修 士 学 位 論 文
題 名
A n a l y s i s o f a c t i n l o c a l i z a t i o n d u r i n g h e a d f o l d f o r m a t i o n i n t h e c h i c k e n e m b r y o s .
ニ ワ ト リ 胚 頭 褶 形 成 時 の ア ク チ ン 局 在 の 解 析 （ 英 文 ）
指 導 教 員 福 田 公 子 准 教 授
平 成 30 年 1月 10 日 提 出 首都大学東京大学院
理 工 学 研 究 科 生 命 科 学 専 攻 学修番号 16881328 氏 名 名 取 澄 香
学位論文要旨（修士（理学））
論文著者名 名取 澄香 論文題名：Analysis of actin localization during head fold formation in the chicken
embryos.
（邦題）：ニワトリ胚頭褶形成時のアクチン局在の解析（英文）
本文
ニワトリを含む有羊膜類の初期胚は背側から外胚葉、中胚葉および内胚葉の 三層が平たく広がったシート状の構造をしている。平面構造であるステージ5 から約4時間後、ステージ6になると胚の前方が背側に盛り上がり、頭褶と呼 ばれるひだを作る。それに伴って内胚葉側に前腸と呼ばれる袋状の構造ができ、
胚は立体的な頭部を持つようになる。そして後方および側方も折れ曲がること で、胚は胚体外から区別された細長い構造をとる。
私はこの過程で胚の立体化の開始となる、前方の折れ曲がりである頭褶がど のようにしてできるのかに興味をもった。そこでシート状の胚が折れ曲がる時 に、どのような力がどこにかかるのかを調べることにした。組織が収縮し立体 構造を作る時にはしばしばアクチンの集積が見られることから、本研究ではニ ワトリ胚を用いて頭褶形成によって変化する構造とともに、どこにアクチンの 集積が見られるかファロイジン染色によって調べた。
まずアクチン局在の変化を見る前に、胚のどの部分が折れ曲がり、頭褶にな るのかを調べた。原腸陥入後のステージ5では前方の正中線に沿って前から脊 索前板(PP)、頭突起(HP)、ヘンゼン結節(HN)が並ぶ。そこでステージ5とステ ージ6の間の胚の腹側前方を正中線に沿ってDiIでラベルし、これをステージ6 まで培養した。その結果、折れ曲がった胚には前腸門と、前腸の最も深い場所 の2点でヒンジポイントが存在し、本研究ではそれぞれ頭褶側ヒンジポイント、
前腸側ヒンジポイントと名付けた。PPの前端の細胞は頭褶側ヒンジポイントと なることがわかった。また、PPの中央付近の細胞は前腸内に寄与し前腸ヒンジ ポイントとなった。
次に胚をファロイジンで染色し、屈曲前後のステージを詳細に分けて重合型 アクチンが局在する場所や胚の厚さの変化を比較した。観察は将来の頭褶側、
前腸側ヒンジポイントを中心に行った。ステージ5では胚はほぼ平面でアクチ ンは全体的に弱かった。しかし発生が進むと徐々にPP上の特に内胚葉頂端側で 斑点状の強い領域が出現し、アクチンが弱く非常に薄いPPの前方と差が生じた。
折れ曲がる直前のPP内は非常にアクチンが強く、その領域は背側に盛り上がり 厚くなった。またPPの後端からHPの先端は外胚葉内胚葉共にアクチンが強く、
胚は少し薄かった。折れ曲がると、アクチンが強い領域は前腸側のヒンジポイ ントに集中し、その後方は斑点状であった。よって前腸形成では、ステージ5 以降にPP付近の内胚葉で強いアクチン局在が見られた。そして平面だったシー トの肥厚と、前腸側ヒンジポイントにおける内胚葉組織の収縮の両方にアクチ ンが関与する可能性がある。また胚が折れ曲がり、前腸が形成され始めてから、
前腸門の背側に位置する頭褶側ヒンジポイントの外胚葉に、アクチンが強く現 れた。このことから頭褶形成では、胚が折れ曲がった後の形状維持にアクチン が関わる可能性が考えられる。
Analysis of actin localization during head fold formation in the chicken embryos.
A thesis
Submitted for degree of Master of Science Tokyo Metropolitan University
By
Sumika Natori
Developmental of Biological Science, Graduate School of Science, Tokyo Metropolitan University
2018
1
Contents
Acknowledgments ………...…2
Summary ………...……3
Introduction ………..…5
Materials and Methods ………8
Results ………10
Discussion ………15
Figures ………20
References ………...32
2
Acknowledgments
I am grateful to Dr. Kimiko Fukuda of Tokyo Metropolitan University for her guidance and encouragement throughout the course of this study. I appreciate Dr.
Naohito Takatori for many advices on the operation of the confocal microscope. I also thank all the members of Laboratory of Developmental Biology for valuable advices.
3
Summary
At gastrulation, early chicken embryo consists of three germ layers, ectoderm, mesoderm and endoderm, spreading into a planer sheet. At neurula stage, anterior part of the embryo is lifted and makes a fold called the head fold. Along with the head fold formation, a sack-like structure, the foregut, is formed on the ventral side and eventually the embryo has a three-dimensional head structure. It has been unclear how the
sheet-like embryo starts to fold. To understand the mechanisms involved in making the head fold, I investigated actin localization during formation of head fold, because actin contraction regulates change of shape in cells or tissues. In this study, I observed the changes of structure and actin accumulation during the head fold formation using chicken embryos.
First, I found that embryo has two folding points. Posterior hinge is future anterior intestinal portal and anterior one will become the deepest part of the foregut. I name them the head fold hinge and the foregut hinge, respectively. Next, original position of each hinge point was investigated by labeling the midline of the anterior embryo with DiI. The cells at the anterior end of the prechordal plate (PP) were found to become the head fold hinge and at the center of PP contributed to the foregut hinge.
From the chicken developmental stages determined by Hamburger and Hamilton (1951) embryos in stage 5 have flat structure and those in stage 6 have the head fold, respectively. To describe folding process fully, I defined the stages between stage 5 and 6 depending on the structure around PP.
4
Based on this stage I analyzed the shape and thickness of embryo and localization of polymerized actin by staining with phalloidin. In stage 5 the embryos were almost flat and actin was weak. Then, strong actin spots appeared on the apical side of the endoderm at PP. Next, embryo around PP became thicker, and actin in the endoderm became much stronger. The cell sheet around PP curved to the dorsal side before folding.
After folding, accumulation of strong actin was found on the endoderm at the foregut hinge. The result of strong actin localization in apical side of the endoderm before and after folding around future foregut hinge suggests a possibility that actin may be very important for folding of foregut hinge. At the head fold hinge, strongly actin appeared in the ectoderm after the head fold formation. Therefore, in the head fold formation, actin may maintain the shape after the embryo is folded.
5
Introduction
In early embryos of chicken three flat germ layers, ectoderm, mesoderm and endoderm, were generated from epiblast during gastrulation (Stern, 2004) at stage 4 (Hamberger and Hamilton, 1951). The endoderm invaginates from epiblast via Hensen’s node to the most ventral layer of the embryo and spreads outward. The mesoderm also invaginates from epiblast and spread between newly synthesized endoderm and
remaining ectoderm. Especially, future axial mesoderm invaginated via Hensen’s node move anteriorly to form head process (HP) at stage 5. In anterior tip of HP an inverted triangle-shaped mesendoderm (differentiate into both endoderm and mesoderm) called prechordal plate (PP) were formed (Bellairs, 1953a; Psychoyos and Stern, 1996).
Even though massive cell movement occurred at stage 5, the embryo still has a sheet-like planar structure. Then after incubating further 4 hours, at stage 6 the embryo becomes a little elongated and the anterior region of the embryo swollen toward the dorsal side, forming a fold called the head fold. Along with this, a sack-like structure called the foregut is formed on the ventral side and finally embryo has a proper head structure. In the results of following posterior and lateral folding, the embryo has three dimensional, elongated body with distinguished from the extraembryonic tissue (Darnell et al., 1999). Therefore, formation of the head fold is the first step to gain proper three-dimensional body in amniotes which are originally planar structure. In this study, I analyzed the dynamic change of embryonic body during formation of the head fold and the foregut.
6
First, I investigated which part of the embryo at stage 5 chicken embryo will fold during the head fold formation. The anterior endoderm cells of the embryo before folding were stained with DiI and incubated until stage 6 to analyze their future position in the embryo. Cells at PP area in stage 5 are known to contribute widely to the foregut (Kirby et al, 2003). In this study, PP endodermal cells along the midline were labeled with DiI and revealed that the anterior tip of PP will give rise to anterior intestinal portal and posterior end of PP will become dorsal foregut.
Actin is one of the major cytoskeletal proteins in the cells. Generally, actin filaments in the cells forms dynamic cytoskeleton to play crucial role for cell polarity and drive cell motility and cell division (Akhshi et al., 2014). Actin filaments
associating with myosin, actomyosin, produces the force to shrink bundle of fibers, and sometime changes shape of cells (Odell et al, 1981). During neural tube formation the neural plate, which is a flat ectodermal tissue in the beginning, were deformed and bent to V-shape structure, a neural groove, before it forms a neural tube (Nagele et al., 1987;
Schoenwolf, 1988; Schoenwolf et al., 1989). At this time, actomyosin accumulates in the surface layer of cells at the midline of the neural plate and contracts to transform the cell into a wedge shape. Such a change in the morphology of the cell is a critical step to form a neural groove (Colas and Schoenwolf, 2001).
In this study, to know mechanism of the head fold formation, I analyzed the local accumulation of actin in the PP by staining with phalloidin as well as change of the shape in the cell sheet near PP. In the foregut endoderm, strong localization of the actin at the apical side is observed before folding, actin accumulation in the head fold
7
ectoderm was found only after the folding. It is suggested that actin is involved in the foregut hinge formation but maintain head fold hinge.
8
Materials and Methods
Chicken embryos
Fertilized eggs were incubated at 38°C for the appropriate time to obtain embryos of the required stage. In this study, incubation time was approximately 24 hours until embryo became stage 5 that has fully formed head process. The embryo was defined based on Hamburger and Hamilton, 1951. Embryos between stages 5 and 6 were observed with a bright field microscope every 30 minutes for further staging (see results).
New culture
Embryos were culture with the modified New method (Stern and Ireland 1981;
Chapman et al, 2001).
Cell labeling with DiI
0.05% Carbocyanine dye DiI, (1,1-dioctadecyl-3,3,3',3'- tetramethyl
indocarbocyanine perchlorate) (DiI-C18; Molecular Probes) in 30mM sucrose was used to label cells with microcapillary pipettes.
Staining of actin filaments in whole embryo with phalloidin
Embryos were fixed overnight with 4% paraformaldehyde in PBS. Fixed embryos were washed 3 times with PBS for 30 minutes each. The phalloidin with Alexa Fluor
9
546 dye or Alexa Fluor 488 dye (Panchuk-Voloshina et al., 1999) diluted 1/200 with PBS was exchanged with the solution and stained with overnight (Chazotte, 2010).
Embryos were washed with PBS three times for 15 minutes each. The embryos were placed in the holes of one or two ring-shaped seals stuck on a slide glass. After PBS was completely wiped off, 3.75 g/ml fructose in PBS was placed on the embryo and covered with a cover glass. After closing the edge of the cover glass with manicure, it was turned over and observed with a confocal microscope (Nikon C2).
10
Results
1. The precordial plate was a central place for formation of the head fold and the foregut in the future.
First, to clarify which part of the embryo participates in formation of the head fold, cells in the stage 5 embryo were labeled. At the stage 5, the prechordal plate (PP), the head process (HP), the Hensen’s node (HN) and primitive streak line up on the midline from anterior to posterior. Cells at slightly anterior to PP (point a), the anterior tip of PP (point b), the center of PP (point c), and the anterior tip of HP (point d) (also most posterior of PP) were labeled with DiI (Fig. 1A, A’ and C). Labeled embryos were incubated for approximately 2 hours until they developed to stage 6. Stage 6 embryo had the head fold/ the foregut at the anterior embryo (Fig. 1B, B’). In the embryo, there were two hinge-like structures in the head fold/ the foregut. One hinge was at the anterior intestinal portal and the ectoderm bent with steep angle (Fig. 1B’ and D Arrow head). Another hinge was at the deepest region of the foregut, and the endoderm was folded tightly (Fig. 1B’ and D Arrow). In this study, I named each as the head fold hinge and the foregut hinge.
Endodermal cells at point a, slightly anterior to PP, contributed to anterior to the head fold hinge (Fig. 1D), outside of the head fold. Cells at point b indicated by the red arrowhead in Fig. A’ was found to be present in the anterior intestinal portal, the head fold hinge. In addition, it was found that cells at point c, the center of PP, contribute to the ventral foregut between the head fold hinge and the foregut hinge. Cells at point d,
11
the anterior tip of HP, contributed to dorsal foregut far posterior to the foregut hinge.
From these results, it was suggested that the PP area contributes to the future head fold and foregut and I focused on observing the shape in PP for further analysis.
2. New stages between 5 and 6 were defined based on the shape of PP.
The stage to be observed is stage 5 (Fig. 2A) to stage 7 (Fig. 2F) before and after folding. In the developmental stage determined by Hamburger and Hamilton, stage 5 embryo is characterized by flat shape with a head process (Fig. 2A), and stage 6 embryo has the head fold/ foregut (Fig. 2E) (Hamburger and Hamilton, 1951). For research following sequential changes from flat embryo to embryo with 3D head (the head fold), I needed finer stages dividing between stage 5 and 6. After observation of many
embryos around the stages 5 and 6 in detail with special attention to the structure of PP and the following results were obtained. Three more stages between the stage 5 to 6 were supplemented.
Stage 5 : Embryo was flat. PP extended half of the primitive streak in length (Fig. 2A).
Stage 5+ : Embryo was still flat. PP stretched almost same length as the primitive streak. Therefore, Hensen’s node was almost in the middle of the embryo (Fig. 2B).
Stage 6-- : PP region of embryo was slightly raised (Fig. 2C).
Stage 6- : This protuberance of PP became prominent and the curve is evident. Still no obvious fold was observed (Fig. 2D).
Stage 6 : The embryo had the head fold. Two hinges were obvious (Fig. 2E).
12
Following observation, I used this stage to describe change of the structure and actin localization.
3. Strong actin localization in the endoderm proceeded the structural changes.
Actin filament is an important cytoskeleton, involved in the many cell processes, such as cell motility, polarity, cell division and cell shape. I hypothesized that cell shape change by contraction force with actin filament contribute to formation of the head process. Actin filaments are stained using phalloidin conjugated with Alexa Fluor 546 or 488. Also the structure of PP in embryos was observed.
In the stage 5 embryo PP was nearly flat and the thickness along with
anterior-posterior axis is similar (Fig. 3A, B and B’). Weak actin staining was observed in the whole PP (Fig. 3B’ blue bracket). Especially actin in the PP ectoderm was weak.
In the stage 5+ embryo, anterior to PP became slightly thinner (Fig. 3D’ yellow and blue brackets), while the thickness in the posterior from PP remained unchanged (Fig.
3D and D’). In this stage salt-and-pepper strong actin staining were observed on the apical side of the endoderm in PP (Fig.3 D and D’). Strongest actin localization was found in the middle of PP (Fig.3 C and D’, arrowheads). It is interesting that shape of the PP remained unchanged in this stage.
13
4. The middle of PP swollen toward the dorsal side and the actin in PP became particularly stronger.
In stage 6-- embryo, PP was thickened (Fig. 4B and B’). Actin localization in the endoderm of the thickened PP became extremely strong (Fig. 4B’ blue bracket). In the ectoderm, actin staining was still weak at PP, but stronger at HP (Fig. 4B’ green bracket).
The strong actin localization was found in entire PP in the endoderm (Fig. 4A blue triangle).
In the stage 6- embryo, PP swelled toward the dorsal side (Fig. 4D and D’, arrowheads). Actin localization in the endoderm became stronger within PP (Fig. 4D’, blue bracket). Actin staining remained weak in the anterior region to PP (Fig. 4D’, yellow bracket). PP became like an open fan and actin was strong in whole PP (Fig. 4C blue fan shape). Within PP particularly strong actin localization was observed in the horseshoe-shaped anterior part of PP (Fig. 4C red horseshoe shape). These results suggested that actin localized strongly in the future folding.
5. Actin is strongly localized at the both hinges after folding.
In the stage 6 embryo, shallow folding was formed at the front end of PP (Fig. 5A, B and B’). Stronger actin localization was observed in the endoderm of the foregut hinge and its posterior region from the region corresponding to the anterior intestinal portal (Fig. 5A blue fan shape and B’ white arrowhead and blue bracket). Also in this
14
stage actin localization in the ectoderm at the head fold hinge appeared (Fig. 5B’ red arrowhead).
In the stage 7 embryo, folding became sharper and cell sheet at this folding became thicker (Fig. 5C, D and D’). The extremely strong actin staining was found in the apical surface of endoderm at the foregut hinge (Fig. 5C and D’ white arrowheads). In the ectoderm of the head fold hinge actin localized strongly (Fig. 5D’ red arrowhead).
6. Protrusions from endodermal cells appeared on the ventral surface of the future foregut.
In the course of this study, I found very interesting structure in the developing foregut at stage 7 (Fig. 6B yellow box). There were bubble-like protrusions on the endoderm surface (Fig. 6A arrowheads). In surrounding endodermal cells, strong actin staining was found on the apical surface (Fig. 6A and C arrows) while whole surface of bubble-like protrusions showed strong actin localization (Fig. 6C arrowheads). Many protrusions were observed only in the folding endoderm at stages 6-, 6 and 7, in which actin was particularly strong. Taken together, it is possible that these protrusions were extruded from endoderm with rapid and strong contraction in apical surface of
surrounding endodermal cells during folding of endodermal cell sheet at the foregut hinge (Fig. 6D). This may permit acute change of shape in the foregut hinge.
15
Discussion
Vertebrates have three-dimensional body elongated along with AP axis. Just after the gastrulation, vertebrate embryo has flat, sheet-like body with three germ layers, ectoderm, mesoderm and endoderm. When the anterior embryo folded, head is raised.
This is the first step to form three-dimensional body for embryo. In this study, change of actin localization was investigated before and after head fold formation to know the mechanism for the head fold formation.
During the course of this study, I defined supplemental developmental stages between stages 5 to 6 described by Hamburger and Hamilton, 1951. Although each stage, 5+，6--，6-, was defined by significant morphological changes, time period between each stages are not same. It is approximately two hours between stage 5 to 5+
and time between stage 6-- and 6 is much shorter, within an hour. This suggests that after elongation of the head process and change of thickness of cell sheet around PP, folding of embryos occurred rapidly.
From my results, I summarized change of the shape of embryos from stage 5 to 6, just before and after the formation of the head fold, respectively. Thickness of embryo at anterior to PP was same as posterior embryo in the stage 5 (Fig. 1B and B’) and then became thinner (Fig. 3D’ yellow bracket) than posterior embryo from stage 5+. This difference in thickness between anterior to PP and PP was maintained to the later stages (Fig. 4B, 4D, 5B and 5D). While no change of thickness in PP was observed in the early stages, stage 5 and 5+ (Fig. 3B’ and D’ blue brackets), PP became thicker in the stage
16
6-- (Fig. 4B’ blue bracket). In stage 6- embryo, PP were swollen on the dorsal side (Fig.
4D and D’, red arrowheads) before sharply folded at the front end of PP in stage 6 embryo (Fig. 5B and B’).
After stage 5+ anterior end of PP is a border of thin anterior and thickened
posterior embryo. This border is future the anterior intestinal portal/ the head fold hinge (Fig. 1C and D, b). It was suggested that this boundary of thickness in the embryo is very important for formation of the head fold. On the simple level, embryo after the stage 5+ is composed of thin anterior plate and thick posterior plate connected with their boundary. If embryos in stage 5-6 is under the converged pressure from posterior to this border and thin anterior plate is solid enough, a pressing force makes thick posterior plate fold at the boundary. Then, this boundary point will become the anterior intestinal portal/ the head fold hinge. There is no report or study mentioning the mechanism involved in thin anterior plate differentiation, it is interesting that this anterior region, proamnion, consists of ectoderm and endoderm without mesoderm (de Melo Bernardo, et al., 2014). This structural specificity may give the anterior plate character.
In the thickened posterior plate, especially the ectoderm became thicker after stage 6-. Even after folding, the vicinity of the foregut hinge became thicker. It is known that the HP and PP, which secrets anti-BMP proteins, affect surrounding ectoderm to differentiate into neural plate (Wilson et al, 2000). Cells in the neural plate were high columnar structures compared to the adjacent ectodermal cells (Schoenwolf, 1985;
Nagele et al., 1987). PP is known to be required for the forebrain induction (Pera and
17
Kessel, 1997). From my study, PP may be essential to fold embryo via change of thickness in the limited region of the ectoderm as well.
Next, I discussed about how the convergent force generated during the head fold formation. In the anterior neural plate, proliferation of the neuroepithelial cells ware very high to provide future forebrain (Smith and Schoenwolf, 1987). Also in the PP endoderm, which is presumptive foregut endoderm shows a high mitotic rate in a report by Bellairs, 1955. Therefore, it seems that cell division can generate the pressure to from posterior to the boundary between anterior and posterior plate. However, I think cell division alone cannot explain rapid folding from stage 5+ to 6, just in two hours.
Cell division in the ectoderm and endoderm should be analyzed in the each subdivided stage as I defined in this study for further discussion.
Another candidate to generate force in the cell plate is apical contraction by actin.
It was shown that actin in endoderm especially at PP localized to the apical side earlier than structural change in stage 5+ (Fig. 3C-D’). This accumulation of apical actin in PP endoderm was getting stronger as swelling and fold in PP (Fig. 4). Finally, strongest apical actin in the foregut hinge at the time of folding from stage 6-- to 6 (Fig. 4C red fan shape and 5B’ white arrowhead) were detected. It is known that strong localization of actin is involved in apical contraction in the rapid morphological change of neural tube cells (Nagele and Lee, 1987; Nagele et al, 1991). Therefore, it is possible that apical actin contraction in the endoderm induces convergence force in the posterior plate. This force may be stronger in the future foregut hinge and give rise to deep angled fold.
18
At the apical surface of the endoderm in the foregut hinge after the head fold formed bubble-like protrusions were found (Fig. 6A). Interestingly, these protrusions were detected only region where extremely strong actin localization was observed. It is possible that they were generated from endodermal cells that squeezed out from
endodermal sheet because rapid and strong contraction during folding. As similar spherical membrane protrusions, the blebs produced by the contractions of the
actomyosin cortex are known (Charras et al., 2008). Reduce in cell number by making bleb during folding in endoderm help to reduce surface area in PP endoderm. This may induce swelling and bending to the dorsal side. It is still unknown how many
protrusions appear and whether they will be retained to the endodermal sheet or proceed to apoptosis during the head fold formation.
No strong localization of actin in ectoderm was detected before or during the head fold formation (Fig. 3 and 4). It appeared in the ectoderm of the head fold hinge
immediately after the head fold was formed (Fig. 5B’ and D’ red arrowhead). From this result, it was suggested that actin in the ectoderm didn’t have a main role for forming the head fold and the head fold hinge. On the other hand, actin in ectoderm might be involved in maintenance of the hinge shape after the embryo was folded. In the stage 7 embryo, remarkably strong actin in the apical surface of the foregut hinge endoderm was also found (Fig. 5C and D’ white arrowheads). Taken together, to maintain head fold structure, apical actin contraction is important in both hinges, foregut hinge and head fold hinge.
19
In this study I observed change of structure and actin localization in the embryonic plate during formation of head fold in detail. I can suggest that endodermal actin in PP is involved in the foregut hinge formation and maintenance, whereas ectodermal actin function only in the maintenance of one of the hinge. With future functional analysis of actin, for example local inhibition of actin polymerization, this study provides vital information the mechanisms of the head fold formation.
20
Figure 1. The precordial plate region bended and formed the head fold and the foregut in the future.
(A) Cells in stage 5 embryo ware labeled with DiI (magenta). (A’) High magnification of (A). Arrowhead shows the anterior tip of prechordal plate (PP) (C, point b). (B) Stage 6 embryo had the head fold and the foregut at the anterior embryo. (B’) High
magnification of (B). Arrowhead indicates a hinge at the anterior intestinal portal.
Arrow indicates another hinge at the deepest region of the foregut. (C) A schematic figure for (A’). Point a (slightly anterior to the PP), b (the anterior tip of PP), c (the center of PP) and d (the anterior tip of HP (head process)) show cells labeled with DiI.
(D) Schematic figure of midline cross section in stage 6 embryo. There were two hinge-like structures as the head fold hinge (arrowhead) and the foregut hinge (arrow).
Scale bars =500 µm.
21 Figure 1.
22
Figure 2. Additional stages between 5 and 6 were defined based on the shape of PP.
Bright field microscope image of chicken embryos observed from the ventral side. The black box show the observation area by the confocal microscope (see figure 3-6). (A) Stage 5 : Embryo was flat. PP extended half of the primitive streak in length. (B) Stage 5+ : PP stretched almost same length as the primitive streak. (C) Stage 6-- : PP region was slightly raised dorsally. (D) Stage 6- : Protuberance of PP became prominent and the curve was evident. (E) Stage 6 : The embryo had the head fold. Two hinges were obvious. (F) Stage 7 : Embryo had first pair of somite. Scale bars =500 µm.
23 Figure 2.
24
Figure 3. Strong actin localization in the endoderm proceeded the structural changes.
(A and C) One of the actin-localized (white) confocal microscopy image stacks in stage 5 (n=5) and 5+ (n=2) embryos (see figure 2A and B black box). (B, B', D and D') The cross section of the midline (A and C yellow line) was reconstructed using all of the Z-axis stack images. The PP region is indicated by blue bracket (B’ and D’) and triangle (C). (A-B’) Weak actin staining was observed in the whole PP. (C-D’) Actin in
endoderm localized to the apical side (white arrowheads). Anterior to PP (yellow bracket) became slightly thinner than in PP. D, dorsal; V, ventral. Scale bars: A and C
=100 µm, B, B’ D and D’ =20µm.
25 Figure 3.
26
Figure 4. The middle of PP swollen toward the dorsal side and the actin in PP became particularly stronger.
(A and C) One of the actin-localized image stacks of stage 6-- (n=4) and 6- (n=4) embryos (see figure 2C and D black box). (B, B', D and D') The cross section of the midline (A and C yellow line) was reconstructed using all of the Z-axis stack images.
The PP region is indicated by blue bracket (B’ and D’), triangle (A) and fan shape (C).
(A-B’) PP was thickened. Actin localization in the endoderm became extremely strong.
In the ectoderm, actin localization was also strong at HP (B' green bracket). (C-D’) PP swelled toward the dorsal side (arrowheads). Within PP particularly strong actin
localization was observed in the horseshoe-shaped anterior part of PP (C red horseshoe shape). In the anterior region to PP, actin staining remained weak (D' yellow bracket). D, dorsal; V, ventral. Scale bars: A and C =100 µm, B, B’ D and D’ =20µm.
27 Figure 4.
28
Figure 5. Actin is strongly localized at the both hinges after folding.
(A and C) One of the actin-localized image stacks of stage 6 (n=3) and 7 (n=2) embryos (see figure 2E and F black box). (B, B', D and D') The cross section of the midline (A and C yellow line) was reconstructed using all of the Z-axis stack images. The PP region is indicated by blue bracket (B’) and fan shape (A). (B’, C and D’) Stronger actin localization was observed in the endoderm of the foregut hinge (white arrowhead). (B’
and D’) In the ectoderm of the head fold hinge, actin localization appeared and became strongly (red arrowhead). D, dorsal; V, ventral. Scale bars: A and C =100 µm, B, B’ D and D’ =20µm.
29 Figure 5.
30
Figure 6. Protrusions from endodermal cells appeared on the ventral surface of the future foregut.
(A) There were bubble-like protrusions on the endoderm surface (arrowheads). (B) The foregut observation region (yellow box) of the stage 7 (C) Whole surface of bubble-like protrusions showed strong actin localization (arrowheads). (A and C) There is strong actin localization in the apical surface of surrounding endodermal cells (arrows). (D) Schematic figure of bubble-like protrusions extruded from endoderm. D, dorsal; V, ventral. Scale bars =20 µm.
31 Figure 6.
32
References
Akhshi, T.K, Wernike, D. and Piekny, A. (2014) Microtubules and actin crosstalk in cell migration and division. Cytoskeleton (Hoboken). 71, 1-23.
Bellairs, R. (1953a) Studies on the development of the foregut in the chick. I. The presumptive foregut area. J. Embryol. exp. Morph. 1, 115-124.
Bellairs, R. (1955) Studies on the development of the foregut in the chick. III. The role of mitosis. J. Embryol. exp. Morph. 3, 242-250.
Chapman, S.C., Collignon, J., Schoenwolf, G.C. and Lumsden, A. (2001) Improved method for chick whole-embryo culture using a filter paper carrier. Dev Dyn. 220, 284-9.
Charras, G. and Paluch, E. (2008) Blebs lead the way: How to migrate without lamellipodia. Nat Rev Mol Cell Biol. 9, 730-6.
Chazotte, B. (2010) Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging. Cold Spring Harb Protoc. 2010, pdb.prot4947.
33
Colas, J.F. and Schoenwolf, G.C. (2001) Towards a cellular and molecular understanding of neurulation. Dev Dyn. 221, 117-45.
Darnell, D. K., Stark, M. R. and Schoenwolf, G. C. (1999) Timing and cell interactions underlying neural induction in the chick embryo. Development. 126, 2505–2514.
de Melo Bernardo, A., Susana, M. and de Sousa Lopes, C. (2014) The involvement of the proamnion in the development of the anterior amnion fold in the chicken. PLoS One. 9:e92672
Hamburger, V. and Hamilton, H.L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92.
Kirby, M.L., Lawson, A., Stadt, H.A., Kumiski, D., Wallis, K., McCraney, E., Waldo, K.L., Li, Y.X. and Schoenwolf, G.C. (2003) Hensen's node gives rise to the ventral midline of the foregut: implications for organizing head and heart development.
Dev Biol. 253, 175-88.
Nagele, R.G., Bush, K.T., Lynch, F.J, and Lee, H.Y. (1991) A morphometric and computer-assisted three-dimensional reconstruction study of neural tube formation in chick embryos. Anat Rec. 231, 425-36.
34
Nagele, R.G., Hunter, E., Bush, K. and Lee, H.Y. (1987) Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation. J Exp Zool. 244, 425-36.
Nagele, R.G. and Lee, H.Y. (1987) Studies on the mechanisms of neurulation in the chick: Morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation. J Exp Zool. 241, 197-205.
Odell, G.M., Oster, G., Alberch, P. and Burnside, B. (1981) The mechanical basis of morphogenesis. I. Epithelial folding and invagination. Dev Biol. 85,446-62.
Panchuk-Voloshina, N., Haugland, R.P., Bishop-Stewart, J., Bhalgat, M.K., Millard, P.J., Mao, F., Leung, W.Y. and Haugland, R.P. (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 47, 1179-88.
Pera, E.M. and Kessel, M. (1997) Patterning of the chick forebrain anlage by the prechordal plate. Development. 124, 4153-62.
Psychoyos, D. and Stern, C. D. (1996) Fates and migratory routes of primitive streak cells in the chick embryo. Development. 122, 1523–1534.
35
Schoenwolf, G.C. (1985) Shaping and bending of the avian neuroepithelium:
morphometric analyses. Dev Biol. 109, 127-39.
Schoenwolf, G.C. (1988) Microsurgical analyses of avian neurulation: separation of medial and lateral tissues. J Comp Neurol. 276, 498-507.
Schoenwolf, G.C., Bortier, H. and Vakaet, L. (1989) Fate mapping the avian neural plate with quail/chick chimeras: origin of prospective median wedge cells. J Exp Zool.
249, 271-8.
Smith, J.L. and Schoenwolf, G.C. (1987) Cell cycle and neuroepithelial cell shape during bending of the chick neural plate. Anat Rec. 218, 196-206.
Stern, C. D. (2004) Gastrulation : From Cells to Embryo. Cold spring harbor laboratory press.
Stern, C.D. and Ireland, G.W. (1981) An integrated experimental study of endoderm formation in avian embryos. Anat Embryol. 163, 245-63.
36
Wilson, S.I, Graziano, E., Harland, R., Jessell, T.M. and Edlund T. (2000) An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo.
Curr Biol. 20, 421-9.
