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Transition of neural crest cells to
mesenchymal cells
during craniofacial development
Kim, Nam Ho
Department of Medical Science
The Graduate School, Yonsei University
Transition of neural crest cells to
mesenchymal cells
during craniofacial development
Directed by Professor Lee, Sang-Hwy
The Master’s Thesis
Submitted to the Department of Medical Science,
the Graduate School of Yonsei University in partial
fulfillment of the requirements for the degree of
Master of Medical Science
Kim, Nam ho
December 2005
Acknowledgements
짧지도 길지도 않았던 2 년이라는 시간 동안 부족한 저의 논문이
나오기까지 도움을 주셨던 많은 분들에 대한 감사의 마음을 이 지면을
통해서 전해드리고자 합니다. 우선 부족한 저를 항상 이끌어주시고,
밤낮으로 같이 고민해 주시고 지도해주시느라 고생하신 이 상휘 교수님께
진심으로 감사드립니다. 연구에 대한 열정과 자세, 그것 이상의 많은
것들에 대한 교수님의 가르침은 제 인생의 큰 이정표가 될 것입니다.
또한, 많은 조언과 도움을 주신 육 종인 교수님과 심사를 위해
수고해주신 김 용욱 교수님께 감사드립니다. 그리고, 저의 대학원 생활에
큰 도움을 주신 이 승일 교수님, 서 정택 교수님, 신 동민 교수님, 정 한성
교수님께도 감사의 마음을 전하고자 합니다. 또한, 경상대학교 김 충원
교수님과 생화학 교실 식구들에게도 감사드립니다.
돌이켜보면 실험실 식구들과 함께 보냈던 많은 시간들이 가장 기억에
남습니다. 큰 형님처럼 저희를 묵묵히 지켜주셨던 박정국 선생님,
실험실의 맏언니인 수현 누나, 친형 같은 태진형, 살림꾼 혜진이, 철없는
은주는 제가 어딜 가더라도 잊지 못할 고마운 사람들입니다. 그리고 항상
제 일을 자기 일처럼 생각하고 도와주었던 재영이형과 친누나 같은 안
정미샘과 정샘, 민석이형과 해형, 정말 고맙습니다. 서로 다른 층에 살지만
항상 식구 같은 조직학 교실 사람들과 약리, 생리학 교실 사람들에게도
고마운 마음뿐입니다. 제 논문을 위해 많은 도움을 주신 병리학 교실의
류주경샘과 차충민 샘, 멀리 영국에서 열심히 살고 있을 두식, 주연과
후배 은주, 쭌이형에게도 감사의 마음을 전하고 싶습니다.
대학원 생활을 하면서 부모님께는 늘 죄송한 마음 뿐이였습니다. 항상
부족한 저를 안아주시고, 제가 힘들때마다 큰 힘이 되어주신 부모님의
사랑은 저에게는 무엇보다 가장 큰 힘이 되었습니다. 그리고 하나뿐인 제
동생에게도 정말 고맙고 미안한 마음이 앞서게 됩니다. 앞으로는
가족들에게 의지가 되고 힘이 되는 사람이 되고 싶습니다.
마지막으로, 어딘가에서 저를 기다리고 있을 그녀에게 고마움과 사랑을
전하고 싶습니다. 모든 이들이 행복하길 바랍니다. 고맙습니다.
i
Table of contents
Abstract ·········································································································· 1
I. INTRODUCTION ······················································································ 3
II. MATERIALS AND METHODS ······························································ 7
1. Materials ·································································································· 7
2. Methods ··································································································· 7
A. Media ··································································································· 7
B. Micromass cultures ·············································································· 8
C. Peanut agglutinin staining ···································································· 8
D. Alcian blue staining ············································································· 9
E. Alizarin red S staining ·········································································· 9
F. Alkaline phosphatase reaction ······························································ 9
G. Immunohistochemistry ······································································ 10
H. Immunocytochemistry ······································································· 10
I. Western blot analysis ·········································································· 11
III. RESULTS ······························································································ 12
1. The cells from H-H stage 15 showed the different morphology with
condensation pattern from those from H-H stage 24. ··························· 12
ii
2. The condensation of cultured cells was occurred at H-H stage 24 more
than at H-H stage 15. ············································································· 17
3. The cultured cells from H-H stage 24 had more capability for cartilage
and/or bone formation than cells from H-H stage 15. ··························· 17
4. Anti-HNK-1-positive cells were decreased in maxilla and mandible with
the increased developmental stages. ····················································· 20
5. The amount of anti-HNK-1 expression was inversely proportional to
that of anti-vimentin as the developments went on. ······························ 22
6. Wnt-3a had an effect on the expression pattern of HNK-1 and E-
cadherin in the cultured cells form H-H stage 15. ································ 25
IV. DISCUSSION ······················································································· 29
V. CONCLUSION ······················································································ 36
REFERENCES ···························································································· 37
Abstract (in Korean) ···················································································· 44
iii
List of figures
Figure 1. Morphological changes of primary cultured cells from H-H stage
15 with elapse of time. ································································ 13
Figure 2. Phase contrast images of primary cultured mandibular and
presumptive maxillary cells after 3 days of culture. ··················· 14
Figure 3. Histologic images of mandibular and maxillary region with H & E
staining. ······················································································· 15
Figure 4. Cell condensation pattern examined with peanut agglutinin (PNA)
staining. ······················································································· 18
Figure 5. Bone and cartilage stainings of micromass cultured mandibular and
maxillary cells after 3days. ························································· 19
Figure 6. The expression pattern of NCC marker, anti-HNK-1 at the different
developmental stages. ································································· 21
Figure 7. Immunocytochemical analysis with anti-HNK-1 antibody after 3
days of culture. ············································································ 23
Figure 8. Immunocytochemical analysis with anti-vimentin antibody.
······································································································ 24
Figure 9. The effect of Wnt-3a on the cellular composition pattern and the
expression of NCC marker from H-H stage 15 for 2 days of
culture. ························································································· 26
iv
Figure 10. The effect of Wnt-3a on anti-E-cadherin expression in the cells
from H-H stage 15 after 2 days of culture. ·································· 27
- 1 -
Abstract
Transition of neural crest cells to mesenchymal
cells during craniofacial development
Kim, Nam Ho
Department of Medical Science
The Graduate School, Yonsei University
(Directed by Professor Lee, Sang-Hwy)
Neural crest cells (NCC), the embryonic precursors of craniofacial
mesenchyme, arrive at the presumptive facial region to form the facial
structures during early developmental stages. Although the roles of neural
crest-derived mesenchymal cells are well known for the formation of
craniofacial bone and cartilage, little information is available about their
transition into mesenchymal cells. So I wanted to find out the temporal
changes of NCC fate in relation to the osetochondrogenic characteristics with
- 2 -
their mesenchymal transition in the in vitro culture system as well as in vivo
model. I also searched for the control mechanism for the maintenance or
differentiation of the NCC fate.
In order to answer these questions, I tried a functional assay of mandibular
and maxillary cells in different stages of chick embryos by micromass culture.
And I examined the NCC-related protein expression profiles, the cell growth
pattern, the osteochondrogenic capacities, and the effect of Wnt for the
maintenance, proliferation, and differentiation of neural crest precursor cells.
The results demonstrated the NCCs in postmigratory developmental stage
of facial region lose their fate as the cells are condensed. Especially in
mandibular cells, as development proceeds, they are mainly confined to the
cellular nodules which will later form the cartilages. And their capabilities to
form bone and/or cartilage are increased as they acquire mesenchymal cell
natures and lose the NCC characteristics. Moreover, Wnt-3a maintains the
characteristics of NCC and suppresses the differentiation of neural crest cells
into mesenchymal cells.
______________________________________________________________
Key words: neural crest cell, mesenchymal cells, craniofacial, Wnt-3a,
development
- 3 -
Transition of neural crest cells to mesenchymal
cells during craniofacial development
Kim, Nam Ho
Department of Medical Science
The Graduate School, Yonsei University
(Directed by Professor Lee, Sang-Hwy)
I. INTRODUCTION
During the development of the vertebrate embryo, neural crest cell (NCC)
is known to contribute to various structures, including the glial cells of
peripheral nervous system, the smooth muscle cells of vascular system, and
the most pigment cells1-3. In addition, NCCs in developing head are unique
pluripotent cells that play a critical role for formation of craniofacial
- 4 -
structures4, 5. In the avian embryo, cranial NCCs start migration around the
Hamburger-Hamilton6 (H-H) stage 9 at the embryonic day (ED 1) to end at H-
H stage 14 (ED 2)7, 8.
The cranial NCC migrates from dorsal neural tube to populate the specific
region of the face and head. And they differentiate into not only the neurons,
glial cells, and connective tissue, but also various mesenchymal components
called the ectomesencyme or mesenchyme9, 10. Later they contribute to the
craniofacial skeleton in the ventral part of the embryonic head11, 12.
Mesenchymal cells are generally derived from the mesoderm13, but
ectomesenchymal cells in craniofacial regions originate from neural crest of
ectodermal origin14. They are known to form the different kinds of structures
including the connective tissue15, cartilage, and bones16, while the mesoderm
contributes the jaw musculature and the cranial base17.
In order to differentiate from NCC to form those craniofacial structures,
many different evolving process is required and the initial step will be the
mesenchymal transition18-20. The transition is followed by the mesenchymal
condensation and differentiation that the mesenchymal transition can be a
prerequisite step for the development of craniofacial structures. Although the
roles of ectomesenchymal cells for the craniofacial chondrogensis and
- 5 -
osteogenesis have been reported in detail21, 22, little information is available
about the mesenchymal transition at their early developments.
Previous studies23 have shown that the chondrogenic as well as osteogenic
capacity is present in mandibular ectomesenchyme of first pharyngeal arch
region in chick embryo. And the mandibular cells acquire their
chondrogenicity only after H-H stage 24 when they reach the stages of
mesenchymal condensation24. In addition their chondrogenicity is unique as
compared with that of other regions25-27, for example, the limb because the the
ectomesenchyme of the mandibular region is composed of two different cell
population, NCC and mesodermal28 and the total area of cultured mandibular
cells occupied by cartilage is increased as development proceeds, while the
amount of cartilage made in cultured cells from limb is independent of stage
of development24.
The members of Wnts are a family of secreted glycoproteins that can work
as an important NCC inducer29. They have been assorted with two groups on
the basis of their activity – canonical and noncanonical30. Wnt-1 and Wnt-3a
are canonical Wnt proteins and the mouse embryos in lack of both proteins
showed a reduced amount of NCC31. Overexpression of either Wnt-1 or Wnt-
3a in whole embryos leads to the increased number of NCCs32 due to their
roles in cell proliferation33. They indicate these genes are not only important
- 6 -
for the induction but also for the proliferation of neural crest precursors. So
Wnt-1 and Wnt-3a protein can influence the transition of NCCs into
mesenchymal cells by the same context to the NCC induction34.
In this study I wanted to observe the cellular features of postmigratory
NCCs from mandibular and presumptive maxillary region of first pharyngeal
arch. And I also wanted to find out the temporal changes of NCC fate with
their mesenchymal transition in relation to the osetochondrogenic
characteristics in the in vitro culture system as well as in vivo model. In
addition, I wanted to know the control mechanism for the maintenance or
differentiation of the NCC fate.
- 7 -
II. MATERIALS AND METHODS
1. Materials
Fertilized White Leghorn chicken eggs were obtained from Yangsung Co.,
Korea. Eggs were incubated at 38℃ and staged according to Hamburger-
Hamilton6.
2. Methods
A. Media
The serum-containing medium was consisted of 1: 1 ratio of F12: DMEM
(Gibco) with 10 % Fetal Calf Serum (FCS, Gibco). And 2mM L-glutamine,
100 units/ml penicillin, 100 ㎍/ml streptomycin, 0.25 ㎍/ml fungizone
(Gibco, Grand Island, NY, USA), 5 ㎍/ml transferrin (bovine, Sigma, St.
Louis, USA), 100 nM hydrocortisone (Sigma), 5 ㎍/ml porcine insulin
(Sigma) and 50 ㎍/ml ascorbate (Sigma) were added.
Wnt-3a medium was consisted of DMEM and 10% FBS, supplemented with
4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 0.4 mg/ml
G-418 (ATCC, Manassas, VA, USA), and was kindly supplied by the Prof.
Yook at the department of oral pathology, Yonsei university, Korea.
- 8 -
B. Micromass cultures
Chick embryos were isolated and dissected in Hank’s Balanced Salt Solution
(HBSS, Sigma) containing 10% fetal calf serum (Gibco). Each tissue
segments were obtained by dissecting out the mandibular and presumptive or
definitive maxillary region of H-H stage 15 (ED 2), stage 20 (ED 3), and stage
24 (ED 4). These segments were placed in 2% trypsin (Sigma) in HBSS for
25 minutes at 4℃ and the epithelia were removed in HBSS with 10% FCS.
Then the cell suspension was prepared by vigorous pipetting.
The 24 well-dish (Nunclon, Denmark) was coated 2 times with 10㎕ of
fibronectin (Calbiochem, La Jolla, CA, USA) with the concentration of 50
㎍/ml in serum-free media and incubated at 37℃ for 1–2 hours.
After cell counting, 10㎕ of a cell suspension containing 2×105 single cells
in micromass were placed in prepared culture dish and were allowed to adhere
for 1 hour before flooding with media to give a final volume of 500㎕. Cells
were maintained in a humidified atmosphere of 5% CO2 and 95% air and the
medium was replaced daily.
C. Peanut agglutinin staining
Micromass cultured cells were fixed with 4% paraformaldehyde for 20
minutes, rinsed with PBS, and incubated with 100 ㎍/ml horseradish
- 9 -
peroxidase conjugated peanut (Arachis hypogaea) agglutinin (PNA, Sigma)
for 30–60 minutes at room temperature. PNA binding was detected by using
the AEC colorimetric substrate (Zymed, CA, USA).
D. Alcian blue staining
The cultured cells were fixed as above and stained with 1% Alcian blue 8GX
(Sigma) in 0.1N HCl, pH 1.0 for 3 hours to detect cartilage-specific sulfated
proteoglycan matrix. Stained cultures were destained with 70% ethanol.
E. Alizarin red S staining
Micromass cultured cells were rinsed with phosphate buffered saline (PBS),
fixed with 4% paraformaldehyde for 10 min, and rinsed with sterilized water.
The cultures were then stained with 40 mM Alizarin red S (Sigma) for 10
minutes at room temperature and washed with sterilized water and PBS (pH
7.2).
F. Alkaline phosphatase reaction
After PBS washing, cells were fixed as described. Alkaline phosphatase
staining solution containing 100 mM NaCl, 100 mM Tris-HCl pH 9.5, 5 mM
MgCl2, 1 mg/ml NBT (Promega, WI, USA), 0.1 mg/ml BCIP (Promega) was
- 10 -
added after washing and incubating at 37℃. The reaction was incubated for 5-
30 minutes at 37℃, stopped by addition of 10 mM EDTA and the cells were
washed with PBS.
G. Immunohistochemistry
Whole-mount immunostaining was carried out using anti-HNK-1 (Leinco
Technologies, St. Louis, USA) antibody. The embryos were fixed in 4%
paraformaldehyde overnight before dehydration in ethanol, followed by the
wax embedding as usual manner. 5㎛ thickness of wax sections were
mounted on silane-covered slides and allowed to dry overnight. Sections were
dewaxed by dehydration through an ethanol series before being quenched
with endogeneous peroxidase and blocked with 20% goat serum for 30
minutes. Sections were incubated overnight at room temperature in primary
antibody HNK-1 in PBS. Binding was visualized according to the
recommended procedure with VECTASTAIN ABC and DAB substrate
solution kit (Vector Laboratories).
H. Immunocytochemistry
Cultured cells were fixed, washed with PBS, and permeabilized with 0.1%
(v/v) Triton X-100. Normal goat serum was used for 30 minutes to block
- 11 -
unspecific reactions. Then, they were incubated with a monoclonal mouse
anti-HNK-1 antibody or a mouse anti-vimentin antibody. Binding was
confirmed by the same kit and method as in section G.
I. Western blot analysis
For immunoblot anaylsis, cells were collected, and then washed out with
PBS twice. Each sample was added with SDS sample buffer (60 mM Tris-
HCl pH 6.8, 4% SDS, 25% glycerol, 14.1 mM 2-mercaptoethanol, 0.1%
bromophenol blue) and run the electrophoresis for 2 hours in 8% SDS-
acrylamide gel. In semi-dry transfer (Bio-Rad, CA, USA), the gel was
transferred onto nitro-cellulose membrane for 30 minutes. The membrane was
blocked in 5% fat-free dry milk-PBST buffer (PBS, 0.1% Tween-20) for 2
hours and washed with PBST 3 times for 5 minutes. Mouse anti-E-cadherin
(1:1000, Zymed) in 3% fat-free dry milk-PBS buffer added transferred
nitrocellulose membrane was shaken for 2 hours and washed with PBST 3
times for 5 minutes. Membrane was incubated for 1 hour in PBS buffer with
anti-rabbit-horseradish peroxidase conjugated-secondary antibody (1:2000).
After washing with PBST buffer, proteins were detected by using enhanced
chemiluminescence (ECL) detection kit (Amersham Biosciences, Piscataway,
NJ, USA).
- 12 -
III. RESULTS
1. The cells from H-H stage 15 showed the different morphology with
condensation pattern from those from H-H stage 24.
Micromass culture was used as a means for verifying morphological
characteristics of the cells from mandibular and presumptive maxillary region
at H-H stage 15 because these cells in preliminary experiments were unable to
be attached to dishes sufficiently at lower densities.
Regardless of regions obtained for primary cultures, the cultured cells were
homogenously round in shape immediately after they were plated to the
dishes (Fig. 1A, 1D). After 12 hours, the shapes of them were changed that
could be classified into two types (Fig. 1B, 1E). One type of them, which
were attached the bottom of dishes, showed the spindle shapes and seemed to
start the condensation from 2 days of culture (Fig. 1C, 1F). Afterwards their
shapes became more stretched to make spindle-shapes with multibularity.
Another cell population, having semitranslucent and round shape, was
detected over underlying spindle shaped cells and their numbers were
decreased as the culture proceeded. When they were aggregated to each other,
these cells formed colonies and could maintain their original shapes up to 10
- 13 -
Figure 1. Morphological changes of primary cultured cells from H-H stage
15 with elapse of time. Cells from mandible (A–C) and presumptive maxilla
(D–F) were cultured. A, D: The cells are round in shape when they were first
plated. B, E: After 12 hours, cells attached to the dish plate, showing spindle
shape with some intermingled round cells. C, F: After 24 hours, the spindle
shaped cells became more stretched with multilobularity, while the round
cells were decreased in number. (scale bar: 100 ㎛).
- 14 -
Figure 2. Phase contrast images of primary cultured mandibular and
presumptive maxillary cells after 3 days of culture. Cultured cells from
mandible of H-H stage 15 (A) showed the spindle shaped multilobular cells,
which were similar to those from presumptive maxillary regions (D) of same
stage. Mandibular cells from H-H stage 20 (B) and 24 (C) were cultured in the
more condensed fashion than those of H-H stage 15. And they form the
several cellular nodules (indicated by white arrows) that were thought to
differentiate into cartilage nodules in the future. There was no cellular nodule
formation at the cells from the maxillary region of H-H stage 20 (E) and 24
(F) (scale bar: 100 ㎛).
- 15 -
Figure 3. Histologic images of mandibular and maxillary region with H & E
staining. Embryos were from H-H stage 15 (A), 20 (B), and 24 (C) and the
mandibular regions were shown at the inserts in higher magnification. A: In
H-H stage 15, the more condensed cells were located in the middle of
mandibular arch, while the maxillary process was not formed yet. B: The
maxilla began to be formed and the condensed cells in the middle of the
mandibular arch became more compact at H-H stage 20. C: In H-H stage 24,
the maxilla was almost completely formed and the cells were highly
condensed in the middle of the first pharyngeal arch (scale bar: 100㎛).
- 16 -
passages (data not shown).
The shape of the cultured cells from H-H stage 15 mandible (Fig. 2A) were
not different from those from presumptive maxilla of the same stage (Fig. 2D).
On sectional images from H-H stage 15 mandible, the core region of the first
branchial arch showed the more condensed pattern of cells than those
surrounding region or the presumptive maxilla (Fig. 3A).
The morphology or the condensation pattern of the cultured cells from H-H
stage 24 mandible or maxilla was clearly different from those from H-H stage
15. The cultured cells from H-H stage 24 also posed the round shape when
they were first plated. However, differences of the shape or condensation
pattern were evident after 12 hours, when they were compared with H-H stage
15. Most of the cells from the mandible were in spindle shapes and some of
them were aggregated to form cell clusters. After 48 hours the cells at the
clusters became more compact and round in shape to form the cellular nodules,
while other adjacent cells were persistently polygonal in shape and condensed
(Fig. 2B, 2C). Also, the similar pattern of more cell condensation was
observed on the sectioned images of the first branchial arch from H-H stage
20 and 24, as compared with those from H-H stage 15 (Fig. 3B, 3C).
The cultured cells form maxilla at H-H stage 20 and 24 also showed the
polygonal shapes and condensed pattern, but no cellular nodule was observed
- 17 -
(Fig. 2E, 2F). These results were similar on the sectioned images from same
stages of maxillary region (Fig. 3B, 3C).
2. The condensation of cultured cells was occurred at H-H stage 24 more
than at H-H stage 15.
The cultured cells from H-H stage 24 were in more condensed pattern than
those from H-H stage 15. In order to confirm this condensation, peanut
agglutinin (PNA) staining was performed. PNA staining was strong in
micromass cultured cells from H-H stage 24 (Fig. 4C, 4D), while those from
H-H stage 15 (Fig. 4A, 4B) were weakly stained with it. And this staining
tendency was the same regardless of region, mandible or presumptive maxilla.
In addition, the cells from mandible of H-H stage 24 (Fig. 4C) exhibited the
highest intensity of PNA, which may be the region of cellular nodules found
in Figure 2C.
3. The cultured cells from H-H stage 24 had more capability for cartilage
and/or bone formation than cells from H-H stage 15.
In order to investigate of capacity for chondrogenesis and/or osteogenesis,
diverse staining was performed on the micromass cultured cells. Alcian blue
- 18 -
Figure 4. Cell condensation pattern examined with peanut agglutinin (PNA)
staining. Images were from the micromass cultured cells of mandible (A, C),
and maxilla (B, D) after 3 days of incubation. Cells from H-H stage 24 (C, D)
showed the positive PNA staining as shown in red, but those from H-H stage
15 (A, B) did not. Especially the cells from mandible of H-H stage 24 (C)
presented the high PNA positive foci, which were in accord with the location
of cellular nodules found in figure 1C. (scale bar: 1 ㎜)
- 19 -
Figure 5. Bone and cartilage stainings of micromass cultured mandibular
and maxillary cells after 3days. Alcian blue (A–D), alizarin red (E–H) and
alkaline phosphatase (I–L) were used for the identification of cartilage and
bone formation. Cultured cells from H-H stage 24 (C, D, G, H, K, L) were
stained in alizarin red and alkaline phosphatase staining, while those of H-H
stage 15 (A, B, E, F, I, J) were not. And alcian blue staining was only positive
in mandibular cells at H-H stage 24 (C). (scale bar: 1 ㎜).
- 20 -
staining, to detect the cartilage matrix, failed to detect any positive
chondrogenesis from cells of H-H stage 15 (Fig. 5A, 5B). On the other hand,
the cells from mandibular region at H-H stage 24 were stained by alcian
blue (Fig. 5C), with a stronger reaction on the cell nodules, although the cells
from maxilla were not stained at all (Fig. 5D).
Alizarin red staining, for the evaluation of calcium-rich deposits, was
performed to investigate the osteogenic activity in cultured. In addition,
alkaline phosphatase staining, which is to identify of osteo-progenitor cells,
also showed the same results as the Alizarin red. The more positive-stained
cells from H-H stage 24 for alizarin red and alkaline phosphatase staining
were found than those from H-H stage 15.
4. Anti-HNK-1-positive cells were decreased in maxilla and mandible with
the increased developmental stages.
To trace the NCCs in vivo, immunohistochemical analysis was performed at
H-H stage 15 and H-H stage 24 with anti-HNK-1 antibody, which are known
to a major neural crest marker. Anti-HNK-1-positive cells were detected in
the vicinity of the eye (Fig. 6A) and in the periphery region of first branchial
arch (Fig. 6B) at H-H stage 15. The anti-HNK-1-positive cells located in first
- 21 -
Figure 6. The expression pattern of NCC marker, anti-HNK-1 at the
different developmental stages. A–B: Expression of anti-HNK-1 postive cells
(as brown colored dots) as the indicator of neural crest origin at H-H stage 15.
A, B: anti-HNK-1 positve NCCs were clearly visualized at the periorbital (A)
and first and second arch (B). C–E: Expression of anti-HNK-1 at H-H stage
24. C: At H-H stage 24, the HNK-1-postivie cells were decreased remarkably
in number. D: Higher magnification view of Fig. C showed that the maxillary
region had low numbers of anti-HNK-1 positive cells. E: NCCs at the H-H
stage 24 mandible were mainly located in the center of first pharyngeal arch,
where the cells were highly condensed (scale bar: 100㎛).
- 22 -
branchial arch were almost occupied under the epithelium and enveloped the
core of branchial arch (Fig. 6B). At H-H stage 24, less anti-HNK-1-
positive cells were observed than H-H stage 15 (Fig. 6C) and occupied the
some center of cell condensation of the first arch and in the part of maxillary
process posterior to the eye (Fig. 6D, E).
5. The amount of anti-HNK-1 expression was inversely proportional to that
of anti-vimentin as the developments went on.
To detect NCCs in the primary cultured in vitro cells, immunocytochemical
analysis was performed with anti-HNK-1 antibody. All of cells used in this
immuno-staining were cultured for 3 days. A number of the anti-HNK-1-
positive cells were detected at H-H stage 15, though it was difficult to find
the differences of expression patterns in mandibular (Fig. 7A) and maxillary
cells (Fig. 7D). In perspective, the anti-HNK-1-positive cells at H-H stage 15
tended to be located at the less condensed regions, regardless of their origins.
The cultured cells from H-H stage 20 and 24 showed the similar pattern of
differentiation. Cartilage nodules were observed from the mandible of H-H
stage 24 as well as of stage 20. At these two stages, anti-HNK-1-positive cells
were shown to be confined to the cartilage nodules (Fig. 7B, 7C). But in
- 23 -
Figure 7. Immunocytochemical analysis with anti-HNK-1 antibody after 3
days of culture. The mandibular (A) or presumptive maxillary cells (D) from
H-H stage 15 showed no difference of anti-HNK-1 expression. Later stages of
H-H stage 20 (B) and H-H stage 24 (C) from the mandible, anti-HNK-1-
positive cells were mainly confined to the cellular nodules. But only a few
anti-HNK-1-positive cells of maxillary region at H-H stage 20 (E) and H-H
stage 24 (F) were positively stained. (scale bar: 100㎛)
- 24 -
Figure 8. Immunocytochemical analysis with anti-vimentin antibody.
Expression of anti-vimentin showed general distribution pattern in both
mandibular (A–C) and maxillary region (D–F). Vimentin was expressed at
some of H-H stage 15 mandibular (A) and presumptive maxillary (D) cells of
spindle shape. Anti-vimentin-positive cells from mandible at H-H stage 20
(B) and stage 24 (C) were sparsely distributed in cellular nodules. And the
expression of anti-vimentin from maxilla of H-H stage 20 (E) and H-H stage
24 (F) showed even distribution without focal concentration. (scale bar:
100㎛).
- 25 -
maxillary cells, the anti-HNK-1-positive cells were rarely detected at H-H
stage 20 and at H-H stage 24 (Fig. 7E, 7F).
Anti-vimentin antibody was used as the mesenchymal marker in micromass
cultures. Among the cells from mandible (Fig. 8A) and maxilla (Fig. 8D) of
H-H stage 15, only some of the cells were positively stained. But it was
difficult to discern the factors that were involved in this difference.
The amount of anti-vimentin expressions from mandible at H-H stage 20
(Fig. 8B) and H-H stage 24 (Fig. 8C) was increased, which were different
from those of anti-HNK-1. So, the amount of anti-vimentin expression was
inversely proportional to that of anti-HNK-1 as the developments went on.
Vimentin was strongly expressed from mandible at these stages, while the
weak expression was observed at the cartilage nodule area. And there were
generally distributed vimentin-positive cells from H-H stage 20 (Fig. 8E) and
stage 24 (Fig. 8F) maxillary cells.
6. Wnt-3a had an effect on the expression pattern of HNK-1 and E-cadherin
in the cultured cells form H-H stage 15.
To investigate the effect of Wnt-3a on NCCs, Wnt-3a treated medium was
added to the micromass culture at H-H stage 15. Morphological changes
- 26 -
Figure 9. The effect of Wnt-3a on the cellular composition pattern and the
expression of NCC marker from H-H stage 15 for 2 days of culture. The
cultured cells from mandible (B) and presumptive maxilla (D) in Wnt-3a-
added medium showed increased number of round shaped cells than those in
control medium (A, C). Anti-HNK-1-positive cells from maxilla with Wnt-3a
(F) were also more frequently observed than control (E) (scale bar: 100㎛).
- 27 -
Figure 10. The effect of Wnt-3a on anti-E-cadherin expression in the cells
from H-H stage 15 after 2 days of culture. Expression of anti-E-cadherin was
not different remarkably in mandibular region between control (A) and Wnt-
3a-added medium (B). In presumptive maxillary regions, there were similar
results of Wnt-3a (D) relative to control (C). Immunoblot analysis of cultured
cells was performed with anti-E-cadherin antibody. From mandible, the
cultured cells in Wnt-3a-added medium displayed decreased levels of anti-E-
cadherin as compared with the control (A). And the maxillary cells showed
similar results of decreased anti-E-cadherin expression (B) (scale bar: 100㎛).
- 28 -
began to be observed after 1 day of Wnt-3a treatment. While the round and
semitranslucent cells were decreased with the elapse of time in control group
(Fig. 9A, 9C), most of cells in Wnt-3a treated medium sustained their round
shape up to 3 days (Fig. 9B, 9D).
The cell population growing under the round cells in Wnt-3a treated
medium was pertained the similar morphology, to those of control group a
multilobular spindle shape.
More expression of anti-HNK-1 in Wnt-3a treated cells were detected (Fig.
9F) as compared with control (Fig. 9E), though not quantitatively measured.
And this observation of Wnt-3a effects was more pronounced in presumptive
maxillary cells than those of mandible.
Although immunocytochemical images for anti-E-cadherin in the control
(Fig. 10A, 10C) and Wnt-3a (Fig. 10B, 10D) did not show the remarkable
differences, the immunoblotting results showed the decreased E-cadherin in
the Wnt-3a treated cells. These results were the same as in the cells of
mandible (Fig. 10E) and presumptive maxilla (Fig. 10F).
- 29 -
IV. DISCUSSION
It has been well known that cranial NCCs play a critical role to
skeletogenesis during the craniofacial development. As many studies about
cranial NCCs have been progressed1, 5, 11, the formation of NCC from neural
ectoderm by the epithelial-mesenchymal transition29, 34 has been well
documented with recent focus.
But there is little information about transformation of NCCs into
mesenchymal cells after their migration in the craniofacial regions. This
postmigratory transition is not only a important step for facial development
but also is imperative for skeletogenesis because only the population of
mesenchymal cells forms an aggregation or condensation35.
In this study, I tried to identify the postmigratory transition of NCCs into
mesenchymal cells in facial region and to characterize these cells as
developments go. In chick embryos H-H stage 15 is a proper stage for the
evaluation of this transition because the migration of cranial NCCs ends at the
pharyngeal arch regions at this stage8, 16, 36.
Micromass cultures have been used to assess the differentiation potential of
the mesenchymal cells25, 37, 38. So, micromass cultures were introduced to this
- 30 -
study to investigate cellular features of postmigratory NCCs from mandibular
and presumptive maxillary region of first pharyngeal arch.
The cells immediately after the single cell segregation in micromass culture
from H-H stage 15 and 24 showed round shape with bright and
semitranslucent cell body in about 10 ㎛ of diameter. They were
homogenous in cell shape or size. Soon after about 1-3 hours of the plating,
some of these cells are adhered to the base to make the changes of shapes.
They became to hold the spindle shapes, like the fibroblast, with
multilobularity, which are the characteristics of mesenchymal cells39.
The culture plates were coated with fibronectin, an extra-cellular matrix, for
the facilitation of the cellular adherence to the plate. And the reason for these
conformational changes into mesenchymal nature may have some relationship
with fibronectin. Without the precoating with fibronectin, the mandibular or
presumptive maxillary cells failed to adhere and to survive (data not shown).
As the culture progress, the spindle shaped cells expand to the periphery.
And the round cells still remains over those underlying multilobular spindle
shaped cells. They preserved their original shape and nature of
semitranslucency, while weakly attached to the underlying cells. They
sometimes form colonies like the embryonic stem cell colony and these
- 31 -
colonies sustain their undifferentiated state during 10 passages of culture
while the expanding into the multilobular spindle shaped cells.
In the sectioned histologic images from H-H stage 15, cells at the core
region of the first branchial arch was more condensed than those of the
periphery (Fig. 3A). Although this condensed region was known to the
mesodermal origin, I could not find clearly whether the cells forming this core
region are multilobular spindle shaped cells or round and semitranslucent cells
in primary cultures. But as the developments went, the intercellular distance
got closer to be more condensed, as observed in Fig. 3A–C.
The cultured cells from H-H stage 15 showed the different morphologies as
well as the cell condensation pattern from those of H-H stage 24 (Fig. 2). The
cells from H-H stage 15 had fibroblast-like spindle shape with decreased cell-
cell contact, while those from H-H stage 24 showed more compact
distribution, as shown in figure 2. In addition, cells from H-H stage 20 were
also similar to cells from H-H stage 24 in morphology and pattern (Fig. 2).
These results suggested that cultured cells after H-H stage 20 get their
information or capability for the condensation, probably through epithelial-
mesenchymal interaction. This finding is in accord with the fact that
epithelium expresses signaling molecules to promote the chondrogenesis in
stage 20 chick embryos40.
- 32 -
Another interesting point from the micromass cultures of H-H stage 24 or
even stage 20 mandibular region is that they could form the cellular nodules
which were overlapped with the cartilage nodules at later stages, which
couldn’t be found at the cells from H-H stage 15 (Fig. 2). These results were
already reported by Mina et al to reveal that the cartilage nodules were
detected in micromass cultured cell from mandibular mesenchyme after H-H
stage 1624. In the histological study, the more condensed region was observed
in first branchial arch after H-H stage 20 than the periphery (Fig. 3B, 3C).
This tendency to form the more condensed cellular nodules at later stages
seemed to have direct relationship with the increased cartilage formation in
mandibular cells. So the chondrogenic capability of the mandibular cells
requires the cell condensation and is acquired only after H-H stage 20 or later.
And some of those chondrogenic cells showed NCC characteristics, from the
fact that anti-HNK-1-positive cells were mainly located in cartilage nodules
(Fig. 6E for in vivo result and data not shown for in vitro result).
The maxillary cultured cells, regardless of their stages, 15 or 24, made no
cartilages or cellular nodules. Moreover, the more condensed regions like
mandible could not be found in maxillary region after H-H stage 20 (Fig. 3B,
3C). But the cells from H-H stage 24 were condensed and positive for PNA
staining. These results indicate that maxillary cells are unable to form the
- 33 -
cartilages in spite of sufficient condensation at H-H stage 24. And this finding
agrees well with the fact there is no cartilage formation in maxillary area from
the in vivo observation or previous reports28, 41 that the maxillary patterning
information is given to maxillary region cells only after H-H stage 2016.
HNK-1-positive cells were observed in high numbers of presumptive
maxillary and periorbital area cells at H-H stage 15 and decreased remarkably
to few numbers at stage 24. Taken together with the mandibular and maxillary
findings, it is possible that the preservation of HNK-1 gene expression has
something to do with the chondrogenic capability, which will be pursued by
later experiments.
The expressions of anti-vimentin antibody were increased as the facial
developments went on. Only some parts of mandibular or presumptive
maxillary cells at H-H stage 15 were positive for this staining, but most of the
cells were positively stained at H-H stage 20 and 24. This rapid increase of
vimentin, one of the representative markers for the mesenchymal cells, was
different from the decreased expression pattern of HNK-1 protein. So I guess
that cranial NCCs are on the transit into mesenchymal cells after or around H-
H stage 15, which will be further clarified. Sparse study about this transition
have been performed by others18-20.
- 34 -
Taken together, cultured cells at H-H stage 15 are not condensed enough to
be able to start chondrogenesis or osteogenesis and may be at the initial phase
of conversion from NCCs to mesenchymal cells. It is already reported that
this stage is characterized to initiate the specification of cell fate35, 42. And the
cells at H-H stage 24 had capacity to be condensed and were already
programmed to develop toward the osteochondrogenesis in mandible or
osteogenesis alone in maxillary region.
In order to find out the factors that influence the postmigratory transition of
NCCs into mesenchymal cells in mandibular and presumptive maxillary
region, Wnt-3a was tried for this study. It has been known as one of the
regulators for maintenance, proliferation, and non-differentiation of neural
crest precursor34.
Wnt-3a conditioned media induced the increased number of round cells,
increased expression of anti-HNK-1 antibody, and decreased E-cadherin
expression. These results could suggest that Wnt-3a maintains the
characteristics of NCCs and suppresses the transition of NCCs to
mesenchymal cells.
Roles of Wnt-3a have been well known as the promotion of neural crest
migration43 , potential modulations the neural crest cell fate on the basis of
their patterns of expression and their ability to alter cell fate during
- 35 -
development44. Exact mechanisms underlying the preservations of round cell
type are beyond the discussion here in this paper, but it should be further
studied. But it is still more likely that Wnt-3a promoted the decrease of E-
cadherin expression, which facilitating the migration of cells, preserving the
nature of NCC fate, and preventing the transition of NCC into mesenchymal
cells.
In summary, I tried to examine the process and influential factors for the
transition of postmigratory cranial NCCs into mesenchymal cells from in vitro
model as well as in vivo. As a result, postmigratory cranial NCCs cultured
from mandible and presumptive/definitive maxilla were observed to be
decreased with developmental stages, while the mesechymal cells were
increased in number. So it is suggested that postmigratory NCCs are on
transition into the mesenchymal cells in mandibular and maxillary region after
H-H stage 15 and that Wnt-related signals can influence this transition.
- 36 -
V. CONCLUSION
The roles of neural crest-derived mesenchymal cells are well known for the
formation of craniofacial bone and cartilage, little information is available
about their transition into mesenchymal cells. So I wanted to find out the
temporal changes and the control mechanism of NCC fate in relation to the
osetochondrogenic characteristics with special reference to mesenchymal
transition.
1. The momicromass cultured cells from H-H stage 15 showed the
mulitlobular spindle-shaped mesenchymal cell like shapes and were not
condensed to start the chondrogenesis or osteogenesis.
2. Postmigratory NCC lost their NCC-specific HNK-1 expression before H-
H stage 20 as they were advanced to more development.
3. The cells after H-H stage 15 showed more vimentin expression, which is
the marker for the mesenchymal cells.
4. Postmigratory mandibular and presumptive maxillary cells showed
increased abilities to form bone and/or cartilage as they acquire mesenchymal
natures.
5. Wnt-3a could influence to maintain the characteristics of NCC and to
suppress the transition of NCCs into mesenchymal cells.
- 37 -
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Abstract (in Korean)
배아배아배아배아 얼굴얼굴얼굴얼굴 발생발생발생발생 단계에단계에단계에단계에 따른따른따른따른 신경능세포의신경능세포의신경능세포의신경능세포의
간엽간엽간엽간엽 세포로의세포로의세포로의세포로의 전환전환전환전환
<지도교수 이상휘>
연세대학교 대학원 의과학과
김 남 호
발생 과정에 있어서 배아의 신경능세포(neural crest cells)는
두개안면골격 형성, 특히 두개안면부에서 얼굴 구조 형성에
결정적인 역할을 하는 세포군으로 알려져 있다. 신경능세포에서
기원된 간엽세포들이 얼굴의 뼈나 연골을 형성하는 데 중요한
역할을 한다는 것은 잘 알려져 있지만, 신경능세포에서
간엽세포로의 전환에 대한 연구는 미흡한 실정이다. 따라서, 본
연구자는 조류 배아에서 발생 단계에 따라 얼굴의 신경능세포가
간엽 세포들로 변화화는 과정을 조직학적인 접근과 함께 세포
배양을 통해 생체 외에서 확인하고, 신경능세포에서 간엽 세포로
전환되는 데에 영향을 미치는 인자를 찾고자 하였다.
이를 위해, 조류 배아의 발생 단계에 따라 하악과 상악을
micromass 방법을 이용하여 배양하였고, 신경능세포와 관련된
단백질들의 변화, 세포 증식 양상, 연골이나 뼈 형성 능력을
확인하였다. 또한, 신경능세포의 유지, 증식, 분화에 관련하여 Wnt-
3a의 영향을 관찰하였다.
그 결과, 얼굴 부위에서 이동이 끝난 신경능세포들은 그들의
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특성을 잃어버리고 서로 응축되기 시작하였다. 특히, 하악의
세포들은 발생 과정이 진행됨에 따라 후에 연골로 분화되는
세포혹(cellular nodules)의 형성 정도가 증가하였다. 또한,
신경능세포가 그들의 특성을 잃어버리고 간엽세포의 특성을
나타냄에 따라 뼈나 연골을 형성할 수 있는 능력이 증가하였다.
Wnt-3a는 신경능세포의 특성을 유지시키고, 간엽세포로의 분화를
억제하였다.
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핵심되는 말: 신경능세포, 간엽세포, Wnt-3a, 얼굴, 발생