was seen in generation 7 (LCL=3. 6%, MCL=

13. 3%;p,. 5-. 3).

The same comparison in the Median line (FIGURE 14) shows a

very significant difference in generation 5 (MCL=19. 40/0, LCL=O%; p=. 0086) with MCL frequency higher than LCL frequency. In the

next two generations, MC L continued to occur with a higher frequency

than LC Land both difference s we re highly significant (p < . 001).

The drop in LCL frequency in the Lateralline between generations

5 and 7 was not due simply to a reduced response to the teratogen

within litters but to a drop in the number ofpositive-reacting litters,

as can be seen in TABLE 10. The frequency of positive-reacting

litters in the Median line increased between generation 5 and

generation 6 and dropped again in generation 7.

3. Spontaneous C left Lip Frequency

One spontaneous lateral cleft lip was found in the Lateral line in

gene ration 7 of se lection, out of a total of 109 embryos of this line

examined on the day of birth up to May 20, 1971. Up to this date, a

total of 111 newborns of the Median line and 132 newborns of the

Cont 1'01 line had been examined on the day of birth. ~o spontaneous

cleft lips .... 'ere found in the Median line, and one spontaneous ~1C L

....;as found in the Control line. There was no significant difference -97-

TABLE 10: Frequency of Litters Showing a C1eft Lip Response,

Generations 5 to 7 (1 3/8 6-AN/N).

GENERATION Total CL LCL MCL Mixed CL *

NO. LINE N CL CL % CL CL

L 7 5 71.4 3 42.8 1 14.3 1 14.3 5 M 7 4 57.1 0 0 4 57.1 0 0

L 23 6 26.1 2 8.7 3 13.0 1 4.4 6 M 15 15 100 0 0 13 86.7 2 13.3

L 15 8 53.3 1 6.7 6 40.0 1 6.7 7 M 18 12 66.7 0 0 10 55.6 2 Il. 1

* Inc1udes 1itters in which at 1east one MCL and one LCL were found. -98-

between the Lateral line spontaneous LC L frequency and that of eithe r the Median line (p=O. 4500) or the Control line (p=. 4523).

The spontaneous MC L frequency in the Control line was not

significantly different from that found in either the Lateral line

(p=. 5477) or the Median line (p=. 5432). No spontaneous cleft lips

we re found in an y of the line s in eithe r gene ration 5 or 6. One

spontaneous LC L had previously been found in gene ration land

three in generation 4 (Hamly, 1971) in the Lateral line.

4. Morphological Stage at the Time of T reatment

The possibility that selection for a diffe rence in the type of

cleft lip re sponse would lead to a change in morphological stage at

the time of treatrnent was studied. Such a difference in morpho­

logical stage rathe r than the postulated diffel"ence in face shape might

be a causative factor in predisposing the embryo to a particular form

of c le ft lip.

One female of the Lateral line and three females of the Median

Une whose response to bAN had been previously determined were

sacrificed on Day 9,10 hours, their litters being of generation t-.

The Lateral line litter, containing 10 embryos, was obtained

from a previously non- reacting female. Three htters with a total

of 2i embryos we re obtained from positive- reacting ~edian hne

fenlales. The embryos were rated for morphological dc~clopmcnt -99-

(Appendix Cl. None of the Lateral line embryos were in the 9A

stage itself, although some were in a late 9A stage, when the overall ratings were examined. This is in agreement with the

results found for the third generation of selection by Carole-Ann

Hamly. The mean morphological rating of the Lateral line embryos was late 8B, while that of the Median line embryos was 9A. The

Lateral line embryos had a mean somite number of 16. 7~. 26, while

the mean somite number of the embryos of the Median line was

16. 3 z. . 26.

5. Embryos Examined on Day 10, 20 hours

Embryos of the two line s whose type of reaction to 6AN was

known were also obtained on Day 10, 20 hours for comparison of

face shape and morphological ratings.

One litter of 10 embryos was obtained from a positive-reacting

mother of the Lateral line, and one Htter of 12 unresorbed embryos

from a non-reacting mother of the same line. Four Htters

totalHng 36 unresorbed embryos were collected from positive­

reacting females of the ~ledian Hne, and one Htter of 13embryos

from a mixed- reacting fe male of that line.

a. Morphological Stage

A difference in morphological stage of the embryo at the time -:101-

70 NAIA&. PUCODI ITA.I ON D1t/2O la M ... l lINIS o l lINI M • M lINI

H

... lMU'fOS

20 1.

NI GYM OVAl oe&ONO cnlClNT COMMA NASAL 'LACODI UA.' -102-

The distribution of tai! somite number of the two lines can be seen in FIGURE 16. Here again. four of the six embryos of the

Median line with less than five tai! somites are found in the one anomalous litter mentioned above.

The mean nasal placode stages in the two lines are not signif­ icantly different (t=. 761. 69 d. f. ;p,. 5-.4) but their variances are

(F=3.064, 48/21 d. f. ;p •. 005-. 001). When the proportion of embryos in the comma stage is compared for the two lines, the Median line is significantly more advanced than the Lateral line (p,. 05-. 02). Thus there appears to be a trend for the presence of more advanced nasal placode s in the Median line.

In addition, comparisons of the means (t=1. 464. 69 d. f. ;P •. 2-. 1) and the variance s (F=l. 440. 48/21 d. f. ;p, . 25-. 1) of tai! somite number in the two line s show no diffe rence s. When the numbe r of embryos of the two lines having fewer than eight tai! somites are compared with embryos having eight or more. a highly significant difference is found (p=. 00105, Fisher's exact ~~). It would appear then that there is a t rend for a portion of the embryos of the

Median line to be more advanced than those of the Late raI line. The

Lateral line embryos had a mean tail somite number of 5. 7 Ê .2lJ,

".. -hile the mean tail somite numbe r of the embryos of the :-"1edian line -103-

FIGURE 16 tG 'AIL SOMlfi NO. ON DIf/H , •••• L UNIS

50 0 L UNI •• UNI

•

fAIL SOMlfi MC). -104-

was 6. 2 ±. 21. b. Face Shape

The hypothe sis that selection for a difference in type of cleft

Hp response will lead to a difference in face shape was tested.

Measurements of the heads at late oblong and crescent stages of embryos between 5 and 7 tail somites from positive- reacting

females of the two lines on Day 10, 20 hours revealed no significant diffe rence (t=. 604, 21 d. f. ;p,. 9-. 5) in the distance between the

anterior limits of the placodes. A significant difference in mean

head width ac ros s the nasal placode region ( t=2. 199, 21 d. f. ;p,

. 025-. 01, I-tailed) was seen with the Lateral group wide r th an the

Median. No difference (t=1. 060, 21 d. f. ;p, 0.4-0.2) was found in

the ratios of he ad width divided into distance between placodes in the

two line s. The re suIts of the se measurements are pre sented in

Appendix E. Thus no difference in face shape between the two line s

could be distinguished. -105-

VI. DISC USSION

A. Effect of 6-Aminonicotinamide on the C57BL Face

The teratogen 6AN affects the rate of development of both the nasal placode and the somites. Somite number is more severely affected than is the rate of differentiation of the nasal placode. The effect of 6AN on the placode is one of retarding both its differentiation and its growth. Although the variance of somite number among treated embryos increases relative to that of the untreated embryos with time

after treament. the same effect is not seen for variance of nasal

placode stage. Treatment with 6AN on Day 9~ results in vertebral

fusions (Goldstein!:.!,. al. 1963) in the C57BL strain of mice. possibly

by its effect on the somites. A relative increase of variance with

time after treatment can be considered to be due to a disturbance of

developmental stability by the teratogen. The lack of a consistent

relative increase in variance of nasal placode stage with time after

t reatment. such as that seen for somite numbe r. togethe r with the

le sse r severity of e ffect of t reatment on the placode s than on the

som.ite s. sugge sts that the state of diffe rentiation of the na saI

placode is not a major locus of action of the teratogen involved in the

production of ~1C L. This is not surprising. since this defect could be

due to a lack of merging of the two medial nasal processes. a -106- quantitative rather than a qualitative effect, and the fact that there

are no obviously abnormal stages lends furthe r weight to this last

idea.

It appears more likely that it is the mass of tissue present in the

placode that is a determining factor leading to MC L. Administration

of 6AN results in absolute decreases in aU parameters measured in

the face and head, both at early nasal placode stages (shallow

indentation and early oval) and at later stages (late oblong and cres­

cent) when the placode is weU differentiated. In addition, in the earlier

stages, the thickness of the nasal placode is more affected than is

the rest of the head. Although the difference, after adjustment,

between the mean placode thickness of treated and controls,

measured as a function of both distance between the placodes and

distance across the placodes, is slight, in the order of only a few

cell diameters, it must be remembered that this difference measures

the relative degree of effect of 6AN on head size and placode thickness.

The absolute difference in thickness between treated and controls is

larger, of the order of 6 to 16 cell diameters.

T reatment with 6 AN doe s not result in a wide r positioning of the

placodes, either early or late (as defined above), and 50 it is unlikely

then that an induced wider face shape is a factor in the pathogenesis -107-

of induced MC L. The already wide face of the C57BL/ 6 embryo, even without treatment, may, of course, interact with the quantitative changes in the nasal placodes resulting from treatment. ln the early face the treatment does, however, affect the mean placode thickness to a greater degree than it does head size, although the change is probably no more than a few ceU layers in this region. The distance ac ros s the placode give s a measure of thickness of the lateral elements of the placode, which will later form the lateral nasal proce ss. Both the mean and the va riance of this parameter are affected by treatment; the mean is reduced and the variance increascd. As the lateral nasal process is not directly in­ volved in the embryology of the cent raI face, it is unlike ly that the se change sare involved in the production of the defect. Of g reate r interest are the changes in both the distance between the placodes, being a measure of the thickness of the medial elements of the nasal placode s. and the p lac ode size, taken to be a function of placode width

measured. The medial elements of the early nasal placodes later

form the medial nasal processes. The reduction of the thickness of the

placode at this position may result in a later deficiency of tissue in

the medial nasal processes. The increase in variance of placode

size could result in a proportion of the treated embryos having a

critically small placode ",.. idth. This, coupled with a smaller -108- placode rnass in these embryos. could place them beyond the threshold for MC L production. As the face remains smaller in treated embryos than in controls at the later stage examined, the

reduction seen in the early face seems to linge r, and not disappear soon afte r treatment. lt appears, then, that MC L can be conside red a quasicontinuous trait, where the threshold is the critical rnass and distribution of tissue in the medial nasal processes

nece ssary for their normal mergence.

The events occurring at and soon after presentation of the

teratogen which manifest themselves later as a critical reduction

in mass and area of the nasal placode in those embryos which will

show the defect are probably of two types. One is an effect on

mitotic rate (Karnofsky, 1964) both of those cells which make up the

ea rly placode, which are of neural cre st origin, and of the cells of

the rest of the head. The other alternative is a retardation of neural

cre st ce 11 mig ration. This latter disturbance of the normal patte rn

of embryogenesis could result in either an insufficiency of these cells

arriving at the placode region, or their arrivaI too late ta react ta

the inductive stimulus to settle and differentiate presented by the

cellular environn... ent in the presurnptive placode region. In sorne

ernbryos, this would lead ta an underpopulation of this re~ion, 50 -109-

that these cells are less densely arranged and are distributed over a smaller area. It is of course possible that although neural crest migration is slowed, the normal complement of cells reach the placode and only then is the disturbance of their cell metabolism after treatment manifested as an arrest or slowing of their mitotic rate.

The re is some evidence that a te ratogen can produce a deficiency of neural crest material. Landauer (1952), working with

Black Minorca chicks, and treating with either insulin, pilocarpine, or boric acid at 96 hours of incubation, caused abnormal down pig­ mentation as well as skeletal defects. Facial skeletal abnorrnalities caused by pilocarpine included shortening of the mandible, often associated with facial clefts, and those associated with borie acid

t reat ment inc Iuded reduction of the mandible, cleft palate, and facial

coloboma. Insulin treatment re sulted in shortening of the maxilla.

1 his stain of chickens has transitory piebald spotting, disappearing

aCte r the fi rst molt, and this condition has been found to re suIt Crom

de fective neural cre st cell mig ration (Schaible, 1968). The un­

treated newborn chicks are normally pigmented on the dorsal body

surface, partially pigmented on the vent raI thoracic surface, and

normally in the extremities except in their most distal parts, while -110- the more ventral surfaces of the body and head and the distal parts of the extremities are unpigmented. After treatment, pigmentation was limited considerably. The severity of hypopigmentation was generally correlated with the seve rit y of skeletal malformation.

When the severity of skeletal malformation was slight, hypo­ pigmentation was limited to parts of the face, the distal portions of the wings, and the scales of the toes. With increasing severity of skeletal defects, there was a tendency for the extent of the pigmentation defect to include the entire head and neck, aU of the wings, most of the hind limbs, and a larger portion of the ventral

surface. Landauer postulated that the pigmentation defect is a

re suIt of interfe rence with neural cre st celI mig ration and! or

melanoblast activity within the affected regions. Administration of

nicotinamide after treatment completely prevented both the skeletal

and the pigmentation defects resulting from insulin treatment, and

lowe red the frequency of defects caused by pilocarpine and boric

ac id. Landaue r sugge sted that the va rious defect s had common

epigenetic pathways, the teratogens acting on carbohydrate

metaboli sm.

In Landauer's study, the treatment could be interacting with the

~enotype of these embryos, p:o:"~ducing hypopigmentation more pro­

nounced than normal. Both Johnston (196L) and Schaib1e 119[8) have -111- found evidence that neural crest ceU mig ration is underway in chicks at 96 hours of incubation, the latter in the Ancona strain.

In the present experiment, nicotinamide is also used as pro­ tection against the teratogenic effects of 6AN. Nicotinamide has been found to completely protect against the teratogenicity of 6AN in the production of cleft palate. It appears that the biochemical action of

6AN may be similar to those of the teratogens used by Landauer in the experiment cited above. As mucopolysaccharides are common to both the skeleton and the medium in which neural crest cells migrate, it is not unlikely that bAN is depressing the rate of synthesis of these substances. In any case, this teratogen has been found to have this effect on mucopolysaccharide synthesis in rat hearts at a developmental age equivalent to that of the mouse embryos at the time of treatment in this experiment (Overman and Beaudoin,

1971). In the present study, neural crest cell migration was found to occur in the head at this time. This finding is corroborated by those of Milaire (l959) and Mulnard (1955).

Newborn mice carrying the dancer (Dcl gene in the homozygous condition, as mentioned earlier, have the LCL defect. As this gene

is thought to cause a neural crest defect, it would be of interest ta use the whole embryo specific staining technique described earlier ta stud')o' the migration of these cells in the heads of mouse embryos -112- of this genotype, as weIl as those of other genotypes which affect neural cre st cell migration. At pre sent, it is not feasible to apply Rawle 5' (1947) and Maye r f 5 (1962, 1967) technique s for studying neural crest cell migration in the mouse to study neural crest defects which are not pigmentation defects. It is also not presently feasible to use isogenic grafts of radioactively-labelled neural tubes to nonlabelled body regions in mice. Following migration with the staining technique pre sented here, although limited, in scope has the advantage s of being both feasible and simple, making mig ration patte rns readily visible as well as rate of mig ration of neural crest cells which form parts of surface structures such as the nasal placode, the visceral arches, or the skeletal elements of the head. One drawback of the technique is that it will stain any structure or ceIl type which has high alkaline phosphatase activity , and this could in some cases give misleading results. An example of such a misleading result in the mistaken identification of migrating primordial ge rm cells as neural cre st cells. A readily apparent effect of 6AN on the treated embryos is their reduction in

size. The finding that the nasal placodes are probably reduced in their thickness could be due, at least in part, to reduced mitotic activity of the placodal mesectodermal precursor cells at this site.

ln orde r to dete rmine whethe r mitotic rate i s diffe rentiall y reduced in -113- the early nasal placode region compared with the rest of the head

after treatment, it would be necessary to compare the mitotic index

in the head and nasal placode region of 6AN-treated and control

embryos. The findings that the mean face shape is not wider and the '. variance of face shape remains unchanged after treatment in the

older embryo support the finding that the positioning of the early

nasal placode is not wider after treatment. Studies by Jacobson

(1963 a, b, c, 1966) on Taricha torosa and Amblystoma punctatum

have shown that the positioning of the nasal placode, as weIl as

that of the eye and ea r ve sic les, is determined by inductive inte r­

actions with other embryonic tissues. The nasal placode position is

dete rmined by inductive inte ractions of the placodal epide rmi s with

first the ante rior endoderm and late r the prechordal plate and neural

folds and finally is maintained by the forebrain. Accordingly,

abnormal positioning of the nasal placodes would require abnormal

positioning of the induce r tissue s or a disturbance in the relative

potencies of their inductive capacities and the competence of the

placoda 1 epithe Hum to re spond. Since no change in position of the

nasal placode was seen in this experiment, it is unlikely that this sort

of disturbance is occurring, at least up to the stage at which positioning

is dete rmined. -114-

Seve raI authors (Le jour-Jeanty, 1966; Deluchatch, 1969), studying induced cleft lip in the mouse and rat embryos, have pointed to this inductive interaction between the nasal placode and its various inducer tissues as the morphogenetic step which is inter­ fe red with by t reatment. Lejour-J eanty (966) noted that t reatment of Wist&r rat embryos with hadicidin, a penicillin derivative, resulted in sudden degeneration of the anterior end of the telencephalon, edematous me soderm, and nec rosis of the nasal placode. This led to late raI c left Hp formation. The effect on the brain and the placode were simultaneous, however, 50 that no true cause and effect

relationship was seen. T reatment could have affected the two independ­ ently.

Deluchatch (1969) treated mouse embryos with X-rays and hypervitaminosis A, and treated rat embryos with a deficiency of

pantothenic acid. MeL resulted, at times associated with exencephaly.

Again the evidence presented is insuHicient to show that inductive

inte ract ion sare aHected.

The biological meaning of "face shape" is vague. Since it

measures the positions of the nasal pits, rathe r than the nasal

processes surroundin~ them, a difference in face shape could be due

to eithe r a difference in the position of the placode 5ui"rûunding the -115-

nasal pit or a difference in the distribution of tissue in the lateral and medial nasal processes about the nasal pit. Such a difference in distribution of tissue would result in an apparent change in the position of the pit relative to maximal head width at the level of the nasal piacodes. Trasler (personal communication) has found that although there is a significant difference in distance between the nasal pits AI J and C57BL mice at the crescent stage of the nasal pit, this diffe rence doe s not exist at the earlie r oblong and oval stage s. This

later difference could thus be due to either a differential shift in placode position in the two strains as the head g rows, or to diffe rence s

between the st rains in eithe r the rate of growth of the late ral and

medial nasal processes relative to one another or in the period of

growth of these processes. Embryos of the two strains could be

examined during this period of growth for quantitative changes in mass

of the lateral and medial nasal processes and for differences in mitotic

rate between the two processes and between the strains. This would

determine whether such a relative change in tissue mass in the two

processes could account for their later difference in face shape.

By examining the nasal placodes of embryos at the oval stage

'with scanning electron microscopy, Verrusio 0971, and personal

communicationl ,,~.. as able to show that the fi r st sign of invagination -115- of the nasal pit was a furrow running lengthwise (antero-posteriorly) within the depression of the placode. If this furrow fixes the later position of the nasal pit on the face of the embryo, and is not placed differently in the AI J and C57BL/6 strains (easily verifiable by measuring such electron mic rographs), then a later diffe rence in face shape could be due to differential growth of the lateral and medial elements surrounding this furrow. A difference at the oval stage in the distance between the furrows in the paired placodes, the distance in A/J being narrower, would show that the actual position of the future pit, and the placode surrounding it, was different in the two stains even at this early stage, but would not rule out an associated diffe rence in the mitotic rate of the tis sue in the nasal proce s se s.

What factors are involved in the production of MC L afte r treatme nt

on Day 9t but LC L afte r treatment on Day 10 113 within the same

st rain? Since the earlie r treatment doe s not see m to alte r the

normal positioning of the placodes, it is difficult to invoke such a

mechanism as a determining factor. The MCL defect may be a result

of an interference with neural crest cell migration and mitotic rate.

The LC L malformation, however, occurs when treatment is given at

a time when the early nasal placodes are already formed. If a11 parts

of the placode are equally disturbed by treatment, it might be supposcd

that the later treatment would result in both types of cleft lip. If, -117- however, different parts of the nasal placode p rolife rate at diffe rent times, and are therefore more sensitive to disruption by an envir­ onmental insult, this would result in retardation of different portions of the placode to different extents. If this is 50, rapid proliferation in the more medial portions of medial nasal process must occur earlier than Day 10 1/3. Such rapid growth would later be present in the more posterior regions of the two processes on Day 10 1/3 which treatment could affect by causing an insufficient mesodermal penetration of the isthmus after breakdown of the double epithelium

separating them when the "zipping up" of these epithelia begins. This would lead to a Le L. Such a difference in mitotic rates could be

examined in conjunction with othe r mitotic index studie s suggested

earlier.

Although it is presently thought (Trasler, 1968) that lack of

contact and subsequent fusion of the epithelia of the posterior rim

of the nasal p lac ode , rathe r than insufficient subsequent me sode rmal

penet ration, is the dis rupti ve step in spontaneous LC L production,

this does not rule out the possibility that such penetration is not

faulty when an induced LC L forms. Verrusio (pe rsonal communication),

has also examined fusion of the lateral and medial nasal processes

with scanning e lect ron mic rosc opy and noted the feature s pre sent -118- when the posterior epithelia of the nasal processes first meet.

Examination of embryos treated on Day 10 1/3 in the same way would show whether it is the conjunction of the se epithelia, rathe r than later penetration by mesoderm, that is disturbed by treatment.

If mitotic rate is indeed implicated in the production of LC L, a large enough dose of the te ratogen even on Day 9i should re suIt in critical reduction of the entire placode and subsequently result in

LC L. This has been found by the present author using a higher dose of

6AN in C57BL/6 embryos. As was previously mentioned, the thickness of the lateral element of the early nasal placode appears to be reduced after treatment relative to head size. and its variance inc reased. A highe r dose than that routine ly used in this expe riment could re suit in a reduction in the tissue of both the late raI and medial nasal processes. and via the mechanism just discussed, produce the LC L phenotype afte r t reatment on Day 9'Î.

B. Success of Selection

The selection experiment, now in its seventh generation, has only partially produced the anticipated responses. The induced cleft

lip re sponse of the Median line in the di rection predicted, that i s, a high :-"IC L frequency and a IoVo" LC L freGuency. Howeve r. a single

spontaneous :-"ICL occurred in the Control line (spontaneous :-"1CL is known to occu r .... e ry ra re ly in the C 5 ïB L st rain) ·... ·hi le none occu r rcd -119- in the Median Hne. This weakens the prediction that selection for a masked trait by using an environmental shock should eventually re sult in it s spontane ou s appearance. A s for the Late raI line, the induced cleft Hp re sponse is in total disagreement with prediction.

The MC L frequency is high, while the LC L frequency is low. As postulated in the Re sults (Section B. 2. ) this is probably due to the infertiHty encountered among positive- re sponding animals of this

Hne in generation 5. Carole-Ann Hamly attributed this poor response to selection against "hole s", ext reme reduction of the premaxilla, in earlier generations. Although she was able to show that selection against "holes" had occurred, the satisfactory positive cleft Hp

response seen in the Lateralline in generation 5 after increasing the dosage suggests that this selection did not remove positive­

reacting animals from this Hne, but merely increased their resistance to 6AN. The subsequent drop in LCL frequency can therefore be attributed to infertility in generation 5 rather than to the earlier

se lection against "hole s ".

Although a significantly larger spontaneous LC L frequency was

found in the Late raI line than in the Median line in gene ration 4, thi s

diffe rence bet'\.... een the two was not found in subsequent generations

up to and inc luding gene ration Î. -120-

No evidence was found that the selection lines differed in embryonic developmental age, a factor which could possibly influence treatment response. Thus no apparent difference s between untreated embryos of the selection lines were seen in morphological stage at the time of treatment, but very few HUers were available for com­ parison, and it was assumed that differences would be at the gross morphologicallevel. Differences such as the state of neural crest migration and the state of induction of the nasal placode were not studied. The former possibility could be studied using the staining technique already discus sed.

In the small number of liUers of generation 7 that were rated for morphological development and somite number at the time of treatment as stated above, no difference was found between the lines.

This does not confirm the results obtained by Carole-Ann Hamly for generation 4, that the Lateral line tended to have fewer embryos which had incomplete closure of the neural tube in the he ad region.

Hel" sample, which was not tested statistically, was taken from the

1'.\"0 line s at random, di 5 rega rding the induced cleft lip re sponse of

the mother in previous pregnancies. The embryos compared by the

present author were taken on the basis of this response, and may

~i\'e a n.ore accurate measure. -121-

Although the tneans of the tnorphological stage on Day 10, 20 hours did not differ significantly, there was tendency for tnore etnbryos of the Median line than the Lateral line to be in the tnore advanced tail of the distribution. This cannot, howeve r, be interpreted as a con­ tinuation of a difference present at the titne of treattnent, as no differences had been found for the earlier titne. Many factors tnay intervene between the two gestational ages, and these tnay in thetn­ selves produce fllch a difference. The lack of a statistical difference between the means of the two groups at this time is in agreetnent with the results obtained by Carole-Ann Hatnly for generation 4.

No difference in face shape was found between the Lateral and

Median lines in generation 7. Independent comparisons of these embryos carried out by Susanna Leong, tnaking measurements from photographs, as was done by Dr. Hamly, were in close agreement with the results obtained by directly measuring the heads with an ocular micrometer. In generation 4, Dr. Hamly found a highly

significant diffe rence in face shape between the lines. Although

fewer embryos were measured in that generation, they were aIl at

the same nasa 1 p lacode stage (c re scent), and thus may have given

a more accurate indication of the existence of any such difference.

It was not stated, ho'\',;ever, what the type of reaction of the mothers -122- of these generation 4 embryos was. AU embryos measured and compared for face shape in generation 7 were taken from mothers

which has previously given the positive reaction appropriate to

their selection Hne. It is of course possible that the drop in

selection response in the Lateral li ne between generation 4 and

generation 7 contributed to this difference in the results obtained by

the two author s.

At present, some females of the Controlline of generation 8

are being tested for their cleft lip response by Susanna Leong. A

few animaIs taken randomly from the Htters of lateral- reacting

females will be used to restock the Lateral Hne with positive-reactors.

Other animaIs taken from the same Htters will be kept in the

Control line 50 as not to disturb its gene pool unduly. lt is suggested

he re that future comparisons between embryos of the two line s not

be made until re lati ve ly high and consi stent positive induced re sponse s

of the appropriate type of c le ft lip occur in both selection line s.

Only then ".. ·ill it be relatively easy to obtain embryos in sufCicient

numbers to make realistic comparisons.

As the Control line was derived from animaIs which gave no

c1eft lip re sponse afte r t reatment in gene ration 0 (F 3). it may not be

a Vc r'i good c ont rol. A bette r c ont roI would have been se lectcd at

random and not on the basis of trcatrr.cnt. The initial group of -123-

C ont roI line animaIs we re in effect a non-cleft lip re sponding group where selection was suspended in subsequent generations. Had such a line actually been se lected for, by choosing female s whose treated

litters gave no cleft lip animaIs but consistently had other malfor­

mations, it might have been inte re sting to examine the face shape of

their embryos, as well as their stage of development at the time of

treatment. Another selection line which would be of interest is a

double-reacting line, i. e. a line in which treated females produced

both LC L and MC L embryos in the same litter. The face shape and

morphological stage of the embryos of this line might be intermediate

between those of the Lateral and Median lines, if differences between

these two lines occur, or it might have a very wide variance to the

point of bimodality.

As lngalls ~ ~ (1964) showed that 6AN can cause chromosomal

breaks and polyploidy, it would be better to modify the present

method of se lecting litte r s of positive- reacting female s. lnstead of

treating in the first parity and selecting in the second, as is no'\\!

clone, the Cirst parity should be raised and then selected on the basis

of the response to treatment of their sibs in the second parity. This

...... ·ould p revent the pos sible accumulation of ch romosomal anoma lie s

as selection proceeded from one gene ration to the next.

Another modification of the present procedure is to treat the -124-

Controllines females so as to use their response, rather than that of the selection line s, as a base against which to compare induced cleft lip frequencies in the selection lines. Selection of the Control line would, of course, remain random, and not be based on their cleft lip re sponse.

If and when the Lateral and Median line s produce embryos of

different face shape or having differences at the time of treatment,

those aspects which differ, at both the gross morphological level

and at the histological level, could be investigated and identified. -125-

VII. SU MMAR Y

1. The early effects of 6-aminonicotinamide treatment leading to median cleft Hp as weIl as selection for differences in type of c1eft lip re sponse to this teratogen were studied.

2. The somite numbers were temporarily more affected than nasal placode stage soon after treatment with 6-aminonicotinamide on

Day 9, 12 hours.

3. The rate of development of both the nasal placodes and the somites was retarded by the treatment.

4. No association could be found between change in nasal placode

stage after 6AN-treatment and the median cleft Hp malformation

caused by 6AN.

5. G reate r dive rgence in the positioning of the incipient nasal

placodes after treatment was not found.

6. Face shape is unaffected by treatment.

7. tAN reduced the absolute size of aH paramete rs measured in the

early and later embryonic head. This suggests that mitotic rate

".. 'as affected soon after treatment.

8. 6A~ appeared to affect the thickne 55 of the nasal placode more

than the size of the re st of the head.

9. The teratogen caused an increase in variance of nasal placode

area. This suggested a threshold model where a critically small -126- placode area and thickness result in insufficient tissue in the medial nasal processes, which would interfere with normal mergence leading to median cle ft Hp.

10. Treatment appeared to interfere with the normal rate of neural

crest cel! migration in the head region. This coupled with a re­

duced mitotic rate in the nasal placode region are possible factors

leading to the median c le ft lip defect.

11. Selection for MC L in response to 6AN treatment in the Median

line is presently successful at generation 7, although a spontaneous

MCL in the Control line casts doubt on this conclusion.

12. Se lection for LC L in the Lateral line is totally unsucce ssful at

pre sent, p robably due to infe rtility among positive- reacting animaIs

in an earlier generation.

13. No difference s in morphological stage at the time of treatment of

the selection lines were seen in generation 6.

14. The Median line embryos tend to be more advanced in morpho­

logical stage than those of the Late raI line on Day 10, 20 hour s.

15. ~o differences in face shape was scen betwcen the t\... ·o selection

line s in gene ration 6, although a diffe rencc had been reported for a

smalt sample in gene ration 4. -127-

VIII. ACKNOW LEDGEMENTS

1 wish to express special thanks to Daphne G. Trasler for he r concern, help and encouragement during the time 1 have known her, as weil as the other members of my supervisory committee, F. Clarke Fraser and Leonard Pinsky. Thanks also go to Hugh Tyson and Kurt Sittmann for advice on the

statistical analysis.

Peggy Turner' s help in producing the figure s and Martina

von Nida' s efforts in preparing the typescript are appreciated.

Thanks go to Susanna Leong for rechecking the measurements,

to lAlcy Shih and Michel Kabay for the loan of sorne mice, and to

Sue Wright for being a beautiful roommate.

Lastly, thanks are expressed to aU those others with whom 1

shared the last few years, both intellectually and socially, for

keeping life interesting in many ways.

Grants-in-Aid of Research from the National Research

Council are gratefully acknowledged. IX. APpmmICRS A-E Appcndix A: l.:ef\ourements of heade of C57 :sr. embryos,treated and oontrol, at the shallow indentation and early oml stBRea of the Msnl plncode.laIeasurements in miorometer units(1 miorometer unit,shown here and in lnter nppendioes as .001,=.01818 mm. ). hend head distanoe between distanoe across plac ode wid th somite ohronol. ~. Il lrentment embryo # length width placodes placodes right left # day~~our

044'1 Control 1 .066 .044 23 10/0 2 .082 .057 .034 .056 .013 .014 26 3 .066 .044 23

04')13 Control 2 .100 .066 .044 .072 .015 .018 27 10/0 1 ~ 3 .090 .060 .038 .062 .015 .015 27 f\) 4 .001 .055 .033 .055 .013 .015 22 Cf 5 .091 .061 .041 .065 .016 .014 26 G .O~O .055 .040 .C65 .018 .016 27 ., .097 .067 .043 .066 .016 .017 26 Ù .069 .059 .037 .061 .015 .013 27 1) .OU3 .058 .035 .058 .015 .014 25 10/8 0044 Control 1 .092 .060 .037 .064 .016 .012 25 2 .090 .061 .034 .013 .013 26 3 .094 .063 .035 :sg~ .016 .016 27 4 .089 .060 .036 .063 .015 .014 26 5 .093 .066 .035 .064 .017 .016 27 (, .0<)2 .062 .034 .064 .017 .015 25 7 .102 .068 .040 .073 .019 .018 29 U ~Ol)7 .063 .035 .066 .017 .016 29 Appondix Ai (Continued) head head distance between distance aoross placode width somite chronol. (. # trentl!\ont embryo # length width placodes placodes tight left no. AGE day/hour

02l) ., (jAN 1 .0613 .041 .022 .047 .015 .016 27 10/20 3 .003 .056 .027 .055 .014 .017 25 4 .057 .034 .022 .039 22 6 .003 .045 .025 .049 23 7 .099 .070 .041 .066 .016 .017 20 13 .096 .066 .034 .064 .017 .019 26 1 .017 .016 ~ ') .001 .050 .029 .055 24 1\) I.Ù 1 0022 GAN 7 .075 .050 .035 .050 .011 .011 22 9/113 13 .072 .046 .030 .045 .012 .013 22 Y .070 .047 .029 .046 .010 .008 22

0150 GAU 13 .070 .050 .028 .050 .009 .011 23 10/U nû2B liAN 2 .071 .04B .027 .044 .010 .011 21 10/14 3 .079 .053 .027 .052 .01; .012 23 4 .081 .055 .032 .053 .015 .015 21 5 .089 .061 .031 .060 .014 .015 21

004.'\ GAN 5 .089 .061 .034 .057 .017 .017 27 10/14 6 .075 .052 .024 .051 .009 .010 24

02132 6Al~ 1 .067 .048 .029 .047 .01; .013 23 10/14 5 .074 .051 .025 .049 .014 .013 22 6 .062 .041 .023 .042 .011 .011 20 7 .067 .042 .021 .039 .01; .010 20 Appcndix 8 Face Measurements of 6-AN-Treated and Control Embryos of the C57 8L/6 Strain at the Late

Oblong and Crcsccnt Stages of Nasal Placode Development. Measurements in micrometer units

ut 2.70x magnification ( 1 micrometer unit • 0.037 mm.).

.:.: no. ~mbryo no. treatment nasal placode stage head width across distance between -maxille nasal anterior posterior process process limita of nasal pits

02)9 6AN oblong .052 .044 .027 .032

2 oblong .052 .044 .027 .032

1 3 oblong .052 .045 .027 .033 ~ VI 6 crescent/comma .057 .048 .026 .034 l'

7 crescent .055 .046 .028 .033

028!. 1 6AN crescent/comma .058 .049 .027 .034

) oblong .052 .044 .027 .031

0271 MN oblong .055 .046 .026 .032

2 oblong .046 .026 .033

0202 Control oblong .056 .048 .030 .035

4 crescent/comma .058 .050 .029 .035

) crescent .055 .048 .029 .035

7 crescent .057 .049 .028 .035 Appcndlx B : (Contlnucd)

'l' no. l'mbr yo no. trcatment nasal placode stane head width across distance between

maxille nasal anterior posterior process process limits of nasal pits

0202 8 Control crcscent .057 .049 .029 .035

9 crcsccnt .057 .049 .029 .035 1 -A VJ -A 0037 4 Control crescent .057 .049 .029 .035 1

5 crescent .055 .048 .029 .034

6 crescent .054 .048 .029 .034

7 crcscent .059 .050 .029 .035

0336 4 Control cre ... cent/oblong .055 .047 .029 .034

8 crescent/oblong .061 .051 .029 .036

031.0 Control crescent/oblong .057 .048 .028 .034

4 crescent .056 .048 .029 .034

5 crescent/oblong .056 .048 .028 .033

(, crescent/oblong .056 .049 .030 .035 Appcndlx 8 ;(Contlnucd)

';' no. ~mbl"yo no. trcatmcnt nasal placode stage he ad width across distance between

MAX ILL. nasal anterior posterior process process limits of nasal pits

0340 7 Control oblong .053 .048 .030 .034

8 oblong .053 .047 .028 .032 ...1 \Jo! 1\) 0327 4 Control crescent/oblong .056 .049 .030 .034 1

7 crescent .056 .029 .035

9 oblong .055 .047 .030 .034 Appcndix C : Morphologieal ratings,Day 9,10 hours.Embryos of Lateral and Median lines,generation 6.

(,) no. line emb.no. neural tube som. no. Milaire rating nasal plaeode previous reaetion

non-reaetor 5172 1. 6 16 9A late (tested twiee)

2 6 18 9B ear1y

3 6 20 9A 1ate

4 4 16 8A

5 6 16 8B 1ate 1 ...& \>1 6 6 16 88 1ate \>1 1 7 4 14 8A

8 6 20 98

9 6 15 88 1ate

11 6 16 88 1ate

5131 1-1 2 6 20 9A 1ate median reaetor

3 6 15 88 1ate

4 6 16 8B

5 6 18 98

6 2' 9 8A

7 4 13 88 Appcndlx C : (Contlnued)

',1 no. Ilnr emb.no. neural tube som. no. Mi1aire rating nasal placode previous reaction

5131 ~I 8 6 15 88 1ate median reactor

9 6 17 9A

10 6 20 98

Il 6 19 98 ....1 Vl ')210 N 1 • 6 18 98 median reactor t 2 6 16 9A

3 6 18 98

4 6 17 98

5 6 18 98

6 6 17 98

7 6 15 9A 1ate

8 6 17 9A

9 6 17 9A

10 6 16 9A App~ndlx C : (Contlnued)

(1) no. Hne cmb.no. neural tube som. no. t-tilaire rating nasal placode previous reaction

)219 N 6 15 8B median reaction

2 5 16 8B

3 6 16 9A

6 5 14 8B 1 -10 7 6 17 9A 1ate VI \on 1 8 4 15 8B

9 6 16 9A

------136-

Appendix D: Morphological ratings, Lateral and Median lines,embryos

taken Day 10,20 hours.

emb. tail som. Umb eye nasal internaI previous ? no. Une no. no. buds placode pit reaction --- 5279 L 1 5 lOB HOLE oblong + lateral 2 6 lOB pinhole early cresc. 3 6 lOB pinhole oblong reactor 4 4 lOB ho le oblong 5 7 lOB very small pinh. cresc. - 6 6 lOB pinhole cres/obI. 7 5 lOB v.sm.pinh. oblong 8 5 lOB v.sm.pinh. oblong 9 2 lOB hole early oblong 10 5 lOB pinhole oblong

5280 L i 7 lOB V.sm.pinh. crescent left+,rt.- non- 2 5 Il pinhole early cres. Jll['eactor 3 6 Il v.sm.pinh. early cres. 4 7 Il v.sm.pinh. late cres/comma + 5 7 Il lens couma + 6 6 Il pinhole oblong 7 7 Il v.sm.pinh. late cres. + 8 7 " v.sm.pinh. cres cent + 10 6 " pinhole oblong 11 7 " v.sm.pinh. oblong 12 5 " pinhole oblong 13 5 " hole oblong

5019 M 2 8 lOB lens early cOtmla + median 4 10 Il lens cotmla + reactor 5 8 Il lens COtmla + 6 8 Il lens COtmla + 7 7 .. small pinh . crescent 8 9 Il lens late COtmla +

nasal 21ac. eye 5218 M 1 4 10A oblong pinhole median 3 5 lOB oblong v.sm.pinh. reactor 4 6 10A late/obI. v.sm.pinh. 11 2 10A oval hole 12 7 loB crescent lens + 13 8 lOB corm:.a lens + -137- Appendix 0: (Continued)

emb. tail som. limb eye nasal internaI previous ? no. line no. no. buds placode pit reaction

median 5211 M 1 8 lOB v.sm.pinh. comma + 2 7 " v.sm.pinh. crescent + 3 8 " v.sm.pinh. crescent + reactor 4 6 " pinhole oblong 5 8 " v.sm.pinh. comma + 6 8 " v.sm.pinh. comma + 7 7 " small pinh~omma + 8 6 " pinhole oblong 9 8 " v.sm.pinh. crescent + 10 6 " pinhole oblong

5240 M 1 5 lOB pinhole oblong median 2 8 " lens comma/cres. + 3 7 " pinhole oblong reactor 4 5 " pinhole oblong 5 5 " pinhole oblong 6 3 10A hole oval 7 8 lOB v.sm.pinh. early comma + 8 0 9B 9B 2 9 3 10A pinhole oblong 10 7 lOB pinhole early cres. + 11 5 lOB pinhole oblong 12 5 lOB pinhole oblong 13 6 lOB v.sm.pinh. 3 14 0 10A early/lOA

5216 M 1 8 lOB lens comma + 2 7 " pinhole crescent + 3 7 " pinhole crescent + 4 7 " v.sm.pinh. crescent + 5 6 " pinho1e crescent + 6 5 " pinhole oblong 7 8 " v.sm.pinh. comma + 8 5 " pinho1e oval 9 6 " small pinh. crescent + 10 6 " v.sm.pinh. crescent + l! 5 " pinhole oblong 12 8 " lens conwna + 13 6 " pinhole crescent + -138-

Appendix E: Face Shape Measurements on Day 10,20 hours of Embryos of

Positive-reacting Females of the Selection Lines.Measure-

ments given in micrometer units at 2.70x magnification.

(1 micrometer unit = 0.037 mm.)

emb. nasal plac. dist.betw.ant.1imits width of head across 9 no. LlNE no. stage of nasal pits placode region

5279 L 1 obI. .028 .045 2 ear1y cres. .029 .047 3 obI. .028 .046 4 obI. .028 .044 5 cresc. .027 .047 6 cres/obI. .029 .045 7 obI. .028 .049 8 obI. .028 .047 10 obI. .029 .046

5019 M 7 cresc. .029 .049

5211 M 2 cresc. .027 .050 4 obI. .029 .050 8 obI. .028 .047 9 cresc. .027 .049 10 obI. .028 .048

5240 M 1 obI. .029 .046 3 obI. .029 .050 4 obl. .029 .048 5 obI. .030 .046 9 obI. .028 .046 10 ear1y cres. .028 .048 11 obI. .030 .045 12 obI. .029 .048 13 obI. .027 .049

5218 X 3 obI. .027 .046 12 cresc. .028 .048 -139-

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