The genesi osf membrane bone in the embryonic chick ... · bone (Fig. 1, Table 1). The epithelium differentiated into a stratified squamous epithelium consisting of a basal layer

/ . Embryo/, exp. Morph. Vol. 56, pp. 269-281, 1980 2 6 9Printed in Great Britain © Company of Biologists Limited 1980

The genesis of membrane bone in theembryonic chick maxilla: epithelial-mesenchymal

tissue recombination studies

By MARY S. TYLER1 AND DAVID P. McCOBB2

From the Department of Zoology, University of Maine

SUMMARYIn the present study, the question of whether a relatively non-specific epithelial require-

ment exists for membrane bone formation within the maxillary mesenchyme was investigated.Organ rudiments from embryonic chicks of three to five days of incubation (HH .18-25)were enzymatically separated into the epithelial and mesenchymal components. Maxillarymesenchyme (from embryos HH 18-19) which in the absence of epithelium will not formbone was recombined with epithelium from maxillae of similarly aged embryos (homotypic-homochronic recombination) and of older embryos (HH 25) (homotypic-heterochronicrecombination). Heterotypic recombinations were made between maxillary mesenchyme(HH 18-19) and the epithelium from wing and hind-limb buds (HH 19-22). Recombinantswere grown as grafts on the chorioallantoic membranes of host chick embryos. Grafts ofintact maxillae, isolated maxillary mesenchyme, and isolated epithelia from the maxilla,wing-, and hind-limb buds were grown as controls. The histodifferentiation of grafted intactmaxillae was similar to that in vivo; both cartilage and membrane bone differentiated withinthe mesenchyme. Grafts of maxillary mesenchyme (from embryos HH 18-19) grown inthe absence of epithelium formed cartilage but did not form membrane bone. Grafts ofmaxillary mesenchyme (from embryos HH 18-19) recombined with epithelium in homotypic-homochronic, homotypic-heterochronic, and heterotypic tissue combinations formedmembrane bone in addition to cartilage. These results indicate that maxillary mesenchymerequires the presence of epithelium to promote osteogenesis and that this epithelial require-ment is relatively non-specific in terms of type and age of epithelium.

INTRODUCTION

Previous studies have shown that epithelial-mesenchymal interactions areinfluential in promoting membrane bone formation within mesenchymal tissue.It has been shown for the developing maxilla (Tyler, 1978), mandible, (Tyler &Hall, 1977), and skull (Schowing, 1968a, b, c; Benoit & Schowing, 1970) of thechick that the mesenchyme requires the presence of an epithelium during aspecific embryonic period early in development in order for subsequent

1 Author's address: Department of Zoology, University of Maine, Orono, Maine 04469,U.S.A.

2 Author's address: Department of Zoology, University of Washington, Seattle, Washing-ton 98195, U.S.A.

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270 M. S. TYLER AND D. P. McCOBB

membrane bone formation to occur. Removal of the epithelium during this timeperiod prevents osteogenesis within the isolated mesenchyme. In the systemsstudied, the presence of the epithelium ceases to be required by the mesenchymeseveral days prior to the actual onset of osteogenesis. In the developing chickmaxilla, epithelial influences are required through Hamburger & Hamilton(1951) stage (HH) 22 (3^-4 days of incubation). By HH 23 (4 days of incubation),removal of the maxillary epithelium will not prevent osteogenesis within themesenchyme. The actual onset of ossification within the maxilla does not occuruntil HH 35 (8±-9 days of incubation) (Tyler, 1978).

The present study examines whether or not the epithelial influence requiredby maxillary mesenchyme through HH 22 is specific for a particular type orage of epithelium. A similar study on the chick mandible indicates that theepithelial requirement for bone formation within the mandibular mesenchymeis relatively non-specific with respect to the type of epithelium that will promotemandibular osteogenesis, but that the age of the epithelium is an importantfactor in determining whether the epithelium will be influential in promotingosteogenesis.

In the present study, tissue recombinations were implemented to test theinfluence of heterochronic and heterotypic epithelia on the genesis of membranebone within the maxillary mesenchyme. Maxillary mesenchyme was isolatedfrom its epithelium at a time when an epithelial influence is required for osteo-genesis, and the mesenchyme was recombined with maxillary epithelium fromsimilarly aged embryos (homotypic-homochronic recombination) and withmaxillary epithelium from embryos beyond the stage at which an epithelialinfluence is required for maxillary osteogenesis (homotypic-heterochronicrecombination). In heterotypic recombinations, isolated maxillary mesenchymewas recombined with epithelia from the wing and hind-limb buds. Theseepithelial regions normally do not participate in promoting membrane boneformation. Recombinations were grown as grafts on the chorioallantoic mem-brane of host chick embryos.

The results indicate that the epithelial requirement for osteogenesis withinthe chick maxilla is relatively non-specific and differ to a certain extent fromthose reported for the chick mandible (Hall, 1978).

MATERIALS AND METHODS

Tissue preparation

Eggs from the common chicken (Gallus domesticus, White Leghorn) wereincubated in a Leahy, forced-draft incubator at 37-5 ± 1 °C and 57 ± 2 %humidity. Chick embryos from eggs incubated for three to five days werestaged according to the Hamburger and Hamilton (1951) staging (HH) series,and organ dissection was carried out in Tyrode's solution. Maxillae fromembryos HH 18-19 and HH 25 (3-3£ and 5 days of incubation), and wing and

Tissue interactions on maxillary osteogenesis 271

hind-limb buds from embryos HH 19-22 (3̂ —4 days incubation) were used in thestudy.

Separation of the epithelium from the mesenchyme of an organ was achievedenzymatically. Organs were placed in a 3 % trypsin-pancreatin solution (3:1(w/w) in calcium- and magnesium-free Tyrode's solution) at 4 °C for 45 min.Following enzymatic treatment, the loosened epithelium was removed from themesenchyme by manipulation with a small-bore pipette and finely sharpenedtungsten needles. Separated tissues were placed in a solution of Tyrode'sand fetal calf serum (1:1, v/v) which served to inactivate any residual enzy-matic solution in the tissues, and tissues were stored in the Tyrode's-serumsolution until use.

Grafting procedures.

Intact maxillae, intact wing and hind-limb buds, the isolated epithelialand mesenchymal components of maxillae, isolated epithelia from the wing andhind-limb buds, and maxillary mesenchyme recombined with epithelia fromthe maxilla or from the wing or hind-limb buds were grafted to the chorio-allantoic membrane of host chick embryos that had been incubated for eightor nine days. As a control to determine whether the enzymatic treatmentinterfered with tissue differentiation, grafts were made of intact maxillaryprocesses that had been enzymatically treated without subsequent mechanicaltissue separation.

Intact organs and isolated tissues were placed on Millipore filter discs(black; 5 mm diameter; 0-45 jam porosity; 125-150jam thick; obtained fromMillipore Filter Corp., Bedford, Massachusetts). The filters served as supportsfor the explanted tissues and facilitated localization of the graft at the time ofharvesting. In tissue recombination experiments, ultra-thin Millipore filterdiscs (white; 5 mm diameter; 0-45 fim porosity; 25 + 5 jam thick) were used toallow observation of the tissues by transmitted light during tissue manipulations.To recombine epithelial and mesenchymal tissues, the mesenchyme was placedon the filter and allowed to adhere to the filter; the epithelium was thenpositioned as a flattened sheet over the mesenchyme. In certain instances, themesenchyme was positioned on top of the epithelium; this, however, was a lesssuccessful method of achieving direct contact between the tissues over a largesurface area. In most instances, recombined tissues were placed in a CO2-humidified incubator for one to two hours prior to grafting; this allowedadhesion of the component tissues prior to any further manipulations.

Tissues on their Millipore filter discs were placed on the chorioallantoicmembrane of host chick embryos such that the grafted tissues were in directcontact with the host tissue. The host embryos were then further incubated foreight days.

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272 M. S. TYLER AND D. P. MCCOBB


Histological procedures.

Grafts were fixed in Bouin's fluid, dehydrated in a graded series of alcohol,cleared in toluene, and embedded in paraffin blocks. The paraffin blocks weresectioned at 5 fim on a Sorval JB-4 rotary microtome. Mounted sections werestained either with van Gieson's stain and alcian blue (pH 2-5-30) (Wilsmanand VanSickle, 1971) or with hematoxylin, eosin, and alcian blue (pH 2-5-3-0)(Pearse, 1960).

The results are based on a total of 131 grafts: of these, 14 were of intactrudiments, 15 were of isolated maxillary mesenchyme, 10 were of enzymaticallytreated intact maxillae, 14 were of isolated epithelia, 17 were of homotypic-homochronic recombinations, 13 were of homotypic-heterochronic recombi-nations, 22 were of heterotypic recombinations with wing-bud epithelium,and 26 were of heterotypic recombinations with limb-bud epithelium.

RESULTS

Intact maxillary processes

The histodifferentiation of intact maxillary processes excised from embryosHH 18-19 and HH 25 and grown as chorioallantoic membrane grafts was

FIGURES 1-6

Figs. 1-2. Photomicrographs of a section through a graft of an intact maxillaryprocess from a HH-19 embryo grown on the chorioallantoic membrane for 8 days.Cartilage (c), derived from the quadrate, has differentiated near membrane bone (b),and a portion of the membrane bone can be seen in Fig. 2 (arrow) to be in closeassociation with the oral region of the maxillary epithelium (ep). The graft, supportedby a Millipore filter (mf), is surrounded by host tissue (ht) from the chorioal-lantoic membrane. Hematoxylin, eosin, and alcian blue, x 51 and x 95, respectively.Fig. 3. Photomicrograph of a section through the aboral region of the grafted maxillashown in Fig. 1. Feather germs (fg), shown in longitudinal section, are in the humpstage of development, x 184.Fig. 4. Photomicrograph of a section through a graft of maxillary mesenchymeisolated from its epithelium at HH 19 and grown on the chorioallantoic membranefor 8 days. Cartilage (c) has differentiated within the mesenchyme, but membranebone did not form, (ht designates host tissue from the chorioallantoic membranewhich surrounds the graft.) Alcian blue and van Gieson's stain, x 95.Fig. 5. Photomicrograph of a section through a graft of maxillary mesenchymeisolated from its epithelium at HH 25 and grown on the chorioallantoic membranefor 8 days. Membrane bone (b) has formed in addition to cartilage (c). Host tissue(ht) from the chorioallantoic membrane surrounds the graft. Hematoxylin, eosin,and alcian blue, x 95.Fig. 6. Photomicrograph of a section through a graft of a maxillary process, re-moved from its embryo at HH 19, that was enzymatically treated without sub-sequent tissue separation and then grown on the chorioallantoic membrane for 8days. Histodifferentiation is similar to that of the grafted intact maxillary processshown in Fig. 1. (b, c, and ep designate membrane bone, cartilage, and epithelium,respectively.) Alcian blue and van Gieson's stain, x 95.


Table 1. Skeletogenesis in intact maxillae and isolated maxillary mesenchymegrafted to the chorioallantoic membrane

Presence ( + ) or absence (—) ofAge ofdonor Cartilage Membrane bone

Intact maxillary processesHH 18-19 + +HH 25 + +

Isolated maxillary mesenchymeHH 18-19 +HH 25 + -I-

similar to that reported for the maxilla in vivo (Tyler, 1978). Bony trabeculaerepresenting the elongate membrane bones of the maxilla differentiated withinthe maxillary mesenchyme, and cartilage, a precursor to the quadrate, anendochondral bone, differentiated usually in close association with membranebone (Fig. 1, Table 1). The epithelium differentiated into a stratified squamousepithelium consisting of a basal layer of mitotically active cuboidal-to-columnarcells, one to two intermediate cuboidal cell layers, and one to three outersquamous cell layers (Fig. 2). The greater number of cell layers occurred ingrafts of maxillae excised from embryos HH 25. In the aboral region of themaxilla, feather germs were distinguishable (Fig. 3). The feather germs were inthe hump stage of development in grafts of maxillae excised from youngembryos (HH 18-19) and were in the elongation phase of development ingrafts of maxillae excised from older embryos (HH 25).

Isolated maxillary mesenchyme

In explants of maxillary mesenchyme isolated from its epithelium duringearly development (HH 18-19) and grown as a graft in the absence of itsepithelium, cartilage differentiated, but membrane bone did not form (Fig. 4,Table 1). These results confirm those of an earlier study (Tyler, 1978) indicatingthat the presence of an epithelium is required at this stage for maxillary mem-brane bone formation. In grafts of maxillary mesenchyme isolated from itsepithelium at a later stage of development (HH 25), membrane bone formed inaddition to cartilage (Fig. 5, Table 1). This confirms an earlier report (Tyler,1978) that at this stage in development, an epithelial influence is no longernecessary for promoting maxillary osteogenesis.

The histodifferentiation of intact maxillae (HH 18-19) that were enzymaticallytreated without subsequent mechanical tissue separation and then grown aschorioallantoic membrane grafts was similar to that of grafted maxillae that hadnot been enzymatically treated (Fig. 6), indicating that the enzymatic separationtechniques do not cause irreparable damage to the component maxillary tissues.


Table 2. Skeletogenesis in maxillary mesenchyme {HH 18-19) recombined withhomotypic and heterotypic epithelium and grafted to the chorioallantoic

membrane

Epithelium

Source

MaxillaMaxillaWing budHind-limb bud

Age ofdonor

HH 18-19HH 25HH 19-22HH 19-22

Mesenchymal differentiationPresence ( + ) or absence ( - ) of

Cartilage Membrane bone

Homotypic recombinations of maxillary mesenchyme and epithelium

In homotypic tissue recombinations, maxillary mesenchyme from youngembryos (HH 18-19) was recombined with maxillary epithelium from similarlyaged embryos (homotypic-homochronic recombination) and with maxillaryepithelium from older embryos (HH 25) (homotypic-heterochronic recombi-nation). The position of the tissues with respect to one another was not accord-ing to their original orientation.

In grafts of homotypic-homochronic recombinants, the histodifferentiationof the recombined tissues was similar to that of grafted intact maxillae. Mem-brane bone formed within the mesenchyme in addition to cartilage (Table 2),and the degree of epithelial differentiation was similar to that of grafted intactmaxillae of a similar cumulative age (initial age + incubation time as graft).Membrane bone formed usually in close proximity to the epithelium. Nospecificity was exhibited in terms of epithelial region with which bone wasassociated; bone was found in association with both oral and aboral regionsof the maxillary epithelium.

In grafts of homotypic-heterochronic recombinants, mesenchymal differenti-ation was similar to that of grafted homotypic-homochronic recombinants(Table 2). Membrane bone formed usually in close proximity to the epitheliumof either the oral or aboral maxillary regions (Fig. 7). Cartilage formed often inclose proximity to membrane bone (Fig. 8). Epithelial differentiation in theserecombinants was similar to that of grafted intact maxillae with a cumulativeage equal to that of the epithelium rather than to that of the mesenchyme of theheterochronic recombinant (Fig. 9).

Heterotypic recombinations of maxillary mesenchyme and epithelium from thewing- and hind-limb buds

Heterotypic tissue recombinations were made between maxillary mesenchymeisolated from young embryos (HH 18-19) and epithelium isolated from wing



and hind-limb buds of embryos HH 19-22, and recombinants were grown aschorioallantoic membrane grafts. Mesenchymal differentiation in each type ofrecombinant was similar to that of homotypic recombinants irrespective ofthe source (wing or hind-limb bud) or the intial age (HH 19-22) of the epithelium(Table 2). Cartilage formed within the mesenchyme of the explant and mem-brane bone was generated usually in close proximity to the epithelium (Fig. 10).In two instances, the chondrogenic region of the maxillary mesenchyme wasnot included in the explant, and in these grafts membrane bone formed inclose association with the epithelium in the absence of cartilage (Fig. 11).

Epithelial differentiation in heterotypic recombinants was similar to that ofgrafted intact wing and hind-limb buds. The epithelium became a stratifiedsquamous epithelium consisting of a cuboidal germinative cell-layer, one to

FIGURES 7-12

Fig. 7. Photomicrograph of a section through a homotypic-heterochronic recom-binant graft. Maxillary mesenchyme, isolated at HH 19, was recombined withmaxillary epithelium, isolated at HH 25, and grown on the chorioallantoic mem-brane for 8 days. Membrane bone (b) has formed in close association with theepithelium (ep) and cartilage is not in the vicinity of the membrane bone. Alcianblue and van Gieson's stain, x 95.Fig. 8. Photomicrograph of a section through a homotypic-heterochronic re-combinant graft similar to that in Fig. 7. In this graft, cartilage (c) is found inclose association with membrane bone (6), and the membrane bone has formed inclose proximity to the epithelium (ep). Alcian blue and van Gieson's stain, x 95.Fig. 9. Photomicrograph of a section through the graft in Fig. 8 showing the aboralregion of the maxillary epithelium. Feather germs (fg), shown in longitudinal sectionat their base and in transverse section more distally, are in the elongation stageof development and are more advanced than those shown in Fig. 3. Alcian blueand van Gieson's stain, x 184.Fig. 10. Photomicrograph of a section through a heterotypic recombinant graft.Maxillary mesenchyme, isolated at HH 19, was recombined with wing-bud epithe-lium, isolated at HH 21, and grown on the chorioallantoic membrane for 8 days.Mesenchymal histodifferentiation is similar to that of homotypic recombinantgrafts as shown in Fig. 8. Epithelial differentiation is similar to that of graftedintact wing buds with a similar cumulative age. Feather germs (fg) are in theelongation stage of development, (c and b designate cartilage and membrane bone,respectively.) Alcian blue and van Gieson's stain, x 95.Fig. 11. Photomicrograph of a section through a heterotypic recombinant graftsimilar to that shown in Fig. 10 except that the chondrogenic region of the mesen-chyme was not included in the explant. Membrane bone (b) has formed in closeproximity to the epithelium (ep) in the absence of cartilage, (ht and mf designatehost tissue and Millipore filter, respectively.) Hematoxylin, eosin, and alcian blue,x 184.Fig. 12. Photomicrograph of a section through a graft of maxillary epithelium (ep)>isolated at HH 19 and grown in the absence of its mesenchyme on the chorioallantoicmembrane for 8 days. The epithelium, underlaid by fibroblasts of host tissue (ht)origin, has differentiated into a stratified squamous epithelium. Regions of theepithelium have formed epithelial whorls (arrow) rather than remaining as aflattened sheet. Feather germs are not present within the graft. Hematoxylin eosin,and alcian blue, x 372.


two intermediate cuboidal cell layers, and one to two outer squamous celllayers. Feather germs in the elongation phase of development were distinguish-able and were at the same level of development as those of grafted intact wingand hind-limb buds of a similar cumulative age (Fig. 10). Differences betweenwing and hind-limb-bud epithelium were not detected.

In approximately 27 % of all recombinant grafts, close association betweenepithelium and mesenchyme was not maintained; in these instances, membranebonefailed to form within the mesenchyme though cartilage did form. Epithelial-mesenchymal contact and consequent osteogenesis were maximized experi-mentally by blanketing the already substrate-adherent mesenchyme with theepithelium and placing recombined tissues in a CO2-humidified incubator for1 to 2 h prior to grafting.

Isolated epithelium from the maxilla, wing-bud, and hind-limb bud.

Isolated epithelia from the maxilla (HH 18-19 and 25) and from the wing-and hind-limb buds (HH 19-22), grown as chorioallantoic membrane graftsin the absence of their mesenchyme, became underlaid by host fibroblastsfrom the chorioallantoic membrane and achieved a limited degree of differ-entiation (Fig. 12). In grafts of each different type of epithelium, theepithelium differentiated into a stratified epithelium consisting of a germinativecuboidal cell-layer and one to five outer cell layers which graded from cuboidalto squamous. Feather germs were not observed, nor were skeletal elements(either cartilage or bone) found within the host tissue associated with thegrafted epithelium.

DISCUSSION

It has been shown in a previous study (Tyler, 1978) and confirmed in thisstudy that during early development the presence of an epithelium is a require-ment for ensuing genesis of membrane bone within the mesenchyme of theembryonic chick maxilla. The results further indicate that this epithelialrequirement is relatively non-specific. In homotypic recombinations, it wasshown that re-establishing the original orientation of the maxillary epitheliumwith respect to its mesenchyme was not necessary for osteogenesis; membranebone formed within the mesenchyme of the recombinants irrespective of theepithelial orientation. The results from heterotypic recombinations establishedthat maxillary mesenchyme does not specifically require maxillary epitheliumto promote osteogenesis; other types of epithelia which in normal developmentare not associated with membrane-bone-forming mesenchyme (epitheliumfrom the wing- and hind-limb buds) were shown to be capable of promotingosteogenesis within maxillary mesenchyme. From heterochronic recombinations,it was shown that the response of maxillary mesenchyme to epithelium is notrestricted to a specific age of epithelium; epithelium removed from maxillaeafter the time during which the epithelium is required for osteogenesis (isolated

Tissue interactions on maxillary osteogenesis 279at HH 25) is still capable of promoting osteogenesis in maxillary mesenchymeisolated from young embryos (HH 18-19).

These results differ to a certain extent from those of a similar study onosteogenesis in the embryonic chick mandible (Hall, 1978); in both studies, how-ever, it is concluded that the epithelial requirements for mesenchymal membranebone formation are relatively non-specific. In the study on mandibular osteo-genesis, hind-limb-bud epithelium was found to promote membrane boneformation in mandibular mesenchyme isolated at an early stage of development(HH 18) (Hall, 1978) as was shown for maxillary mesenchyme in the presentstudy. In contrast to our results, however, the results in the mandibular studyindicate that neither wing-bud epithelium nor homotypic(mandibular) epitheliumpromotes osteogenesis within mandibular mesenchyme (isolated at HH 18)in either homochronic or heterochronic recombinations. The epithelial require-ments for osteogenesis in the developing chick mandible, therefore, appear to bemore restrictive than those in the developing chick maxilla. Whether the resultsreflect intrinsic differences in the two osteogenic systems or whether the differ-ences between the two studies are a reflection of the different techniques usedfor growing the tissues (organ culture, Hall, 1978; chorioallantoic membranegraft, this study) is still to be determined. It has been shown that both organculture and the chorioallantoic membrane of host embryos promote normalhistogenesis of intact organ rudiments; however, the two environments havebeen shown to differ in the amount of tissue growth that each supports and inthe type of organ morphogenesis that occurs within each (Tyler and Hall,1977). Further studies of maxillary and mandibular osteogenesis, therefore, arebeing made to determine the osteogenic potential of maxillary tissue recombi-nations in organ culture and mandibular tissue recombinations grown aschorioallantoic membrane grafts.

In earlier histological studies of membrane bone formation it was suggested,based on the proximity of cartilage to the mandibular membrane bones, thatcartilage is necessary for membrane bone formation (Frommer & Margolies,1971). This suggestion has yet to be substantiated, and results from the presentstudy, in which membrane bone formed within maxillary mesenchyme in theabsence of cartilage in two heterotypic recombinant grafts, indicate that thepresence of chondrogenic centers (beyond HH 18) is not required for maxillaryosteogenesis. This conclusion is supported by results from an earlier study(Tyler, 1978).

The results from grafts of epithelium separated from the maxilla, wing,-and hind-limb buds and grown in the absence of its mesenchyme confirm earlierreports (Tonegawa, 1973; Tyler & Hall, 1977; Tyler, 1978) that host fibroblastsfrom the chorioallantoic membrane are sufficient to maintain a germinativecell layer within an epithelium and to support a limited degree of epithelialhistodifferentiation. That the host mesenchymal tissue did not participate infeather formation suggests that there are specificity requirements for the type


of mesenchyme that will support feather formation within an epithelium.This suggestion is supported by other recombination studies of feather- andnon-feather-forming tissues (e.g. Rawles, 1963; Dhouailly, 1978). The factthat the grafted epithelium, though capable of promoting osteogenesis inmaxillary mesenchyme, did not induce membrane bone formation in the hosttissue associated with it indicates that the presence of epithelium, though arequirement for maxillary membrane bone formation, is not a sufficient conditionfor inducing bone formation in normally non-osteogenic mesenchyme.

In summary, the results of this study indicate that in the developing chickmaxilla, reciprocal epithelial-mesenchymal interactions are necessary fornormal histodifferentiation and that the epithelial requirement for genesis ofmembrane bone within maxillary mesenchyme is relatively non-specific withrespect to the source and age of the epithelium.

The authors are grateful to Mr David C. Warner for his skilled technical assistance. Thisinvestigation was supported by Research Grant 1 R23 DEO4859-02 from the NationalInstitute of Dental Research.

REFERENCES

BENOIT, J. A. & SCHOWING, J. (1970). Morphogenesis of the neurocranium. In Tissue Inter-actions During Organogenesis (ed. E. Wolff), pp. 105-130. New York: Gordon andBreach.

DHOUAILLY, D. (1978). Feather-forming capacities of the avian extra-embryonic somato-pleure. / . Embryol. exp. Morph. 43, 279-287.

FROMMER, J. & MARGOLIES, M. R. (1971). Contribution of Meckel's cartilage to ossificationof the mandible in mice. / . dent. Res. 50, 1260-1267.

HALL, B. K. (1978). Initiation of osteogenesis by mandibular mesenchyme of the embryonicchick in response to mandibular and non-mandibular epithelia. Archs Oral Biol. 23,1157-1161.

HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in development of thechick embryo. / . Morph. 88, 49-92.

PEARSE, A. G. E. (1960). Histochemistry, Theoretical and Applied, 2nd ed. Boston: Little,Brown & Company.

RAWLES, M. E. (1963). Tissue interactions in scale and feather development as studied indermal-epidermal recombinations. / . Embryol. exp. Morph. 11, 765-789.

SCHOWING, J. (1968a). Influence inductrice de l'encephale embryonnaire sur le developpe-ment du crane chez le poulet. I. Influence de l'excision des territoires nerveux anterierssur le developpement cranien. / . Embryol. exp. Morph. 19, 9-22.

SCHOWING, J. (19686). Influence inductrice de l'encephale embryonnaire sur le developpe-ment du crane chez le poulet. II. Influence de l'excision de la chorde et des territoiresencephaliques moyen et posterieur sur le developpement cranien. / . Embryol. exp. Morph.19, 23-32.

SCHOWING, J. (1968 c). Influence inductrice de l'encephale embryonnaire sur le developpe-ment du crane chez le poulet. III. Mise en evidence du role inducteur de l'encephale dansl'osteogenese du crane embryonnaire du poulet. / . Embryol. exp. Morph. 19, 83-94.

TONEGAWA, Y. (1973). Inductive tissue interactions in the beak of a chick embryo. Develop.Growth and Differentiation 15, 57-72.

TYLER, M. S. (1978). Epithelial influences on membrane bone formation in the maxilla of theembryonic chick. Anat. Rec. 192, 225-234.

Tissue interactions on maxillary osteogenesis 281TYLER, M. S. & HALL, B. K. (.1977). Epithelial influences on skeletogenesis in the mandible

of the embryonic chick. Anat. Rec. 188, 229-240.WILSMAN, N. J. & VANSICKLE, D. C. (1971). Histochemical evidence of a functional hetero-

geneity in neonatal canine epiphyseal chondrodytes. Histochem. J. 3, 311-318.

{Received 10 August 1979, revised 7 November 1979)

Documents

The genesi osf membrane bone in the embryonic chick ... · bone (Fig. 1, Table 1). The epithelium differentiated into a stratified squamous epithelium consisting of a basal layer