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Stuart Hosie Æ Michael Farag Æ Maria Vena

Andreas Holloschi Æ Karl-Herbert Schafer

The role of contractile microfilaments in the morphogenesisof the developing foregut of chick embryos

Accepted: 28 November 2001 / Published online: 24 September 2002� Springer-Verlag 2002

Abstract The cellular processes that lead to changes inshape (morphogenesis) and organ formation (organo-genesis) are poorly understood. Local contraction ofmicrofilaments can change cell shape and lead tochanges of tissue shape. To clarify the role of contactilemicrofilaments in the foregut morphogenesis of chickembryos, 2- to 4-day-old embryos were exposed tocytochalasin D (CD), which is known to disruptmicrofilaments. Untreated age-matched embryos wereused as controls. Sections of treated embryos and con-trols were stained with phalloidin, which binds to actin,and examined with a fluorescence microscope. Micro-dissected specimens were examined using a scanningelectron microscope (SEM). Immunofluorescent stainingshowed a bright signal belt toward the apical cell regionof the foregut epithelium in controls. This signal was notevident in CD-exposed embryos. SEM micrographs ofthe controls showed the cranial foregut as a smooth,even, cylindrical structure in all stages studied. The lu-men was narrow and perfectly straight, the ventral anddorsal walls were in close apposition. The foregut ofCD-exposed specimens, however, showed a wide lumenand the walls were separated from each other. Thestructure seemed atonic and appeared conical, curved,or tilted. We observed a dense microfilament network

toward the apical cell pole of the epithelial foregut cellsof controls that was no longer evident after CD expo-sure. This network seems to play an important role inforegut morphogenesis, since actin-filament disruptionby CD causes loss of the normal shape.

Keywords Embryology Æ Foregut Æ Chick embryo ÆMorphogenesis

Introduction

The foregut in early stages is a uniform tubular structureconsisting of endodermal cells that adhere to one an-other. During further development this structure differ-entiates and a system of folds is generated, creating thepharynx, larynx, esophagus, and tracheobronchial tree[1, 2]. The cellular mechanisms that lead to these mor-phologic changes are unknown, as are disorders leadingto an abnormal shape, and hence, to a congenital mal-formation.

Cells contain an internal filamentous framework, thecytoskeleton, that stabilizes cell shape. Cytoskeletalcontractile microfilaments generate active tension that istransferred to neighboring cells via junctional complexes(adherens junctions, desmosomes) [3]. Local contractionof microfilaments can change the cell shape and lead tochanges of tissue shape, as has been demonstrated forthe optic vesicle, lens, alveoli, salivary glands, and neuraltube [4–6]. The aim of this study was to clarify the role ofthe contractile microfilaments in the foregut morpho-genesis of chick embryos.

Materials and methods

Fertile white leghorn eggs were incubated at 38.5 �C in a humidi-fied atmosphere for 2 to 4 days to obtain embryos ranging betweenHamburger and Hamilton (HH) stages 14 and 24 [7]. Whole em-bryos were exposed for 60 min to cytochalasin D (CD) 20 lg/ml(Fluka, Deisenhofen, Germany). Controls were maintained inRinger’s lactate solution.

Pediatr Surg Int (2002) 18: 611–614DOI 10.1007/s00383-002-0867-3

Supported by grant 55/96, Medical Faculty Mannheim, HeidelbergUniversity

S. Hosie (&) Æ M. Farag Æ M. Vena Æ K.-H. SchaferDepartment of Pediatric Surgery,University Hospital Mannheim,Heidelberg University, Theodor-Kutzer Ufer,D-68167 Mannheim, GermanyE-mail: stuart.hosie@kch.ma.uni-heidelberg.deFax: +49-621-383-3823

A. HolloschiDepartment of Molecular Biology andCell Culture Technology,Mannheim University of Applied Sciences,Mannheim, Germany

The embryos were fixed with 3.8% paraformaldehyde inphosphate-buffered saline (PBS) for 24 h. After paraffin embed-ding, sections 3 lm thick were obtained. The samples were depa-raffinized in xylol for 10 min and hydrated through a series ofethanols (100%, 95%, 90%, 75%, 50%, 25%). After rinsing withPBS, the tissues were treated with trypsin 5 mg/ml (GIBCO) for30 min at 37 �C. Following three rinses with PBS, nuclear stainingwas obtained using 4,’6-diamidino-2-phenylindol (DAPI, Sigma,Deisenhofen) 2 lg/ml for 5 min at room temperature. Afterwashing, the sections were incubated with phalloidin (Sigma) 5 lg/ml for 20 min at room temperature. Controls were incubated withPBS. Tissues were rinsed with PBS once again and examined with aZeiss fluorescence microscope equipped with a confocal imagingsystem (Openlab 1.7.5).

Embryos were fixed in 2.5% glutaraldehyde (Fluka) for 24 h.After microdissection, samples were dehydrated in graded series ofacetone for 10 min each (20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99.5%) and critical-point dried. After gold sputteringfor 60 s, the samples were examined using a DSM 950 scanningelectron microscope (SEM) (Zeiss, Jena, Germany).

Results

Immunofluorescent staining with phalloidin, which spe-cifically binds to actin, showed a similar pattern incontrols at all stages studied. The cylindrical epithelialcells of the foregut displayed a bright, regular, evenlydistributed fluorescent band at the apical region facingtoward the lumen. The cell region facing the basalmembrane and the adjacent mesenchyme showed amuch lower intensity of fluorescence and an irregular,uneven distribution. CD-treated embryos lacked thebright band at the apical region (Figs. 1 and 2).

Fig. 1 Actin-specific immunofluorescent staining with phalloidinin a 3.5-day-old chick embryo (HH stage 21). Note marked positivestaining at apical cell region of foregut endoderm

Fig. 2 Phalloidin staining in a 3-day-old CD-exposed chickembryo (HH stage 20) lacking bright signal at apical region offoregut cells

Fig. 3 Scanning electron mi-crograph of sagittal section of4-day-old chick embryo (HHstage 23). Inset shows detail ofcranial primitive foregut, whichis narrow and cylindrical

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SEM was performed on 2- to 4-day-old embryos (HHstages 14 to 24) after microdissection. The cranial part ofthe foregut was easy to identify, comprising the buccalcavity, primitive pharynx, primitive larynx, and tra-cheoesophageal space, from which the esophagus andtrachea are derived. The pharyngeal pouches and bran-chial arteries are clearly visible landmarks as well.Noteworthy is the observation that the cranial foregutrepresents a smooth, even, regular cylindrical structurein all stages studied. The lumen is narrow and perfectlystraight, the ventral and dorsal walls are in very closeapposition (Figs. 3 and 4).

In contrast, CD-treated specimens showed a differentpicture: the foregut had a wide lumen, the walls wereseparated from each other or even diverging, and thewhole structure appeared flaccid and atonic. It had lost

its straight, tubular configuration and appeared conical,curved, or tilted (Figs. 5 and 6).

Discussion

The cellular processes leading to changes in shape(morphogenesis) and organ formation (organogenesis)are poorly understood. His described a process in 1887in which lateral ridges appear in the lateral walls, fuse,and create a septum dividing the foregut. An anteriorrespiratory and posterior digestive tract are formed [8].Zaw-Tun questioned the existence of the tracheoesoph-ageal septum [9], and is supported by SEM studies ofKluth et al. [1, 2], who described a system of folds in theprimitive foregut demarcating the tracheoesophageal

Fig. 4 Scanning electron mi-crograph of sagittal section of3-day-old chick embryo (HHstage 19). Pharyngeal pouchesand branchial arteries are easyto identify. Note straight, nar-row cranial foregut

Fig. 5 Scanning electron mi-crograph of 3-day-old chickembryo (HH stage 18) afterexposure to cytochalasin D.Note wide, conical, flaccid cra-nial foregut

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space cranially and caudally. Movements of these foldscreate a separation from which the trachea and esoph-agus are formed.

We were intrigued as to the cellular mechanisms thatcould create mechanical forces capable of changing ormaintaining shape. Contractile microfilaments havebeen implicated in morphogenesis in a variety of organsand structures such as the optic vesicle, lens, alveoli,salivary glands, and neural tube. Inhibition of actinwas followed by loss of normal shape in these structures[4–6]. Our finding of a positive actin-specific immuno-fluorescent signal on the apical region of the foregutendoderm showed a similar pattern of localization anddistribution as in the epithelia of the above-mentionedstructures. Since CD disrupts actin filaments, the brightapical immunofluorescent signal was absent in the epi-thelial cells of treated embryos.

Microdissection and SEM of treated and controlembryos enables three-dimensional imaging of parts ofthe foregut. Our results show that microfilaments have atonifying effect on the foregut segments studied. Theypreserve its cylindrical, straight, narrow morphology,since actin-filament disruption causes a wide, atonic,dysmorphic structure.

This experimental design shows only the momentaryeffect of microfilament disruption on the morphology of

the foregut, since CD is highly toxic and kills the em-bryos immediately. Morphogenesis occurs over a longertime course, and the long-term effect upon inhibition ofcytoskeletal microfilaments using other strategies shouldbe addressed in future studies.

References

1. Kluth D, Habenicht R (1987) The embryology of usual andunusual types of esophageal atresia. Pediatr Surg Int 2: 223–227

2. Kluth D, Steding G, Seidl W (1987) The embryology of foregutmalformations. J Pediatr Surg 22: 389–393

3. Ingber DE (1997) Tensegrity: the architectural basis of cellularmechanotransduction. Ann Rev Physiol 59: 575–599

4. Wessells NK, Spooner BS, Ash JF, Bradley MO, Luduena MA,Taylor EL, Wrenn JT, Yamada KM (1971) Microfilaments incellular and developmental processes. Science 171: 135–143

5. Maloney C, Wakely J (1982) Microfilament patterns in the de-veloping chick eye: their role in invaginations. Exp Eye Res 34:877–886

6. Schoenwolf GC, Folson D, Mol A (1988) A reexamination ofthe role of microfilaments in neurulation in the chick embryo.Anat Rec 220: 87–102

7. Hamburger V, Hamilton HL (1951) A series of normal stages inthe development of the chick embryo. J Morph 8: 44–92

8. His W (1887) Zur Bildungsgeschichte der Lungen beim mens-chlichen Embryo. Arch Anat: 89–106

9. Zaw-Tun HA (1982) The tracheo-esophageal septum – fact orfantasy? Acta Anat 114: 1–21

Fig. 6 Sagittal section of 3.5-day-old chick embryo (HHstage 22) after exposure tocytochalasin D. Note wide,atonic, bent cranial foregut

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