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Three-dimensional reconstructions of the primary palate region in normal human embryos.

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THE ANATOMICAL RECORD 238:108-113 (1994)
Three-Dimensional Reconstructions of the Primary Palate Region in
Normal Human Embryos
FRANK P. RUDfi, LEIGH ANDERSON, DAVID CONLEY, AND RAYMOND F. GASSER
Department of Orthodontics and Curriculum i n Genetics, University of North Carolina,
Chapel Hill, North Carolina (F.P.R.); Department of Oral Biology, School of Dentistry
(L.A.) and Department of Biological Structure, School of Medicine (D.C.), University of
Washington, Seattle, Washington; Department of Anatomy, School of Medicine, Louisiana
State University Medical Center, New Orleans, Louisiana (R.F.G.)
ABSTRACT
Our knowledge of the precise spatial relationships of human primary palate morphogenesis remains poorly defined. This is due to
intrinsic difficulties that exist in the study of the subject matter and a lack
of adequate methodologies. We present a novel new method to allow precise three-dimensional (3-D) visualization of developing embryonic structures in previously sectioned embryos. In our study we focus on human
primary palate development. Five normal human embryos from the Carnegie collection were used. 3-D reconstructions appear similar to scanning
electron micrographs (SEM); however, unlike in SEM studies, the original
specimen has been previously sectioned histologically. 3-D reconstruction
from serial sections involved 1) histological preparation of specimen, 2)
projection onto digitizing board, 3) digitization, 4) automated reassembly,
and 5 ) relay to interactive optical disc recorder. Detailed observations of
each reconstruction were then made. Data generated in this manner may
also be used in the near future for quantitative morphometrics. Thus, 3-D
reconstruction techniques presented in this paper generated precise spatial information on the development of the human primary palate.
0 1994 Wiley-Liss, Inc.
Key words: Computer, Reconstruction, Primary palate, Human
The human primary palate is formed during the
sixth week of embryological development with the
merging of the maxillary, medial, and lateral nasal
prominences, and the proliferation of their underlying
mesenchyme (Warbrick, 1960; Pourtois, 1972; O’Rahilly, 1978, Hinrichsen, 1985).In humans, clefts of the
primary palate result in clefts of the lip and anterior
maxilla. Such clefts are among the most common congenital malformations observed in live births (O’Rahilly, 1978; Fraser, 1980; Biddle and Fraser, 1986:
Wedden and Tickle, 1986: Sulik et al., 1988; Rowe et
al., 1991). Much is known about the many complex
cellular interactions leading to primary palate formation. These include epithelial bridging (Forbes and
Steffek, 1989), programmed cell death (Sulik et al.,
1988), and mesenchymal proliferation (Igawa et al.,
1986; Minkoff, 19911, among others. However, to fully
comprehend the impact of these cellular events on primary palate formation we must study them in the context of the known regional growth patterns of the developing midface. During primary palate formation
several major morphological events occur simultaneously. These morphogenetic relationships merit our
attention and will increase our comprehension of the
exact mechanisms controlling primary palate formation. For example, i t is known that profound changes in
craniofacial morphology occur in response to growth of
0 1994 WILEY-LISS, INC.
the developing brain (Desmond and O’Rahilly, 1981;
Diewert, 1985; McCann et al., 1991).
Our knowledge of primary palate morphogenesis remains poorly defined. The collection and processing of
spatial data in the field of embryology has been limited
by two main factors. These are the intrinsic difficulties
which exist in the study of the subject matter, and a
lack of adequate methodologies. The intrinsic difficulties include size and complexity of the object, opacity,
and lack of native contrast. Several of these factors
have been overcome, in part, by existing techniques.
Each, however, has its practical limitations. Histological staining and fixation techniques allow individual
structures to be studied in a two-dimensional plane of
section (Gasser, 1975). Serial section techniques have
then been employed. However, it remains difficult to
infer the precise spatial relationships of objects prepared in this manner. Electron microscopy (EM) remains a valuable tool in the study of primary palate
formation (Waterman and Meller, 1973; Sulik and
Received February 5, 1993; accepted July 30, 1993.
Address reprint requests to Dr. Frank P. Rude, CB#7090, 312 Taylor Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC
27599.
3-D RECONSTRUCTIONS O F HUMAN PRIMARY PALATE
Schoenwolf, 1985; Steffeck and Lenke, 1987). SEM
studies provide images of high magnification and resolution. However, the specimen prepared for SEM is
very fragile after critical point drying and can be easily
damaged prior to embedding and sectioning. This is
particularly disadvantageous in the study of human
embryos where collections remain scarce. New techniques are needed to make detailed three-dimension
studies of each available specimen while allowing further study of the original embryo using different techniques. This is particularly desirable in the field of
developmental toxicology where one would like to compare data generated in histological, molecular, and 3-D
studies on the same specimen and when rare human
embryos with particularly interesting malformations
are found.
Recent advances in computer science have provided
new insight in the study of primary palate morphogenesis. In a recent morphometric analysis of human
embryos in the Carnegie collection, regional growth
patterns in the face and brain were identified using
finite-element methods (Diewert and Lozanoff, 1986).
However, no high resolution 3-D reconstructions of the
primary palate region have been made to date.
In this paper we present a novel new method to allow
precise visualization of developing embryonic structures. These computer generated reconstructions are of
similar quality as scanning electron micrographs
(SEM); however, unlike in SEM studies the original
specimen has been previously sectioned histologically.
High resolution graphical reconstructions of the primary palate region of five human embryos were made.
These were taken from the fourth to sixth week of development in utero.
MATERIALS AND METHODS
Specimens
Five human embryos of excellent quality from the
Carnegie collection were used. All specimens were
staged according to the Streeter-ORahilly criteria
(O’Rahilly, 1978, 1979; O’Rahilly and Muller, 1987)
which is based on a composite point scoring of external
and internal developmental features. These include
head-body proportions, development of face, eyes, ears,
limbs, and internal organ systems. Crown-rump length
measurements had been taken of each embryo after
fixation but prior to sectioning. The lengths and developmental stage of each embryo are as follows: stage 14,
7 mm; stage 16, 13.5 mm; stage 17, 14 mm; stage 18,
14.5 mm; and stage 19, 16.5 mm. According to ORahilly and Muller (19871, stages 14 to 19 have a n estimated postovulation age of 32 to 48 days. All of the
embryos had been fixed in Bouin’s solution, embedded
in paraffin, serially sectioned a t 10 pm, and stained
with hematoxylin and eosin.
Software and Hardware
Digitization, coordinate reassembly, and graphics
software were all written by J. Prothero for the Digital
Anatomist Program, Department of Biological Structure, University of Washington, Seattle, and are described in detail in the literature (Prothero and Prothero, 1989; Conley et al., 1992). An IBM compatible
personal computer and a Scriptel digitizing pad were
used for the digitization process using the Morpho soft-
109
ware package. This generates numerical descriptions
of the objects. Different embryonic structures were separated into subfiles at this time. The numerical data
generated during digitization were networked to a
workstation with high resolution graphic production
capabilities (Silicon Graphics IRIS 4D/70 GT workstation). 3-D reconstructions were then made using the
Skandha software package. The images created in this
manner were relayed to a n optical disc recorder for
interactive image display (Panasonic TQ-3031F). Computer facilities used in this study were housed a t the
department of Biological Structure computer imaging
lab and the Regional Clinic Dental Research Center,
School of Dentistry, University of Washington, Seattle.
Reconstruction Process
Three-dimensional reconstruction from serial sections involves 1)histological preparation of specimen,
2) projection onto a digitizing board, 3) digitization, 4)
automated reassembly, and 5 ) relay to a n interactive
optical disc recorder. Each embryo section was projected onto a digitizing board using a trisimplex microprojector. Constant focal length and magnification
were maintained at all times to eliminate magnification artifact. A micrometer was used to determine the
exact magnification of each section prior to digitization. This was entered into the computer at the time of
digitization to allow for quantitative measurements.
The various epithelial, mesenchymal, neural, vascular,
and calcified components were traced manually and
then digitized by guiding a digitization device over the
contours. During digitization these components were
entered into distinct subfiles to allow future manipulation, i.e., color coding, adding or subtracting objects,
rotating objects in real time, and making surface objects transparent to reveal deeper structures. To realign the serial sections in precise register the sections
may be superimposed by employing a best-fit method
or by the use of internal fiducials (Prothero and Prothero, 1986).
The x and y coordinates generated during digitization were subsequently reassembled to form a numerical description of the object. These data were then used
to form 3-D reconstructions using the Skandha software package. The reconstructions were relayed to a n
interactive optical disc recorder. In this manner they
could be rotated on any axis of the x, y, and z planes of
space in real time. Separate embryos could be displayed simultaneously in precise relative scale a t any
desired magnification. Data generated in this manner
may be used in the near future for quantitative morphometrics. High resolution 3-D reconstructions were
made of the primary palate region, one for each stage.
RESULTS
Each 3-D reconstruction was observed in detail from
multiple views. The overall morphology was similar to
that previously reported as was expected. When the
frontonasal prominence (elevation or swelling) elongated vertically and narrowed, the distance between
the nasal pits decreased and the maxillary prominence
enlarged and elongated in the frontal direction
(Diewert and Shiota, 1990). In addition to these previously reported findings, detailed specific information
was obtained. Particularly interesting observations
110
F.P. RUDE ET AL.
Fig. 1. Three-dimensional reconstructions of the stage 14 embryo. inences. C : Close-up, frontal view of upper face of the same view
A Frontal view of the face showing the nasal pit (np) and the fron- shown in A with the mandibular and second arch prominences removed. Arrowhead points to the site of the epithelial entrapment as in
tonasal cfn), medial nasal (rnn), lateral nasal (In),maxillary (m),
mandibular (mn),and second arch (10prominences. B: Lateral view of B. D Caudal (inferior) view of the primary palate region resulting
the face resulting from a + 75" rotation in the y-axis of the view shown from a -30" rotation on the x-axis of the view shown in A. The relain A. Abbreviations are the same as in A. Arrowhead points to site of tionship of the nasal and maxillary prominences are better visualized.
epithelial entrapment between the medial nasal and maxillary prom-
were made when comparing the reconstructions of the
embryos a t different stages. The medial nasal, lateral
nasal, and maxillary prominences were easily distinguished from one another and their relationships to
each other as well as to the frontonasal prominence and
internal structures was evident. Both the epithelial
and mesenchymal components of this region could be
clearly delineated.
In the youngest embryo (stage 141, the epithelium of
the nasal pits and facial prominences were readily
identified and their relationship observed (Figs. 1A-D,
2A). The precise location and arrangement of the epithelial entrapment were observed between the medial
nasal and maxillary prominences (Fig. lB,C). As the
head expands between stages 14 and 16 the nasal
prominences move forward. the epithelium entrapped
by their forward growth formed epithelial seems called
the nasal fins that separate the mesenchyma underlying the nasal and maxillary prominences (Fig. 2B, ar-
rows a and b). A medial fin (arrow a)separated lateral
nasal and maxillary mesenchyma and a lateral component of the fin (arrow b) separated lateral nasal and
maxillary mesenchyma. The medial fin extended dorsally (posteriorly) without interruption to the site of
the oronasal membrane that separated the primitive
oral and nasal cavities. the position and extent of the
medial fin is indicated by a surface groove at stage 19
(Fig. 3A,B).
The oronasal membrane has ruptured in the stage 17
reconstruction thereby forming the primary choana
(Fig. 2C, arrowhead b). In the stage 18 reconstruction a
localized dissruption of the medial epithelial fin was
present resulting in the formation of a mesenchymal
isthmus that joined the mesenchyma in the medial nasal prominence with that in the maxillary prominence
(Fig. 2D). As the region continued to enlarge between
stages 18 and 19 the isthmus expanded and was accompanied by mesenchymal proliferation. The expansion
3-D RECONSTRUCTIONS OF HUMAN PRIMARY PALATE
111
Fig. 2. Inner surface of the nasal pit epithelium a t four different
stages. A Stage 14. Inner surface of the right nasal pit epithelium
(np)is shown from its left lateral and slightly caudal aspect when the
view shown in Figure 1A was rotated 220" on the y-axis. When the
intervening mesenchymal tissue is made translucent the contour of
the inner surface of the epithelium is revealed. B Stage 16. Dorsal
view of the inner surface of the left nasal pit epithelium (np).Nasal
fins (aand b arrows) separate medial nasal (mn)and lateral nasal (In)
mesenchyma from the mesenchyma in the maxillary prominence
(m).
C: Stage 17. Dorsal view of the inner surface of the left nasal pit
epithelium (np).Left nasal fin separating mesenchymal tissue in the
medial nasal (mn)and maxillary (mz)prominences remains intact
(arrow a).Site of earliest rupture of oronasal membrane is shown on
the right side a t arrowhead b. D. Stage 18. Dorsal view of the inner
surface of the right nasal pit epithelium (np).Mesenchymal isthmus
on the right side (arrowhead a)was formed by localized disruption of
the nasal fin epithelium thereby joining the medial nasal (mn)and
maxillary (m)mesenchyma. Compare Figure 3C. Primary choana
(arrowhead b) is located a t the site of the former oronasal membrane.
was not symmetrical but predominated in the rostrocaudal direction.
When the size of the primary palate region was compared in the stages studied a marked increase in the
volume of mesenchymal tissue became evident. The increase occurred as nasal cavity volume decreased relatively.
The lateral palatine process that forms the secondary
palate could be identified in the stage 17 to 19 reconstructions. It extended dorsally (posteriorly) and caudally (inferiorly) from the site of the oronasal membrane/primary choana (Fig. 3). The arrangement is
similar to that reported by others (Tamarin, 1982). The
mesenchyma in the process was continuous rostrally
with that in the maxillary prominence (Fig. 3C).
In future studies morphometric analysis being devel-
oped in the Digital Anatomist Program will allow
quantification of these observations. Both Meckel's cartilage and the osseous structures of the developing
mandible were digitized in the older embryos. The developing telencephalon was also digitized in each specimen. Thus, its position relative to developing primary
palate could be seen at each stage.
DISCUSSION
The 3-D reconstruction techniques described in this
paper provide images of high magnification and resolution of serially sectioned embryos. These images are
similar to those generated in SEM studies. However, at
present, the resolution of these reconstructions is limited by the thickness of the plane of section and, therefore, is lower than that of SEMs. Also, since the origi-
112
F.P. RUDE ET AL.
Fig. 3.Three-dimensional reconstructions of the stage 19 embryo. is indicated by arrowheads. C: Caudal view of the primary palate
A: Close-up, frontal view of the nasal and primary palate regions region resulting from a 180"rotation on the y-axis of the view shown
showing the medial nasal (rnn),lateral nasal (In),and maxillary (m)in A. Arrowhead points to the former site of the left oronasal memprominences. The left lateral palatine process @p) extends dorsally brane. D: Close-up, caudal view of the primary palate region similar
(posteriorly) from the oronasal membrane (arrowhead). B Caudal to that shown in C. When the intervening mesenchymal tissue is
view of the primary palate region resulting from a -40" rotation on made translucent, the relationship of the right oronanasal membrane
site (arrowhead) to the nasal pit epithelium ( ~ pand
) lateral palatine
the x-axis of the view shown in A. Relations of the right primary
choana are better visualized in this view. Medial border of the choana process (pp) are better visualized.
nal specimens is sectioned, dimensional changes
incurred during tissue preparation and data processing
can introduce artifact (McLean and Prothero, 1991).
In the field of developmental biology one would like
to compare data generated in histological, molecular,
and 3-D studies on the same specimen when rare human embryos with particularly interesting malformations are found. Although SEM specimens can be
scanned first, and then embedded and sectioned, these
specimens are extremely fragile after critical point drying and can be easily damaged. This problem is obviated using our technique since the original specimen is
preserved histologically.
Several questions can be addressed in future studies
using the computer imaging techniques described in
this paper. The first question represents temporal, a s
well as structural, aspects of our work. How does primary palate growth proceed after isthmus formation?
In our study, a n increase in the volume of the primary
palate region was seen to closely match isthmus expansion in the stage 17-19 embryos. In fact their growth
appeared to be in direct proportion. This occurred, however, a t the time when the distance between the nasal
pits was decreasing. Further studies are needed to examine this relationship.
The second question that could be addressed involves
secondary palate development. In our study the early
events of secondary palate formation were seen in relation to primary palate formation. Further studies
could provide spatial data on the position of the palatine shelves and developing tongue a t key developmental stages. Relative positions of maxillary and mandibular structures could also be determined.
Finally, high resolution 3-D reconstructions can address the question of how normal primary palate formation compares with that observed in early human
3-D RECONSTRUCTIONS OF HUMAN PRIMARY PALATE
fetuses exhibiting cleft lip with and without cleft palate. In serial section studies of human embryos with
cleft lip a reduced thickness of mesenchymal bridging
between the median nasal and maxillary prominences
is seen in combination with a n excessive amount of
epithelium at the junctions between these processes
(Diewert and Shiota, 1990). In future studies, accurate
visualization of these changes in three-dimension will
be possible using methods described in our study.
In conclusion, high resolution, 3-D computer reconstructions of five normal human embryos from the Carnegie collection were made. They represent critical
stages in normal human primary palate development
and help improve our comprehension of the spatial relationships of the precise anatomical components of the
developing primary palate. Future studies should increase this understanding by comparing our findings to
3-D models of early fetuses with cleft lip and or palate
or other significant craniofacial malformations.
ACKNOWLEDGMENTS
The authors would like to make the following acknowledgments. John Prothero, John Sundsten, and
Kenneth Kastella for training in the 3-D reconstruction technique. Cornelius Rosse, Digital Anatomist
Program, Department of Biological Structure, University of Washington, Seattle, for providing the 3-D reconstruction software. Roy Page, and Lars Hollender,
Regional Clinical Dental Research Center, University
of Washington, Seattle, for computer facilities. Thomas
Sadler, University of North Carolina at Chapel Hill,
for critical review of the manuscript. This study was
funded by a n AADR student research fellowship, NLM
grant LM04925-03, and BRSG grant RR05346. It was
presented at the 1991 Edward H. Hatton awards national competition of the American Association of Dental Research, and was awarded its pre-doctoral award.
(J. Dent. Res. 70 (special issue):381, 1991).
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