Mesenchymal metaplasia of the chick chorioallantoic membrane; a non-specific response to selected stimuli

96

Experimental

MESENCHYMAL CHICK

METAPLASIA

CHORIOALLANTOIC RESPONSE R. R. RICH,2 Department

MEMBRANE; TO SELECTED

D. K. ROGERS,?

of Pharmacology, Kansas

University City, Kansas,

Received February

Cell Research 40, 96-l 03 (I SC5)

OF THE A NON-SPECIFIC STIMULI1

and F. E. LEADERS

of Kansas Medical School, U.S.A. 5, 19653

THE

control of cellular differentiation and, in particular, the process of induction, has long interested developmental biologists. The chorioallantoic membrane (CAM) of the chick has been especially attractive to those investigating experimental inductive systems since it offers, in an iI1 uivo context, a large, easily accessible area of relatively undifferentiated cells of all three germ layers. There was, therefore, considerable interest \vhen van Haeften [7] reported in 1958 that cell-free homogenates of chicken skeletal muscle could induce, Lvithin the CAM mesoderm, the development of elongated, longitudinall> oriented, non-striated “myocytes”. This worker interpreted the appearance of these “myocytes” as a completely specific response of the CAM mesenthyme to inductive sub-cellular particles within the homogenate. Shortly thereafter Ebert [2] observed the development of similar “muscle and muscle-like” cells within the CAM following inoculation with an RNA extract of chicken cardiac muscle and Rous sarcoma virus. Ranzi et nl. [5] also reported the induction of “muscle-like” cells in the CAM following inoculation with muscle ribonucleoprotein. However, in contrast to the observations of van Haeften and Ranzi, Ebert noted the development of cross-striations in a few membranes. Ebert [2] postulated that the virus RN,4 served as a vehicle to transport the large muscle RSh molecules into the cell, within which the muscle RNA could specifically direct the difrerentiation of multipotential mesenchymal cells into muscle tissue. The possibility remained that van Haeften had observed the proliferation of undetected cells in the “cell-free” homogenate rather than a response of 1 This work was SuaDorted in Dart by U.S.P.H.S. Grant NB 04392-03 and bv the National Aeronautics and Space kdministration &ant NSF-298 to the University of Kansas. 2 U.S.P.H.S. Student Training Grant and Lederle Student Training Grant awardecs. 3 Revised version received May 4, 1965. Experimental

Cell Research 40

Jletaplnsia the membrane

itself.

However,

97

of the chorioallantois the studies

of Wilt

and

Stolz

[S j and

Rich

and Filosa [6] have confirmed van Haeften’s observations. Wilt and Stolz used homogenates of mouse rather than chicken muscle. On the basis of staining differences betvveen the cells of these two species they concluded that the non-striated, longitudinally oriented, elongated cells found within the CAN mesenchyme were of host rather than graft origin and thereby represented a genuine response of the CAM to components of the muscle homogenate. Nonetheless, these investigators also pointed to the considerable lability of the CAM and questioned the specificity claimed for the response of the CAY mesenchyme to these “inductive” stimuli. The present investigation vvas designed to clarify this phenomenon and to direct attention particularly to the question of its specilicitv.

MATERIALS

AND

METHODS

Muscle homogenates were prepared aseptically. Chicken or mouse skeletal muscle was ground with 2 ml of sterile Ringer solution/g of tissue for 5 min in a cooled Waring Blender. This was followed by homogenization in a cooled motor-driven Tefton-inglass microhomogenizer for 15 min. Histological examination of this preparation revealed occasional nuclear fragments but no intact cells were seen. Portions of homogenate were centrifuged at 10,000 g and the supernatant was then lyophilized to approximate the consistency of the original homogenate. Aliquots (0.3-0.5 ml) of the homogenate, or the lyophilized supernatant, were pipetted onto the CAM of nine-dayold White Leghorn eggs using a false air-sac technique. After resealing the eggs were returned to the incubator. Additional groups of eggs were treated similarly except that various other stimuli were substituted for muscle homogenate or supernatant. The following stimuli were used: Inoculation of a gel composed of colloidal silica (Cab-O-Sib Cabot Chemical Co.) and Ringer solution; inoculation with lyophilized egg albumin; fenestration without resealing to allow exposure to the incubator atmosphere; inoculation of controls with Ringer solution. Five days after treatment the eggs were removed from the incubator and the chorioallantoic membranes were recovered and fixed in a solution of formalin in saline. All tissues were coded to allow subsequent unbiased examination. Serial, tangential sections (8 p thick) of the treated areas of all membranes were prepared. These were stained with hematoxylin and eosin, with phosphotungstic acid hematoxylin, and with Gomori’s trichrome stain [4].

RESULTS The results of various stimuli on the CAkI Histological examination of the CAN of embryos 7 ~ F51817

are presented receiving only E.xperimental

in Table I. Ringer soluCell Hesearch 40

98

R. R. Rich,

Il. Ii’. Rogers,

and F. E. Leaders

tion revealed no metaplastic or morphogenetic changes (Figs. 1 and 2). In all cases these membranes were comparable to the normal condition as described by other investigators [1 j. In other experimental groups, holyever, extensive changes were noted in all three germ layers. The responses deTABLE

I. Results

of’ rmrious

stimrlli

on ihe

chick

chorioallnntoic

Survivors exhibiting elongatrd, longitudinally oriented mesoderm cells

Survivors Stimulus

Inoculated

SO.

membrrrne.

%

so.

;;

(41) (36) (33)

20 24 44 41 17 1 0

(48)

Muscle homogenate \Yhole Lyophilized supernatant Total Silica gel Atmospheric exposure Lyophilized albumin Ringer solution

103 98 201 79 52 22 40

42 35 77 49 29 15 33

(62) (56)

(68) (83)

(69) (57) (84) (5% ( 7) ( (9

scribed in numerous studies and considered non-specific by ran Haeften [7] and Ebert [2] were again noted. These included marked vascular invasion of the treated area, proliferation and stratification of both ectodermal and endodermal epithelial surfaces, ectodermal keratinization and erosion, esteninvasion of the sive hyperplasia of mesoderm, and occasional epithelial of chorionic epithelial cells. mesenchyme by keratinized cysts or “pearls” Of particular interest in the present context was the metaplastic response of the mesenchymal cells since this reaction has been considered a specific inductive process in other studies [2, 5, 71. This response was the derelopment of elongated, longitudinally oriented cells \t\-ithin the mesoderm of the Fig. l.-Tangential section of a chorioallantoic membrane treated only with The normal morphologic features of the membrane are demonstrated. x 72. Fig. 2.-Primitive mcsenchymal cells of a chorioallantoic membrane treated solution. Characteristic stellate configuration of the cells is seen. v 1080. Fig. 3.-Ectodermal and mesodermal metaplasia Elongated longitudinally oriented mesenchymal Fig. 4.-Cellular nate. x 1080. Experimental

morphology

Cell Research

of longitudinally

40

Ringer only

with

solution. Ringer

in a membrane treated with muscle homogenate. cells are apparent. x 72. oriented

mesoderm

treated

with muscle homoge-

Metnplnsin

of the chorionllantois

Experimental

Cell Reseurch 40

100

R. R. Rich, Il. R. Rogers, and F. E. Leaders

CAM. These cells bore a strong histological resemblance to primitive muscle cells; however, they were seen not only in membranes treated with muscle homogenate or a lyophilized supernatant of homogenate (Figs. 3 and 4) but also in membranes treated with Cab-O-%1 silica gel (Figs. 5 and S), those exposed to the incubator atmosphere (Figs. 7 and S), and, in one instance, a membrane treated with lyophilized albumin (Table I). Staining with Gomori’s tri-chrome stain [3] accentuated the resemblance of the elongated mesoderm cells to primitive muscle. In all experimental groups these cells exhibited staining characteristics compatible mith primitive non-striated muscle. Staining both \vith hematoxylin and eosin and phosphotungstic acid hematoxylin at no time revealed cross-striations in the elongated cells. DISCUSSION The reaction of cells and tissues to injury, long a fundamental study of pathologists, is of necessity of interest to all investigators who experimentally manipulate living material. The chick CAM is a living system of exceptional but similarly, to a wide range of irritative lability, responding strikingly, Two groups of virologists il, 91, in attempting to establish the stimuli. importance of adequately controlled experiments, have reported similar histological responses to both physical and chemical insult to the CARI. Camain ef al. [l] noted particularly the proliferation of elongated, linearly oriented cells of mesenchymal origin; these investigators believed that this represented the differentiation of fibroblasts from primitive mesenchyme in response to a variety of irritative experimental procedures. It has been noted that staining both with hematoxplin and eosin and phosphotungstic acid hematoxylin at no time revealed cross-striations in the elongated, longitudinally oriented cells. This observation is consistent with other studies utilizing muscle homogenates and extracts [%Sj but is in contrast with Ebert’s report [aj of cross-striations in a few membranes to lvhich an extract of cardiac microsomes and Rous sarcoma virus was

Fig. 5.--Mesenchymal Fig. G.--Elongated gel. x 1080.

mctaplasia longitudinally

in a membrane oriented

treated

mesodermal

with silica gel. x 72. cells in a membrane

Fig. T.-Longitudinally oriented mesodermal cells which developed the choriollantoic membrane to incubator atmosphere. x 72. Fig. X.-Mesenchymal atmosphere. x 1080. Experimental

metaplasia

Cell Research 40

following

treated

with

silica

in response to exposure

exposure of the chorioallantoic

of

membrane to incubator

101

Mettrcplasia of the chorioallantois

Experimental

Cell Research 40

102

R. R. Rich, D. Ii. Rogers, and F. E. Lenders

applied. Further investigation is required to determine whether or not Ebert, in these instances, has observed a phenomenon qualitatively different from that noted in the majority of his preparations and that reported in the present study. Throughout this investigation both the mortality rate and the percentage of survivors exhibiting longitudinally oriented elongated mesoderm cells were higher than those reported by other investigators. This may be due to the relatively greater trauma, e.g. more concentrated homogenates in larger amounts utilized in the present experiments. This further supports the importance of physical factors in eliciting the reactions whic,h have been described. Thus it appears that a fundamental phenomenon, the response of a primitive cellular system to injury, has resulted in a diversity of interpretations when studied in several contexts. Despite their histological similarity to muscle cells, the actual nature of the longitudinally oriented elongated mesodermal cells remains uncertain. The present data indicate that regardless of their true cellular type they do not represent the response of receptive undifferentiated cells to a specific inductive stimulus. Instead, the chick CAR1 appears to he a particularly reactive experimental medium which is capable of a limited range of responses to a wide variety of traumatic stimuli. Since the artifacts of trauma may be exceptionally deceptive, we suggest that considerable caution and elaborate controls arc necessary requisites of subsequent histological study of this system.

SUMMARY

The hypothesis that sub-cellular components of chicken muscle homogenates can induce the differentiation of primitive muscle cells \vithin the chick chorioallantoic membrane (CAM) has been examined. Sterile cell-free homogenates of chicken and mouse skeletal muscle lvere applied to the CAM utilizing a false air-sac technique. Other experimental groups received, in lieu of muscle homogenate, Cab-O-S1 silica gel, lyophilized albumin, exposure to incubator atmosphere, or Ringer solution. Histological examination five days after treatment revealed the longitudinalI\ oriented elongated mesoderm cells considered primitive muscle by other investigators. However, these cells were noted not only in membranes treated with muscle homogenate but also in all other experimental groups except that receiving only Ringer solution. It is therefore concluded that regardless of the true cellular nature of the Experimental

Cell Research 40

Metaplasiu

103

of the chorionllmtois

longitudinally oriented elongated mesenchymal derivatives described, they do not represent a specific inductive phenomenon but rather a component of the general reaction to injury of an exceptionally labile system. Caution, consequently, is urged in subsequent studies of the chick CAM which utilize present histological techniques. We are indebted to Drs Frank Mantz and Craig Fischer of the Department of Pathology for their assistance in interpretation of the histological material, and to Mr Herbert Lindsley for critical reading of the manuscript. The technical assistance of Mr. William Rich is also gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6.

R., BRBs, P. and PLAGNOL, H., Ann. Inst. Pusfew. 98, 846 (1960). EBRT, J. D., Expff Zool. 142, 587 (1959). GOMORI, G., Am. J. Cfin. Pafhof. 20, 661 (1950). MOSCONA, A., Deuel. Biol. 1, 1 (1959). RANZI, S., GAYAROSI, G. and CITTERIO, P., Experienfia 17, 395 (1961). RICH, R. and FILOSA, M., Nature 196, 1329 (1962). 7. V.4x HAEFTEN, F., Acfa Physiol. Phnrmacof. Nderf. 7, 1 (1958). 8. \VILT, F. and STOLZ, T., Expfl Cell Res. 27, 189 (1962). 9. \VYLER, R. and v.4~ TOSGERES, H. A. E., J. Prcfhof. Bncferiof. 74, 275 (1955). CAMEAIS,

Experimental

Cell Research 40