DEVELOPMENTAL DYNAMICS 235:1219 1229, 2006

REVIEWSA PEER REVIEWED FORUM

Genes, Forces, and Forms: Mechanical Aspects

of Prenatal Craniofacial Development
Ralf J. Radlanski* and Herbert Renz

Current knowledge of molecular signaling during craniofacial development is advancing rapidly. We know
that cells can respond to mechanical stimuli by biochemical signaling. Thus, the link between mechanical
stimuli and gene expression has become a new and important area of the morphological sciences. This eld
of research seems to be a revival of the old approach of developmental mechanics, which goes back to the
embryologists His (1874), Carey (1920), and Blechschmidt (1948). These researchers argued that forces play
a fundamental role in tissue differentiation and morphogenesis. They understood morphogenesis as a
closed system with living cells as the active part and biological, chemical, and physical laws as the rules.
This review reports on linking mechanical aspects of developmental biology with the contemporary
knowledge of tissue differentiation. We focus on the formation of cartilage (in relation to pressure), bone (in
relation to shearing forces), and muscles (in relation to dilation forces). The cascade of molecules may be
triggered by forces, which arise during physical cell and tissue interaction. Detailed morphological
knowledge is mandatory to elucidate the exact location and timing of the regions where forces are exerted.
Because this nding also holds true for the exact timing and location of signals, more 3D images of the
developmental processes are required. Further research is also required to create methods for measuring
forces within a tissue. The molecules whose presence and indispensability we are investigating appear to be
mediators rather than creators of form. Developmental Dynamics 235:1219 1229, 2006.
2006 Wiley-Liss, Inc.
Key words: craniofacial morphogenesis; tissue differentiation; forces; developmental movements
Accepted 30 December 2005

INTRODUCTION
Molecules, Tissues, and
Forces
Our current knowledge of molecular
signaling during craniofacial development is advancing rapidly (Thorogood,
1993; Thorogood et al., 1998; Depew et
al., 2002; Francis-West et al., 2003).
The homeobox code has been shown to
be crucial in determining the polarity
of the head and the patterning of the
arrangement of the anatomical fea-

tures that make up the craniofacial

complex (Sharpe, 1995). There is,
however, a deep gap between the
rapid progress in elucidating the regulative interdependencies within molecular cascades and the availability
of detailed knowledge on the morphological level (Radlanski, 2003).
Research reports focusing on molecular signaling often exclude the morphological aspect. However, no tissue
grows alone; thus, the neighborhood of
tissues may be the decisive factor in

creating organic form. It is obvious

that these changes also involve forces
and mechanical interactions of the
neighboring tissues. This aspect has
been virtually neglected in recent
years.

MECHANICS AND
EMBRYOLOGY
The discussion has now been reopened
on an interdependency between mechanical stimuli, molecular signals,

Charite - Campus Benjamin Franklin at Freie Universitat Berlin, Center for Dental and Craniofacial Sciences, Department of Oral
Structural Biology, Berlin-Wilmersdorf, Germany
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Ra 428/1-3
*Correspondence to: Ralf J. Radlanski, Charite - Campus Benjamin Franklin at Freie Universitat Berlin, Center for Dental
and Craniofacial Sciences, Department of Oral Structural Biology, Amannshauser Str. 4-6, D-14197 Berlin-Wilmersdorf,
Germany. E-mail: ralfj.radlanski@charite.de
DOI 10.1002/dvdy.20704
Published online 2 February 2006 in Wiley InterScience (www.interscience.wiley.com).

2006 Wiley-Liss, Inc.

1220 RADLANSKI AND RENZ

Fig. 1. The Blechschmidt Collection of Human Embryos in the Anatomical Institute of Gottingen
University (Germany).

and tissue differentiation (Henderson

and Carter, 2002; Benjamin and
Hillen, 2003; Brouzes and Farge,
2004; Curtis, 2005; de la Fuente and
Helms, 2005). It was as early as in
1874 when Wilhelm His sen. (His,
1874) explained our body form as a
consequence of mechanical forces exerted by tissues growing in a conned
space. Eben J. Carey was also convinced that forces act during morphogenesis when he explained the formation of the annular muscles of the
colon and esophagus (Carey, 1920a,b,
1935).
In the 1950s, Blechschmidt created
a collection of three-dimensional (3D)
reconstructions (Blechschmidt, 1954)
of human embryos in Gottingen (Germany), which is still on display in
large showcases (Fig. 1). This enabled
him to compare the different stages of
development at the same scales. He
was very specic in describing growth
changes in terms of local increments
of tissue volume, displacement of tissue, local recession of tissue mass, invagination, and evagination. From his
observations, he drew conclusions on
the differential growth of different tissues in different regions. He found
that expansion in one region was accompanied by the formation of curvatures, folds, bulges, brims, and
crimples in adjacent or even distant
regions (Blechschmidt, 1960). Some
cells or tissues were in a favorable
condition and continued to grow,
whereas others were displaced or were
conned and compressed within a limited space. He described these ontoge-

netic processes as developmental

movements of the tissues. Blechschmidt examined local changes on
the level of cellular differentiation for
a correlation with the overall changes
of body shape, and he established general embryological rules (Blechschmidt, 1948). For example, he found
that cartilage develops in regions of
dense mesenchyme (densation elds),
whereas bone arises in regions of detraction and muscles in regions of dilation (Blechschmidt, 1978, 2004).
The mechanical view of developmental anatomy suggests that forces
play a fundamental role in tissue differentiation
and
morphogenesis.
Blechschmidt (1948) could only assume molecular interactions, as they
have only been demonstrated in recent years. We now know that cartilage is formed under the inuence of
SOX9 (Akiyama et al., 2002), which is
found in regions of pressure, and that
bone is formed under the inuence of
RUNX2 (Mundlos et al., 1997), which
is found in regions of tensile strain
(Carter et al., 1998; de la Fuente and
Helms, 2005). We think the return to
this view of developmental mechanics,
including forces, cell forms, and their
location, will enable better understanding of the morphological aspects
of craniofacial development.

OUTCOME OF GROWTH
When cells grow, and particularly
when they increase in number, they
cannot stay in place and, thus, inevitably will exert an inuence on adja-

cent cells and on the extracellular matrix in their neighborhood. This

extracellular matrix is viscoelastic
and will be distorted by the movement
of the cells it contains. Distortion of
the extracellular matrix may trigger
further cell movement. Cells can respond in terms of chemotaxis and galvanotaxis. They can also move down a
density gradient (diffusion), move up
an adhesive gradient (haptotaxis), be
guided and inhibited by contact, or
just oat away (convection; Murray,
2000). Monolayer in vitro cultures
showed that chemotactic responsiveness was highly dependent on cell
density (Gormar et al., 1990; Szabo et
al., 2001).
Response to mechanical stimulation
is a basic biological phenomenon.
Nearly all cells process mechanical input and respond to it by inducing and
modulating biochemical pathways. If
the average mechanical load is increased in tissues, some tissues can
increase their performance and often
increase their bulk by cell division
(Jones et al., 1995). There is more detailed evidence that cells can respond
to mechanical forces (Chen and Grinnell, 1997). Cells that are stretched
proliferate in vitro (Kippenberger et
al., 1999, 2000). Investigations on the
molecular level revealed that mechanical forces at the interface between the
cell and the extracellular matrix can
activate stretch-induced changes in
ion channels. Cell contractions at adherence junctions activate cell membrane-associated secondary messenger pathways and lead to growth
-factor-like activities that inuence
cellular proliferation and protein synthesis (Silver et al., 2003). The diversity of responses reect the genotype
of the cell and the mechanical demands of the resident tissue (Banes et
al., 1995).
The link between mechanical stimulus and gene expression represents a
new and important area of morphological sciences. Stretch is an important
mechanical signal for the production
of more actin and myosin laments
and the addition of new sarcomeres in
series and in parallel (Goldspink,
1999). That mechanical aspect as one
of the main factors responsible for creating organic form can be found in the
older anatomic literature (His, 1874;
Carey, 1920b; Blechschmidt, 1948).

GENES AND FORCES 1221

Fig. 2. Diagram of the trapezius muscle, right

half. The orientation of the muscle bers reects
vertical growth of the cervical, thoracic, and
lumbar spine and transverse growth of the
shoulder (arrows). Taken from Blechschmidt,
2004, with permission of North Atlantic Books.

Blechschmidt regarded the whole of

human morphogenesis as a closed system with living cells as the players
and biological, chemical, and physical
laws as the rules. He was convinced he
could explain how the form of the
whole body arises and how tissues differentiate by mechanical forces.

Tissue Differentiation as a
Consequence of Unequal
Growth
Numerous varying conditions of tissue
differentiation have been described
(Blechschmidt, 1960). Three examples
will be given to illustrate the special
features of the regions in which cartilage, bone, and muscles form, as
viewed by Blechschmidt (1948, 2004).

Cartilage Formation
Cartilaginous formation of early skeletal components is a good example of
outsideinside differentiation: The
outer form inuences what happens

inside. Deeper-seated mesenchymal

tissue undergoes a loss of water from
the intercellular substance (Blechschmidt, 1948). This loss is because, in
that early stage, the outer cells have
better access to nutrients, grow better,
and put pressure on the central cells.
The mesenchymal cells, thus, are condensed. Cells in these so-called densation elds can be identied as precartilage cells. These condensed cells,
however, impede themselves more
and more in terms of cellular uptake
and release of molecular metabolites
the larger the densation eld becomes.
As a consequence, these cells develop
a high osmotic pressure, because their
intracellular catabolites have a high
molecular weight. As water now starts
to ow into these cells from their surroundings, they differentiate into
globular chondrocytes. Because these
cells are lined up in the center of a
mesenchymal bulge, they assume a
longitudinal arrangement. This structure also extends in a longitudinal direction, termed Stemmkorperfunktion in German, which can be
translated as piston-like function, as
suggested by Freeman (Blechschmidt,
2004). This nding is a general process that takes place during early
skeletal development of the arms,
legs, ribs, and spinal column. Meckels
cartilage in the rst visceral arch also
arises in this way. Further growth
stretches not only the cartilaginous
formation itself but also the adjacent
tissue by exerting forces as it pushes
on either end (pressure). This push
gives rise to a shearing force.

Muscle Formation
Possibly the most striking example of
embryonic development as developmental movements of tissues against
each other is the orientation of muscle
bers. It is apparent that all the arm
and leg muscles run in a longitudinal
direction, reecting the main direction of the skeletal components underneath. There is one muscle in the
arm, however, the quadrate pronator
muscle, which runs transversely,
stretched out between the ulna and
radius. It should be obvious that the
orientation of the muscles reects the
direction of growth of the underlying
skeletal elements, in this case, the
moving apart of the ulna and radius.
If we go through the whole body, we
always nd a correlation between the
muscle ber direction and the developmental movements of the underlying skeletal components. For example,
the autochthonous muscles of the vertebrae reect the vertical, transverse,

Bone Formation (Desmal

Ossication)
Desmal ossication is observed when
an expanding cartilaginous core slides
against its surrounding tissue, thus
exerting a shearing force. This process
occurs during formation of the mandibular bone next to Meckels cartilage and the long bones of the arms
and legs, and it also applies to the
frontal and parietal bones, when expanding brain tissue slides against
the overlying skin. Blechschmidt
(2004) called these regions where expanding tissues slide against each
other detraction elds.

Fig. 3. Diagram of the head and neck region.

The orientation of the sternocleidomastoid
muscle reects growth of the cervical spine.
The increasing angle of the mandible (arrow)
during the descent of the viscera is concomitant with the formation of the masseter and the
medial pterygoid muscles. Taken from Blechschmidt, 1960, with permission of Karger,
Basel.

1222 RADLANSKI AND RENZ

Fig. 4. A,B: The muscles of mastication (B)

take on the same orientation as the growth
vectors of the skull (A). The image in B (without
arrows) is taken from (Sobotta, 1993) with permission of Urban and Schwarzenberg, Munich.

and oblique expansion of each vertebra. The trapezius muscle shows the
expansion of the shoulder area in a
transverse direction as well as the
vertical expansion of the upper vertebral column (Fig. 2). Also, if we superimpose the direction of the masticatory muscles, we may as well draw the
growth vectors of the enlarging skull
(Figs. 3, 4A,B).
Blechschmidt (2004) drew the conclusion that muscles arise in regions
of dilation. Muscle bers arise wherever mesenchymal tissue is available
that can be dilated by adjacent expanding tissue (Carey, 1920a,b, 1921,
1936).

Folding, Molding, and Cell

Form as Consequences of
Unequal Growth
Developmental movements of tissues
lead to unequal growth. The consequent forces created lead to differentiation of tissues. Unequal growth was
held responsible for the creation of
form during embryogenesis (His,
1874; Carey, 1920b; Blechschmidt,
2004). Different growth rates of adjacent tissues are thought to cause folding and bulging.
As a consequence, there are no really at surfaces in the human body.
True cuboidal or columnar epithelial
cells are rarely found, except in textbook diagrams (Blechschmidt, 2004).
When an epithelium expands in a
conned space, it can bulge upward or
downward and invaginate into the
mesenchyme. In doing so, the cells at
the margin of the curvature become
wedge-shaped. This epithelial tissue
is thus called a wedged epithelium
(Blechschmidt, 1948, 1955, 1960,

2004). It is obvious that the cells exert

mutual pressure in a lateral direction,
which leads to bulging. Typical wedge
epithelia are dental lamina, the buds
of the dental primordia (Fig. 5AC), or
the acini of glands. In other regions,
e.g., the colon or esophagus, unequal
growth leads to stretching of the outer
cell layers (Carey, 1920b).

CRANIOFACIAL GROWTH
In craniofacial development, a typical
example of active morphogenetic
forces is exure of the head during
early embryonic development. Rapid
growth of neuronal tissue and the expanding anlage of the brain in the cranial region cause the early head to
bend forward. The tissue in the facial
region must now grow in a conned
space between the anlagen of the forebrain and the heart (Fig. 6A,B). Thus,
the early human face is characterized
by a series of bulges that run predominantly in an horizontal direction. We
identify the maxillary bulge, the rst
visceral arch, which will give rise to
the mandible, and further below, we
see the subsequent visceral arches.
According to the embryological
rules of Blechschmidt (2004), we
should nd a mesenchymal, and later
on, a cartilaginous condensation in
the center of these arches. The nasal
capsule arises in the maxillary arch,
and cartilaginous centers in the rst,
second, and subsequent visceral
arches give rise to Meckels cartilage
and the anlagen of the hyoid bone and
larynx. These cartilaginous brackets,
following their orientation in early
stages, reect the outer form and extension of the bulge of the visceral
arch. This nding corroborates Blech-

Fig. 5. A: Horizontal section through the region

of the dental lamina of a human embryo of 21-mm
crownrump length (CRL; Carnegie stage 20).
The numbers 13 indicate the epithelial condensations of the dental primordia at the bud stage.
Hematoxylineosin (HE) stained. VL, vestibular
lamina; OC, oral cavity; T, tongue. B: At the
bulges of the tooth buds, a wedge epithelium (W)
dominates, while the epithelium of the dental lamina in the regions between the tooth buds shows
a cuboidal (C) form. HE stained. C: A three-dimensional reconstruction of the mandibular region of the same embryo of 21-mm CRL (Carnegie stage 20). The vestibular lamina (Lv) and the
dental lamina (green) with the dental primordia in
their bud stages (i1, i2, c, m2) grow in a space
conned anteriorly by the lip and posteriorly by
Meckels cartilage (CM). Mand, mandible. Taken
from Radlanski, 1993, with permission of Quintessence Publishing Group. Scale bars 500 m in
A,C, 200 m in B.

GENES AND FORCES 1223

Fig. 6. A: Human face of an embryo of 16-mm crownrump length (CRL; Carnegie stage 19). The
face is characterized by bulges and furrows running predominantly in a horizontal direction.
B: Thus, they reect pressure exerted by the expanding brain (b) and the expanding heart (h), with
the face in between. Taken from Blechschmidt, 1960, with permission of Karger, Basel.

schmidts principle of outsideinside

differentiation (Blechschmidt, 2004).
When we compare earlier stages of
craniofacial morphogenesis (Fig. 7A)
with later stages (Fig. 7B), it becomes
obvious that the facial region is initially much smaller and that the expansion of the brain tissue dominates.
We may draw conclusions as to forces
compressing the facial region while
observing growth proportions at different stages. Densated mesenchymal
tissue in the center of the bulges gives
rise to precartilaginous cell condensations. Cartilage has the ability to expand rapidly, while maintaining the
basic form. In this stage of development, the cartilaginous structures
take control, and while expanding,
they counteract the compression force
that originally formed them.
It is undisputed that the mesenchymal cells in the center of the visceral
arches live in different environmental
conditions than those close to the epithelial surface. Light microscopy
shows increased tissue density in the
center of the bulges, but a pressure
increase in the center would be difcult to measure. However, the facial
region straightening and enlarging
relative to the brain region presumes
pressure factors are operating.

Formation of the Mandible

When Meckels cartilage develops into
mature expanding cartilage, it moves

along its longitudinal axis, increasing

rapidly in anteroposterior length (Radlanski et al., 1994). A triple curvature of Meckels cartilage occurs,
which gives it a swinging outline
(Kjaer et al., 1999; Radlanski et al.,
2003). This wavy outline of Meckels
cartilage can be interpreted as a reaction to further elongation against a
limiting outer skin. At its anterior
end, the mesenchymal tissue clearly
appears compressed and the cells are
longitudinally arranged with the appearance of being oated away (Fig.
8). Taken together, these ndings suggest a force originating from the longitudinal expansion. Although there
are many morphological indications of
forces molding cells and tissues, we
would only obtain satisfactory evidence if they could be measured in
vivo.
The rst bone formations of the
mandible occur at the buccal sides of
Meckels cartilage (Fig. 9A). Our 3D
reconstructions of the early development of the mandible (Radlanski et
al., 2003) show that this region may
well be characterized by a sliding action of the elongating Meckels cartilage against the skin with the mesenchymal tissue in between being
sheared (Fig. 9BD). According to
Blechschmidt (2004), mesenchymal
tissues form bone in these regions of
detraction. Anterior and posterior expansion of the mandible along Meck-

els cartilage proceeds as long as

shearing forces continue to be exerted
there. Also, the encroachment of bony
tissue around Meckels cartilage with
its partial embrasure is concomitant
with detraction of the mesenchyme
caused by expansion of the cartilaginous frame.
During these developmental stages,
the mandible alters its shape. It elongates anteroposteriorly, while expanding transversally. The gonial angle and the ramus of the mandible
form, when the whole embryo
straightens. Muscle tissue is formed
from mesenchymal tissue that is not
detracted but dilated under the direct
mechanical inuence of the expanding
mandible (Carey, 1920b; Blechschmidt, 1948, 2004). The arrangement of muscles of the oor of the
mouth during early prenatal stages
(Fig. 10A,B) exactly reects these
changes in size and form (Radlanski et
al., 2001). Thus, bers of the mylohyoid muscle extend in a transversal
direction as the mandible becomes
wider; the geniohyoid and digastric
(anterior belly) muscles reect the forward thrust of the mandible, and the
genioglossus muscle bundles reect
the vertical stretch with the increase
of the tongue. Blechschmidt (1948,
2004) was convinced that the epithelium is one of the driving forces that
create form and that the underlying
mesenchyme is under the inuence of
the developing movements (and
forces) of the epithelium. The increased size and the formation of the
tongue, rising from the oor of the oral
cavity as a protrusion, thus, is dependent on the molding function of the
bulging epithelium.

Formation of the Oral

Cavity
An embryo with a CRL of 14 mm
(early sixth week, Carnegie stage 18)
has an oronasal cavity with a gentle
in- and out-contour of the epithelium
lining the oronasal capsule (Fig. 11A).
The epithelium grows faster than the
oronasal cavity itself, and the spatial
impediment causes elevations and invaginations to increase and become
more accentuated (Fig. 11B). As soon
as bulging alleviates the spatial impediment, the epithelial bulges expand into the oronasal cavity or the

1224 RADLANSKI AND RENZ

underlying mesenchymal tissue. The

central elevation is the tongue, which
expands in vertical, transverse, and
anteroposterior directions. The mesenchyme is dilated, thus giving rise to
the formation of the intralingual muscles in corresponding directions. On
either side of the tongue, the epithelium grows inward, leading to formation of the sublingual and the submandibular glands. Further laterally,
another invagination of the oral epithelium leads to formation of the dental and vestibular laminae. At the
buccal corner, another epithelial duct
turns inward for the parotid gland.

In the maxillary region, another

band of invagination forms the upper
dental and vestibular lamina. Palatal
processes arise through further expansion of the epithelium that turns
toward the oronasal cavity. In addition, a perpendicular epithelial fold at
the top of the oronasal cavity gives
rise to the nasal septum. Again, the
epithelium grows faster than the cavity, so it bulges again and forms the
prominences of the nasal conchae.
Also, the lowest prominences, the palatal processes, become larger. As soon
as the embryo stretches, when vertical
growth increases in the neck (medi-

Fig. 7. Outlines of the head of a human embryo of 21-mm crownrump length (CRL; Carnegie
stage 20) and a fetus of 40-mm CRL. Blue, cartilaginous skeletal components of the craniofacial
region. Brown, bone. Yellow, brain. Black, contour of the head. A: The development of the
expanding brain (arrow) leads to a spatial impediment of the tissue within the visceral arches, in
whose centers the cartilaginous skeleton forms. B: Later, facial height, length, and width are
increased by rapid expansion of the cartilaginous structures, predominantly Meckels cartilage and
the nasal capsule (arrows). Thus, the cartilaginous parts of the facial skeleton reverse the action that
led to their formation.

ated by expansion of the cervical vertebral column), the infrahyoidal muscles arise as well as the gonial angle,
and the tongue is pulled away from
the oronasal cavity. Further growth
and the elasticity of the palatal processes (Ferguson, 1981) allow them to

Fig. 8. Vertical, parasagittal section through

the mandibular region of a human embryo,
37-mm crownrump length (CRL). The enlarged
cells of the expanding Meckels cartilage (MC)
cause the surrounding mesenchymal cells to
oat around its anterior end. i1, dental primordium of the deciduous central incisor; VL, vestibular lamina; M, early formation of mandibular
bone. Scale bar 100 m.

Fig. 9. A: The computer-aided three-dimensional reconstruction shows a human embryo of 21-mm crownrump length (CRL; Carnegie stage 20) with
the components of the craniofacial region made visible through the transparent skin. BD: Meckels cartilage, mandibular bone, and the alveolar
inferior nerve in a human embryo of 21 mm CRL (B), 25-mm CRL (Carnegie stage 22; C), and 66 mm CRL (D), seen from an anterior, lateral, 45 degree
oblique view. The image in A is taken from Radlanski, (2003), with permission of Blackwell Munksgaard, Copenhagen, and those in BD are taken from
Radlanski et al., (2003), with permission of Springer Verlag, Berlin. Scale bar 1,000 m.

GENES AND FORCES 1225

Fig. 10. A,B: Mandibular region with the muscles of the oor of the mouth in a human embryo of 25-mm crownrump length (CRL; A), cranial view,
and a human fetus of 68 mm CRL (B), viewed from a posterior direction. The orientation of the muscle bers matches the growth pattern of the
mandible, which is in a sagittal, transverse, and vertical direction. Taken from Radlanski et al., (2001), with permission of Gustav-Fischer-Verlag, Jena.
Scale bar 500 m.

swing up and fuse in the center of the

palate (Fig. 11C).

Location of Maxillary Bone

Formation

Fig. 11. AC: Histological frontal sections through the oral cavity of human embryos of 14-mm
crownrump length (CRL; Carnegie stage 18; A) and 26-mm CRL (Carnegie stage 22; B), and a
fetus of 68-mm CRL (C). In B, the lines lead to the primordium of the nasal septum (a), to Meckels
cartilage (b), to the mandibular bone (c), to the masseter muscle (d), to the mylohyoid muscle (e),
and to the fusion of the mylohyoid muscles (f). A and B are taken from Blechschmidt, (1960), with
permission of Karger, Basel, and C is from the Radlanski collection. Scale bar 1 mm.

There are also indications that Blechschmidts concepts of developmental

mechanics may apply in the maxillary
region, although he himself did not
describe them. In the center of the
(epithelial) nasal septum, the mesenchymal tissue is likely to be condensed
in such a way that a condensation
eld gives rise to a cartilaginous septum that expands according to cartilage behavior in a vertical and anteroposterior direction. This process
denitely contributes toward enlarging the midfacial region, and it also
shears the mesenchymal tissue next

Fig. 12. A: Maxillary bone formation (ochre) in a human fetus of 54-mm crownrump length (CRL), seen from an oblique 45 degrees posterior, cranial,
and lateral view. P, palatal bone formation. Computer-aided three-dimensional reconstruction. The outlines of the eyes (e) and the nasal capsule (n)
are superimposed to show the spatial relationships. B: Diagram of the craniofacial components in a human fetus of 42-mm CRL, frontal view. Red
arrows show the direction of growth and expansion of the tissues (eyes, gray; nasal capsule and Meckels cartilage, blue; oral cavity, pink) next to bone.
They thus can exert shearing forces (red dotted arrows), which may cause the mesenchyme to react by bone formation (brown). Scale bars 500 m
in A, 1 mm in B.

1226 RADLANSKI AND RENZ

to it and under the septal epithelium.

This determines the bone-forming region. Further condensation centers
developing in the region of the future
nasal conchae contribute toward the
formation of the nasal capsule. This in
turn adds to expansion of the midface
in corresponding directions. Furthermore, the region of initial maxillary
bone formation can be characterized
as detraction elds of the expanding
nasal capsule, eye bulbs, and oral epithelium shearing the remaining mesenchyme. The early maxilla, as shown
by 3D reconstructions (Radlanski et
al., 2000), has the form of a small pyramid, wedged exactly into the remaining space just described (Fig. 12A,B).
The adjacent mesenchyme shows
signs of owing motion (possible
shearing forces) parallel to the expanding organs mentioned.

DISCUSSION
Pure Mechanics?
At rst glance, it is difcult to give
credence to the very unusual mechanical interdependencies between cell
growth, spatial impediment and subsequent folds and bulges (His, 1874),
and the concept of tissue differentiation in elds of specic mechanical
stress (Carey, 1920a,b, 1921; Blechschmidt, 2004). It seems too mechanistic and too simplistic. Where are
the intrinsic biological capacities of
the cells, and how does this concept t
into our contemporary knowledge of
molecular signaling?
The changes of form and differentiation of tissues can be seen as biological responses of the cells, not as pure
mechanics. The key idea, however, is
that the cells do not follow an intrinsic
program of differentiation during
morphogenesis but are able to react to
the different situations into which
they have been maneuvered by their
own growth and that of their neighboring cells (Carey, 1920a,b, 1921;
Blechschmidt, 2004). The genetic code
provides the basis for condensed mesenchymal cells to react by forming cartilage. Other cells subjected to shearing forces react by forming bone, and
still others respond to being stretched
by forming muscle bers.

Cartilage Formation
We know that many molecular signals
or transcription factors are indispensable during formation of cartilage.
Sox9 is a downstream mediator for
chondrogenic commitment, as shown
in zebrash (Yan et al., 2002) and
chickens (Healy et al., 1996), and it
can be counteracted by Msx2 (Takahashi et al., 2001), as shown in mice.
Dlx5 also seems to be necessary, because Dlx5 knockout mice have hypoplastic nasal capsules (Depew et al.,
1999).
There may be many more signaling
molecules expressed during differentiation of cartilage tissue, and further
studies may elucidate their indispensability. These molecules should be
seen as mediators of cartilage formation, whereas the creation of cartilage
form under the inuence or at least in
the presence of these molecules has
not been addressed by signaling studies. Is there anything that contradicts
the concept of mechanics and development? It is maintained that mesenchymal cells in specic locations (i.e.,
where mesenchyme is being condensed by growth of adjacent tissue)
will form cartilage, and the form of the
cartilage is determined by the spatial
arrangement of the neighboring tissue. In terms of differentiation, cartilage is formed in response to pressure
changes within the tissue (Copray et
al., 1986; Francis-West et al., 2003).
The differentiation of cartilage in
these locations can be identied by the
molecular signals just discussed. How
and where does it start? Is Sox9 expression typical for mesenchymal cells
being compressed in the center of a
visceral arch or in any other places
where cartilage is being formed? It
would be benecial if we could measure the forces in play at precisely the
time when SOX9 is expressed.

Bone Formation
A major regulator found during bone
formation is Runx2 (Mundlos et al.,
1997; Kundu et al., 2002; Komori,
2003; Lian and Stein, 2003). Bone formation is a very complex process with
many more signals active during matrix formation (Sodek et al., 2002; Holliday et al., 2003) and mineralization
(Linkhart et al., 1996; Yamashiro et
al., 2004). Again, our knowledge of

molecular cascades per se does not

give us any information about the control of bone form. However, we may
specify our questions with the aim of
bridging the gap between molecular
biology and morphogenesis: How do
mesenchymal cells that start to form
bone differ from those that do not form
bone?
They differ in many points, one being location. Thus we can ask whether
location may have an inuence on
these mesenchymal cells. The start
signal to form bone in these specic
regions may be conveyed as a watersoluble protein. On the other hand,
the start signal may also be transmitted by the physical conditions; for
bone, these conditions would be the
shearing forces exerted on the mesenchyme by the developmental movements of adjacent tissues. There is evidence that mesenchymal cells about
to be transformed into osteoblasts are
arranged in such a way as to oat in
the mesenchyme under the inuence
of a shearing action (our own histological ndings and Blechschmidt, 1948,
1960). The mesenchymal cells in these
regions may react to this specic condition by expressing Runx2, for example, to start the cascade that leads to
ossication. In the absence of one or
more molecular signaling factors in
knockout mice, the cells subject to the
forces that trigger bone formation cannot do their bone-forming job.
Other putative factors, e.g., the
mandatory presence of nerves in the
region of early bone formation (Kjaer,
1998), can also be explained as a detraction between the more or less taut
nerves leashing and shearing the mesenchymal tissue, which responds with
the bone-forming cascade at that specic spot.
Of interest are not only the starting
points of ossication of a bone but also
the points where the form of the bone
is initially created. When examining
the formation of the mandible in morphological detail, we see that bone extends along Meckels cartilage in an
anterior and posterior direction; it
spreads underneath Meckels cartilage
and ows over Meckels cartilage in an
oral direction, thus forming a gutter
between an inner and an outer bony
partition (Radlanski and Klarkowski,
2001; Radlanski et al., 2003). This
nding may be explained by ongoing

GENES AND FORCES 1227

shearing forces, which can be envisioned when the different developmental stages of the mandible are
compared in 3D at the same scale. It is
difcult, however, to explain the early
formation of the coronoid and condylar processes. The condylar process as
an Anlagerungsgelenk (There is no
English term; it may be translated as
approaching joint) will remain more
enigmatic (Youdelis, 1966; Burdi,
1992; Naidoo, 1993; Radlanski et al.,
1999a), whereas the coronoid process
may result from muscle insertion and
traction, as in later developmental
stages (Spyropoulos, 1977; Uchida et
al., 1994).

Muscle Formation
Whereas the molecular interactions
leading to muscle formation in the
limbs have been unraveled in greater
detail, there are very few data on the
craniofacial muscles (Francis-West et
al., 2003). Among many others, Myf5
has been recognized as a major myogenic determination factor controlled
by distinct promoters in the head and
trunk (Noden et al., 1999; Hadchouel
et al., 2000; Summerbell et al., 2000).
There also seems to be evidence that
cranial neural crest cells migrating to
sites where they will form the corresponding muscles are preprogrammed
(Noden, 1983, 1988, 1991; Vaglia and
Hall, 1999; Francis-West et al., 2003).
There are transplantation experiments that corroborate this concept,
while others lead to an unclear judgment (Wahl and Noden, 2001), questioning whether cranial neural crest
cells migrate actively at all.
The literature does not indicate to
what extent the direction and arrangement of muscle bellies might be
regulated by signals (Robson, 1993).
The cause of the developmental movement of the muscles is unknown, e.g.,
extension of the rectus lateralis muscle of the eye (Francis-West et al.,
2003). The review suggests that the
direction of that muscle might be a
result of cranial exing (Wahl et al.,
1994). In human development, the direction and extension of the muscles
responsible for eye motility have been
seen as a consequence of the expansion of the growing eyeball (Blechschmidt, 1960), and it has been reported more recently that stretch of

mesenchymal cells leads to formation

of muscle bers (Goldspink, 1999). It
is not clear, however, why the muscle
bellies concentrate as four bundles adjacent to the eye.
There is not much overlap between
our knowledge of the molecular signals associated with differentiation of
muscle tissue and the control of orientation of the muscle bers and muscle
bellies. The arrangement of the muscles of the oor of the mouth recently
described for human prenatal development (Radlanski et al., 2001) correlated well with the concept of dilated
mesenchymal tissue. Our measurements of the growing mandible closely
matched the orientation of muscle bers in any direction: anteroposterior,
transverse, or vertical (Radlanski et
al., 2003).

PERSPECTIVES
There has been signicant progress in
understanding the signaling network
controlling the patterning and development of the face (Francis-West et
al., 1998; Mina, 2001). It is believed
that specic signaling molecules dispersed in the mesenchymal regions
command the cells to differentiate
(Mina et al., 1991). Most recent results describing human gene expression changes that occur during early
stages of development with particular
emphasis on craniofacial development
can be found at http://hg.wustl.edu/
COGENE. It is now obvious that
studies elucidating the molecular signaling cascades during tissue differentiation do not explain the morphological aspect involved in creating the
form of the organs. It seems as though
the molecules are mediators rather
than creators of form.
Knockout experiments demonstrate
the essential requirement of the signaling molecules (Young et al., 2000;
Gimara et al., 2004), and rescue experiments show that it is possible to restore
the signaling cascade (Johnston and
Nusslein-Volhard, 1992; Pispa et al.,
1999; Bei et al., 2000). This nding
does not affect the credibility of the
concept that cells react to irritation by
mechanical forces (Banes et al., 1995;
Carter et al., 1998; Goldspink, 1999;
Murray, 2000; Szabo et al., 2001; de la
Fuente and Helms, 2005). The biochemical signal cascade, which is part

of the differentiation process, can be

initiated by mechanical forces.
We do not know how genes are
switched on and off in nature. We assume that forces generated by growth
play a role. However, not much detailed information is available on the
changes in form during morphogenesis. Future studies should elucidate the
interdependency between mechanical
stimuli and molecular signals and
how they lead to differentiation. They
should identify mechanosensors that
may be responsible for this signal
transduction and develop novel methods for measuring such forces within a
tissue (de la Fuente and Helms, 2005).
For this purpose, it is also necessary
to create 3D reconstructions showing
the growth, the proportional changes,
the developmental movements, the
spatial arrangement, and the surroundings of the evolving craniofacial
complex, preferably for human development (Radlanski et al., 1999b; Radlanski, 2003). We think that an increased number of studies will be
devoted to comparative embryology.
An interspecies comparison can be
made in studies on craniofacial development in terms of form, 3D arrangement, mechanical interactions, and
signaling cascades. This knowledge
will enable us to gain a greater insight
into the way in which similar signaling cascades create different forms.
This approach may help to bridge the
gap and correlate the morphological
changes with the molecular signaling.
When it becomes possible to independently extract signal cascades
from the force systems to which they
are linked in nature, molecular medicine may become practicable in the
eld of craniofacial development, particularly in preventing malformation
syndromes of the head and neck at
early developmental stages.

EXPERIMENTAL
PROCEDURES
The human histological material was
obtained from legal and spontaneous
abortions according to German law.
The material was dehydrated, embedded in parafn, sectioned at 10 m,
stained with hematoxylin eosin, and
reconstructed in 3D using the software AnalySIS (SIS, Munster, Germany).

1228 RADLANSKI AND RENZ

ACKNOWLEDGMENTS
We thank Mrs. B. Danielowski and
Mrs. I. Schwarz for their skillful technical assistance in processing the embryos and the 3D reconstructions.
This work has been partially funded
by the Deutsche Forschungsgemeinschaft (Ra 428/1-3), and the studies
have been conducted within the frame
of the COST actions B8 and B23.

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