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/ . Embryo/, exp. Morph. Vol. 56, pp. 269-281, 1980
Printed in Great Britain © Company of Biologists Limited 1980
269
The genesis of membrane bone in the
embryonic chick maxilla: epithelial-mesenchymal
tissue recombination studies
By MARY S. TYLER 1 AND DAVID P. McCOBB 2
From the Department of Zoology, University of Maine
SUMMARY
In the present study, the question of whether a relatively non-specific epithelial requirement 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. Maxillary
mesenchyme (from embryos HH 18-19) which in the absence of epithelium will not form
bone was recombined with epithelium from maxillae of similarly aged embryos (homotypichomochronic recombination) and of older embryos (HH 25) (homotypic-heterochronic
recombination). Heterotypic recombinations were made between maxillary mesenchyme
(HH 18-19) and the epithelium from wing and hind-limb buds (HH 19-22). Recombinants
were grown as grafts on the chorioallantoic membranes of host chick embryos. Grafts of
intact maxillae, isolated maxillary mesenchyme, and isolated epithelia from the maxilla,
wing-, and hind-limb buds were grown as controls. The histodifferentiation of grafted intact
maxillae was similar to that in vivo; both cartilage and membrane bone differentiated within
the mesenchyme. Grafts of maxillary mesenchyme (from embryos HH 18-19) grown in
the absence of epithelium formed cartilage but did not form membrane bone. Grafts of
maxillary mesenchyme (from embryos HH 18-19) recombined with epithelium in homotypichomochronic, homotypic-heterochronic, and heterotypic tissue combinations formed
membrane bone in addition to cartilage. These results indicate that maxillary mesenchyme
requires the presence of epithelium to promote osteogenesis and that this epithelial requirement is relatively non-specific in terms of type and age of epithelium.
INTRODUCTION
Previous studies have shown that epithelial-mesenchymal interactions are
influential 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 the
chick that the mesenchyme requires the presence of an epithelium during a
specific 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, Washington 98195, U.S.A.
l8
EMB 56
270
M. S. TYLER AND D. P. McCOBB
membrane bone formation to occur. Removal of the epithelium during this time
period prevents osteogenesis within the isolated mesenchyme. In the systems
studied, the presence of the epithelium ceases to be required by the mesenchyme
several days prior to the actual onset of osteogenesis. In the developing chick
maxilla, 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 the
mesenchyme. The actual onset of ossification within the maxilla does not occur
until HH 35 (8±-9 days of incubation) (Tyler, 1978).
The present study examines whether or not the epithelial influence required
by maxillary mesenchyme through HH 22 is specific for a particular type or
age of epithelium. A similar study on the chick mandible indicates that the
epithelial requirement for bone formation within the mandibular mesenchyme
is relatively non-specific with respect to the type of epithelium that will promote
mandibular osteogenesis, but that the age of the epithelium is an important
factor in determining whether the epithelium will be influential in promoting
osteogenesis.
In the present study, tissue recombinations were implemented to test the
influence of heterochronic and heterotypic epithelia on the genesis of membrane
bone within the maxillary mesenchyme. Maxillary mesenchyme was isolated
from its epithelium at a time when an epithelial influence is required for osteogenesis, and the mesenchyme was recombined with maxillary epithelium from
similarly aged embryos (homotypic-homochronic recombination) and with
maxillary epithelium from embryos beyond the stage at which an epithelial
influence is required for maxillary osteogenesis (homotypic-heterochronic
recombination). In heterotypic recombinations, isolated maxillary mesenchyme
was recombined with epithelia from the wing and hind-limb buds. These
epithelial regions normally do not participate in promoting membrane bone
formation. Recombinations were grown as grafts on the chorioallantoic membrane of host chick embryos.
The results indicate that the epithelial requirement for osteogenesis within
the chick maxilla is relatively non-specific and differ to a certain extent from
those reported for the chick mandible (Hall, 1978).
MATERIALS AND METHODS
Tissue preparation
Eggs from the common chicken (Gallus domesticus, White Leghorn) were
incubated 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 were
staged according to the Hamburger and Hamilton (1951) staging (HH) series,
and organ dissection was carried out in Tyrode's solution. Maxillae from
embryos 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 (3^—4 days incubation) were used in the
study.
Separation of the epithelium from the mesenchyme of an organ was achieved
enzymatically. 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 the
mesenchyme by manipulation with a small-bore pipette and finely sharpened
tungsten needles. Separated tissues were placed in a solution of Tyrode's
and fetal calf serum (1:1, v/v) which served to inactivate any residual enzymatic solution in the tissues, and tissues were stored in the Tyrode's-serum
solution until use.
Grafting procedures.
Intact maxillae, intact wing and hind-limb buds, the isolated epithelial
and mesenchymal components of maxillae, isolated epithelia from the wing and
hind-limb buds, and maxillary mesenchyme recombined with epithelia from
the maxilla or from the wing or hind-limb buds were grafted to the chorioallantoic membrane of host chick embryos that had been incubated for eight
or nine days. As a control to determine whether the enzymatic treatment
interfered with tissue differentiation, grafts were made of intact maxillary
processes that had been enzymatically treated without subsequent mechanical
tissue separation.
Intact organs and isolated tissues were placed on Millipore filter discs
(black; 5 mm diameter; 0-45 jam porosity; 125-150jam thick; obtained from
Millipore Filter Corp., Bedford, Massachusetts). The filters served as supports
for the explanted tissues and facilitated localization of the graft at the time of
harvesting. In tissue recombination experiments, ultra-thin Millipore filter
discs (white; 5 mm diameter; 0-45 fim porosity; 25 + 5 jam thick) were used to
allow observation of the tissues by transmitted light during tissue manipulations.
To recombine epithelial and mesenchymal tissues, the mesenchyme was placed
on the filter and allowed to adhere to the filter; the epithelium was then
positioned as a flattened sheet over the mesenchyme. In certain instances, the
mesenchyme was positioned on top of the epithelium; this, however, was a less
successful method of achieving direct contact between the tissues over a large
surface area. In most instances, recombined tissues were placed in a CO2humidified incubator for one to two hours prior to grafting; this allowed
adhesion of the component tissues prior to any further manipulations.
Tissues on their Millipore filter discs were placed on the chorioallantoic
membrane of host chick embryos such that the grafted tissues were in direct
contact with the host tissue. The host embryos were then further incubated for
eight days.
18-2
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M. S. TYLER AND D. P. MCCOBB
Tissue interactions on maxillary osteogenesis
273
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 were
sectioned at 5 fim on a Sorval JB-4 rotary microtome. Mounted sections were
stained either with van Gieson's stain and alcian blue (pH 2-5-30) (Wilsman
and 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 intact
rudiments, 15 were of isolated maxillary mesenchyme, 10 were of enzymatically
treated intact maxillae, 14 were of isolated epithelia, 17 were of homotypichomochronic recombinations, 13 were of homotypic-heterochronic recombinations, 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 embryos
HH 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 maxillary
process 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 close
association with the oral region of the maxillary epithelium (ep). The graft, supported
by a Millipore filter (mf), is surrounded by host tissue (ht) from the chorioallantoic 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 maxilla
shown in Fig. 1. Feather germs (fg), shown in longitudinal section, are in the hump
stage of development, x 184.
Fig. 4. Photomicrograph of a section through a graft of maxillary mesenchyme
isolated from its epithelium at HH 19 and grown on the chorioallantoic membrane
for 8 days. Cartilage (c) has differentiated within the mesenchyme, but membrane
bone did not form, (ht designates host tissue from the chorioallantoic membrane
which surrounds the graft.) Alcian blue and van Gieson's stain, x 95.
Fig. 5. Photomicrograph of a section through a graft of maxillary mesenchyme
isolated from its epithelium at HH 25 and grown on the chorioallantoic membrane
for 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, removed from its embryo at HH 19, that was enzymatically treated without subsequent tissue separation and then grown on the chorioallantoic membrane for 8
days. Histodifferentiation is similar to that of the grafted intact maxillary process
shown in Fig. 1. (b, c, and ep designate membrane bone, cartilage, and epithelium,
respectively.) Alcian blue and van Gieson's stain, x 95.
274
M. S. TYLER AND D. P. McCOBB
Table 1. Skeletogenesis in intact maxillae and isolated maxillary mesenchyme
grafted to the chorioallantoic membrane
Presence ( + ) or absence (—) of
Age of
donor
Cartilage
Membrane bone
Intact maxillary processes
+
+
+
+
Isolated maxillary mesenchyme
HH 18-19
+
HH 25
+
-IHH 18-19
HH 25
similar to that reported for the maxilla in vivo (Tyler, 1978). Bony trabeculae
representing the elongate membrane bones of the maxilla differentiated within
the maxillary mesenchyme, and cartilage, a precursor to the quadrate, an
endochondral bone, differentiated usually in close association with membrane
bone (Fig. 1, Table 1). The epithelium differentiated into a stratified squamous
epithelium consisting of a basal layer of mitotically active cuboidal-to-columnar
cells, one to two intermediate cuboidal cell layers, and one to three outer
squamous cell layers (Fig. 2). The greater number of cell layers occurred in
grafts of maxillae excised from embryos HH 25. In the aboral region of the
maxilla, feather germs were distinguishable (Fig. 3). The feather germs were in
the hump stage of development in grafts of maxillae excised from young
embryos (HH 18-19) and were in the elongation phase of development in
grafts of maxillae excised from older embryos (HH 25).
Isolated maxillary mesenchyme
In explants of maxillary mesenchyme isolated from its epithelium during
early development (HH 18-19) and grown as a graft in the absence of its
epithelium, cartilage differentiated, but membrane bone did not form (Fig. 4,
Table 1). These results confirm those of an earlier study (Tyler, 1978) indicating
that the presence of an epithelium is required at this stage for maxillary membrane bone formation. In grafts of maxillary mesenchyme isolated from its
epithelium at a later stage of development (HH 25), membrane bone formed in
addition to cartilage (Fig. 5, Table 1). This confirms an earlier report (Tyler,
1978) that at this stage in development, an epithelial influence is no longer
necessary for promoting maxillary osteogenesis.
The histodifferentiation of intact maxillae (HH 18-19) that were enzymatically
treated without subsequent mechanical tissue separation and then grown as
chorioallantoic membrane grafts was similar to that of grafted maxillae that had
not been enzymatically treated (Fig. 6), indicating that the enzymatic separation
techniques do not cause irreparable damage to the component maxillary tissues.
Tissue interactions on maxillary osteogenesis
275
Table 2. Skeletogenesis in maxillary mesenchyme {HH 18-19) recombined with
homotypic and heterotypic epithelium and grafted to the chorioallantoic
membrane
Epithelium
Source
Maxilla
Maxilla
Wing bud
Hind-limb bud
Mesenchymal differentiation
Presence ( + ) or absence ( - ) of
Age of
donor
HH
HH
HH
HH
Cartilage
Membrane bone
18-19
25
19-22
19-22
Homotypic recombinations of maxillary mesenchyme and epithelium
In homotypic tissue recombinations, maxillary mesenchyme from young
embryos (HH 18-19) was recombined with maxillary epithelium from similarly
aged embryos (homotypic-homochronic recombination) and with maxillary
epithelium from older embryos (HH 25) (homotypic-heterochronic recombination). The position of the tissues with respect to one another was not according to their original orientation.
In grafts of homotypic-homochronic recombinants, the histodifferentiation
of the recombined tissues was similar to that of grafted intact maxillae. Membrane bone formed within the mesenchyme in addition to cartilage (Table 2),
and the degree of epithelial differentiation was similar to that of grafted intact
maxillae of a similar cumulative age (initial age + incubation time as graft).
Membrane bone formed usually in close proximity to the epithelium. No
specificity was exhibited in terms of epithelial region with which bone was
associated; bone was found in association with both oral and aboral regions
of the maxillary epithelium.
In grafts of homotypic-heterochronic recombinants, mesenchymal differentiation was similar to that of grafted homotypic-homochronic recombinants
(Table 2). Membrane bone formed usually in close proximity to the epithelium
of either the oral or aboral maxillary regions (Fig. 7). Cartilage formed often in
close proximity to membrane bone (Fig. 8). Epithelial differentiation in these
recombinants was similar to that of grafted intact maxillae with a cumulative
age equal to that of the epithelium rather than to that of the mesenchyme of the
heterochronic recombinant (Fig. 9).
Heterotypic recombinations of maxillary mesenchyme and epithelium from the
wing- and hind-limb buds
Heterotypic tissue recombinations were made between maxillary mesenchyme
isolated from young embryos (HH 18-19) and epithelium isolated from wing
276
M. S. TYLER AND D. P. MCCOBB
Tissue interactions on maxillary osteogenesis
277
and hind-limb buds of embryos HH 19-22, and recombinants were grown as
chorioallantoic membrane grafts. Mesenchymal differentiation in each type of
recombinant was similar to that of homotypic recombinants irrespective of
the 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 membrane bone was generated usually in close proximity to the epithelium (Fig. 10).
In two instances, the chondrogenic region of the maxillary mesenchyme was
not included in the explant, and in these grafts membrane bone formed in
close association with the epithelium in the absence of cartilage (Fig. 11).
Epithelial differentiation in heterotypic recombinants was similar to that of
grafted intact wing and hind-limb buds. The epithelium became a stratified
squamous epithelium consisting of a cuboidal germinative cell-layer, one to
FIGURES
7-12
Fig. 7. Photomicrograph of a section through a homotypic-heterochronic recombinant graft. Maxillary mesenchyme, isolated at HH 19, was recombined with
maxillary epithelium, isolated at HH 25, and grown on the chorioallantoic membrane for 8 days. Membrane bone (b) has formed in close association with the
epithelium (ep) and cartilage is not in the vicinity of the membrane bone. Alcian
blue and van Gieson's stain, x 95.
Fig. 8. Photomicrograph of a section through a homotypic-heterochronic recombinant graft similar to that in Fig. 7. In this graft, cartilage (c) is found in
close association with membrane bone (6), and the membrane bone has formed in
close 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 aboral
region of the maxillary epithelium. Feather germs (fg), shown in longitudinal section
at their base and in transverse section more distally, are in the elongation stage
of development and are more advanced than those shown in Fig. 3. Alcian blue
and 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 epithelium, isolated at HH 21, and grown on the chorioallantoic membrane for 8 days.
Mesenchymal histodifferentiation is similar to that of homotypic recombinant
grafts as shown in Fig. 8. Epithelial differentiation is similar to that of grafted
intact wing buds with a similar cumulative age. Feather germs (fg) are in the
elongation 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 graft
similar to that shown in Fig. 10 except that the chondrogenic region of the mesenchyme was not included in the explant. Membrane bone (b) has formed in close
proximity to the epithelium (ep) in the absence of cartilage, (ht and mf designate
host 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 chorioallantoic
membrane for 8 days. The epithelium, underlaid by fibroblasts of host tissue (ht)
origin, has differentiated into a stratified squamous epithelium. Regions of the
epithelium have formed epithelial whorls (arrow) rather than remaining as a
flattened sheet. Feather germs are not present within the graft. Hematoxylin eosin,
and alcian blue, x 372.
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M. S. TYLER AND D. P. McCOBB
two intermediate cuboidal cell layers, and one to two outer squamous cell
layers. Feather germs in the elongation phase of development were distinguishable and were at the same level of development as those of grafted intact wing
and hind-limb buds of a similar cumulative age (Fig. 10). Differences between
wing and hind-limb-bud epithelium were not detected.
In approximately 27 % of all recombinant grafts, close association between
epithelium and mesenchyme was not maintained; in these instances, membrane
bonefailed to form within the mesenchyme though cartilage did form. Epithelialmesenchymal contact and consequent osteogenesis were maximized experimentally by blanketing the already substrate-adherent mesenchyme with the
epithelium and placing recombined tissues in a CO2-humidified incubator for
1 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 wingand hind-limb buds (HH 19-22), grown as chorioallantoic membrane grafts
in the absence of their mesenchyme, became underlaid by host fibroblasts
from the chorioallantoic membrane and achieved a limited degree of differentiation (Fig. 12). In grafts of each different type of epithelium, the
epithelium differentiated into a stratified epithelium consisting of a germinative
cuboidal cell-layer and one to five outer cell layers which graded from cuboidal
to squamous. Feather germs were not observed, nor were skeletal elements
(either cartilage or bone) found within the host tissue associated with the
grafted epithelium.
DISCUSSION
It has been shown in a previous study (Tyler, 1978) and confirmed in this
study that during early development the presence of an epithelium is a requirement for ensuing genesis of membrane bone within the mesenchyme of the
embryonic chick maxilla. The results further indicate that this epithelial
requirement is relatively non-specific. In homotypic recombinations, it was
shown that re-establishing the original orientation of the maxillary epithelium
with respect to its mesenchyme was not necessary for osteogenesis; membrane
bone formed within the mesenchyme of the recombinants irrespective of the
epithelial orientation. The results from heterotypic recombinations established
that maxillary mesenchyme does not specifically require maxillary epithelium
to promote osteogenesis; other types of epithelia which in normal development
are not associated with membrane-bone-forming mesenchyme (epithelium
from the wing- and hind-limb buds) were shown to be capable of promoting
osteogenesis within maxillary mesenchyme. From heterochronic recombinations,
it was shown that the response of maxillary mesenchyme to epithelium is not
restricted to a specific age of epithelium; epithelium removed from maxillae
after the time during which the epithelium is required for osteogenesis (isolated
Tissue interactions on maxillary osteogenesis
279
at HH 25) is still capable of promoting osteogenesis in maxillary mesenchyme
isolated from young embryos (HH 18-19).
These results differ to a certain extent from those of a similar study on
osteogenesis in the embryonic chick mandible (Hall, 1978); in both studies, however, it is concluded that the epithelial requirements for mesenchymal membrane
bone formation are relatively non-specific. In the study on mandibular osteogenesis, hind-limb-bud epithelium was found to promote membrane bone
formation in mandibular mesenchyme isolated at an early stage of development
(HH 18) (Hall, 1978) as was shown for maxillary mesenchyme in the present
study. In contrast to our results, however, the results in the mandibular study
indicate that neither wing-bud epithelium nor homotypic(mandibular) epithelium
promotes osteogenesis within mandibular mesenchyme (isolated at HH 18)
in either homochronic or heterochronic recombinations. The epithelial requirements for osteogenesis in the developing chick mandible, therefore, appear to be
more restrictive than those in the developing chick maxilla. Whether the results
reflect intrinsic differences in the two osteogenic systems or whether the differences between the two studies are a reflection of the different techniques used
for growing the tissues (organ culture, Hall, 1978; chorioallantoic membrane
graft, this study) is still to be determined. It has been shown that both organ
culture and the chorioallantoic membrane of host embryos promote normal
histogenesis of intact organ rudiments; however, the two environments have
been shown to differ in the amount of tissue growth that each supports and in
the type of organ morphogenesis that occurs within each (Tyler and Hall,
1977). Further studies of maxillary and mandibular osteogenesis, therefore, are
being made to determine the osteogenic potential of maxillary tissue recombinations in organ culture and mandibular tissue recombinations grown as
chorioallantoic membrane grafts.
In earlier histological studies of membrane bone formation it was suggested,
based on the proximity of cartilage to the mandibular membrane bones, that
cartilage is necessary for membrane bone formation (Frommer & Margolies,
1971). This suggestion has yet to be substantiated, and results from the present
study, in which membrane bone formed within maxillary mesenchyme in the
absence of cartilage in two heterotypic recombinant grafts, indicate that the
presence of chondrogenic centers (beyond HH 18) is not required for maxillary
osteogenesis. 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 earlier
reports (Tonegawa, 1973; Tyler & Hall, 1977; Tyler, 1978) that host fibroblasts
from the chorioallantoic membrane are sufficient to maintain a germinative
cell layer within an epithelium and to support a limited degree of epithelial
histodifferentiation. That the host mesenchymal tissue did not participate in
feather formation suggests that there are specificity requirements for the type
280
M. S. TYLER AND D. P. MCCOBB
of mesenchyme that will support feather formation within an epithelium.
This suggestion is supported by other recombination studies of feather- and
non-feather-forming tissues (e.g. Rawles, 1963; Dhouailly, 1978). The fact
that the grafted epithelium, though capable of promoting osteogenesis in
maxillary mesenchyme, did not induce membrane bone formation in the host
tissue associated with it indicates that the presence of epithelium, though a
requirement for maxillary membrane bone formation, is not a sufficient condition
for inducing bone formation in normally non-osteogenic mesenchyme.
In summary, the results of this study indicate that in the developing chick
maxilla, reciprocal epithelial-mesenchymal interactions are necessary for
normal histodifferentiation and that the epithelial requirement for genesis of
membrane bone within maxillary mesenchyme is relatively non-specific with
respect to the source and age of the epithelium.
The authors are grateful to Mr David C. Warner for his skilled technical assistance. This
investigation was supported by Research Grant 1 R23 DEO4859-02 from the National
Institute of Dental Research.
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