Ultrastructural changes at gap junctions between lesioned crayfish axons

Summary In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segment retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal region usually contains gap junctions and 50–60 nm vesicles near the axolemma of both SLGAs. From 1–14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find.These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic — pinocytotic action of 50–60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.This work was supported by NIH research grant NS-14412 and and RCDA 00070 to G.D.B.     

Neuritic growth cone and ependymal gap junctions in the feline subfornical organ during early development

Summary Intercellular contacts in the subfornical organ (SFO) of kittens 3, 16, and 29 days old were studied in thin sections and by the freeze-etch method. Gap junctions appeared between growing nerve processes and target cells. The junctions were interspersed between immature synapses lacking mitochondria as well as full preand postsynaptic membrane specializations. Gap junctions were seen on filopodia as well as on more mature processes. The morphology of these junctions was typical of those described earlier but they were of small size (0.2–0.3 m).Gap junctions of peculiar form were also seen between ependymal elements in the SFO at 16 days. These were of large size (0.5–0.8 m) and were often of segmented character. This segmentation consisted of bands 3–4 particles in width with a center-to-center spacing of 90 nm with particle free corridors between corresponding to the width of about two rows of particles. The margin of the group might be circumscribed by a row of particles. Although gap junctions of large size were seen between ependymal cells in thin section, features corresponding to the particle free corridors have not been observed to date.On leave of absence from the National Institute of Neurological and Communicative Disorders and Stroke, Section of Functional Neurosurgery, Branch of Clinical Neuroscience, Bethesda, Maryland 20014, USAThis work was supported by grants from the Swiss National Foundation for Scientific Research Nos. 3.636.76 and 3.611.0.75, the EMDO Stiftung and the Dr. Eric Slack-Gyr Stiftung     

Prismatic cristae and paracrystalline inclusions in mitochondria of myocardial cells of the oyster Crassostrea virginica Gmelin

Summary Several types of unusual mitochondrial configurations were found in myocardial cells of the oyster Crassostrea virginica Gmelin. These mitochondria include, in order of frequency, prismatic cristae, filamentous paracrystals in honeycomb and herringbone configurations, and paracrystals composed of rows of electron dense particles. The long, parallel, evenly spaced prismatic cristae are square or rhomboidal in cross section. In the space between the prismatic cristae are rodlike structures (4–6 nm in diameter) that are regularly spaced about 12nm apart and appear to pass between adjacent cristae. Filamentous paracrystals are observed in slender, elongated mitochondria. The filament spacing and form of these paracrystals suggest that they are composed of the intercristal rods. Alternatively, filamentous paracrystals might be tangential sections of prismatic cristae and intercristal rods. Particulate paracrystals which consist of dense lines or rows of particles are the least frequent type of unusual configuration. The particles are triangular, possibly pyramidal, in shape; their bases are 10–12 nm thick and repeat in rows every 17–18 nm. There is a close association between particulate paracrystals and prismatic cristae plus intercristal rods. Although similar mitochondrial configurations have been associated with disease or altered metabolism in a number of species, we have found no such association in the oyster as yet.Supported in part by the Mississippi-Alabama Sea Grant Consortium, through NOAA, Dept. of Commerce under grant no. NA 79AA-D-0049We wish to thank Ms. Barbara M. Hyde, Ms. Patricia A. Vermiere and Mr. Robert Allen for their technical assistance     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

Intermitochondrial junctions in the heart of the frog,Rana esculenta

Summary In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4±0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3±0.29 nm. There are 65.3±2 periodic structures per m of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11±0.3 nm thick and are separated from each other by 16.5±0.46 nm, their density being 65±2.28 per m of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Neuroepithelial bodies in the lung of the tree frog,Hyla arborea L.

Summary The endocrine-like cells (ELC), which together with nerve endings form the neuroepithelial bodies, are located on primary and secondary septa in the non-ciliated epithelium of the lung of Hyla arborea. ELC protrude markedly toward the lumen of the lung and are surrounded by pneumocytes, which separate ELC from the lumen by thin cytoplasmic processes. ELC possess a light cytoplasm containing two types of granules: (i) numerous small granules, 50–110 nm in diameter, and (ii) single large granules, 290–860 nm in diameter. Numerous nerve fibers, often forming synaptic junctions, can be observed in contact with ELC.This study was supported by a grant No. 476/II from the Polish Academy of Sciences     

Interneuronal and glial-neuronal gap junctions in the lamina ganglionaris of the compound eye of the housefly,Musca domestics

Summary The cell-body layer of the lamina ganglionaris of the housefly, Musca domestica, contains the perikarya of five types of monopolar interneuron (L1–L5) along with their enveloping neuroglia (Strausfeld 1971). We confirm previous reports (Trujillo-Cenóz 1965; Boschek 1971) that monopolar cell bodies in the lamina form three structural classes: Class I, Class II, and midget monopolar cells. Class-I cells (L1 and L2) have large (8–15 m) often crescentshaped cell bodies, much perinuclear cytoplasm and deep glial invaginations. Class-II cells (L3 and L4) have smaller perikarya (4–8 m) with little perinuclear cytoplasm and no glial invaginations. The midget monopolar cell (L5) resides at the base of the cell-body layer and has a cubshaped cell body. Though embedded within a reticulum of satellite glia, the L1–L4 monopolar perikarya and their immediately proximal neurites frequently appose each other directly. Typical arthropod (-type) gap junctions are routinely observed at these interfaces. These junctions can span up to 0.8 m with an intercellular space of 2–4 nm. The surrounding nonspecialized interspace is 12–20 nm. Freezefracture replicas of monopolar appositions confirm the presence of -type gap junctions, i.e., circular plaques (0.15–0.7 m diam.) of large (10–15 nm) E-face particles. Gap junctions are present between Class I somata and their proximal neurites, between Class I and Class II somata and proximal neurites, and between Class II somata. Intercartridge coupling may exist between such monopolar somata. The cell body and proximal neurite of L5 were not examined. We also find that Class I and Class II somata are extensively linked to their satellite glia via gap junctions. The gap width and nonjunctional interspace between neuron and glia are the same as those found between neurons. The particular arrangement and morphology of lamina monopolar neurons suggest that coupling or low resistance pathways between functionally distinct neurons and between neuron and glia are probably related to the metabolic requirements of the nuclear layer and may play a role in wide field signal averaging and light adaptation.     

Ultracytochemistry of cholera-toxin binding sites in ciliary processes

Summary Cholera toxin reduces the rate of formation of aqueous humor in concentrations (10^–11 M) that do not disturb the morphology of the aqueoushumor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30–40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.Supported in part by USPHS grant # EY-00237, the Connecticut Lions Eye Research Foundation, Inc., and Research to Prevent Blindness, Inc.     

Gap junctions between astrocytes during growth and differentiation in organ culture systems

Summary Fetal rat neocortex maintained in organ culture systems with the use of sponge foam matrices and millipore filter platforms undergoes growth and cytodifferentiation along classical neuronal and glial lines up to 36 days in vitro (DIV). Astrocytic differentiation is characterized by accumulation of 80–90 Å glial filaments in the cell bodies and processes of astrocytes. Gap or nexus junctions closely resembling those formed in mammalian brain in situ are identified by 15 DIV. By 36 DIV, interastrocytic gap junctions are numerous and frequently join extensive lengths of adjacent glial plasma membranes. The results suggest that these organ culture systems may provide a favorable environment for the study of cellular structure and function of coupled neuroglia.This work was supported by a research grant from the Veterans Administration. Skilled technical assistance was provided by Robin L. Isaacs and Marilyn Woodward     

Membrane junctions between salivary gland cells ofChironomus thummi

Summary Cells ofChironomus salivary glands communicate through intercellular connections of high permeability. Electron micrographs of salivary glands show two kinds of junctions between the membranes of adjacent cells, which may be responsible for cell coupling: septate junctions and close membrane junctions.A large fraction of lateral cell surfaces is occupied by septate junctions, while the area of close membrane junctions appears to be very small. Consequently septate junctions have been considered as likely sites for intercellular coupling. There are however some indications that intercellular communication is provided by structures which seem to be unstable. As osmotic effects are among the factors which can disrupt cellular communications, we have tried to eliminate possible effects of the fixing solutions on the ultrastructure of intercellular connections by using isoosmotic fixatives. Under these conditions large regions of close membrane junctions of the nexus kind have been observed to occur between gland cells. They are of similar size as septate junctions. It seems to be possible that as in other communicating cell systems nexus could be the sites for intercellular coupling of salivary gland cells.The authors would like to thank Prof. Dr. H. Leonhardt, Institut für Anatomie I, Homburg, for the use of his electron microscope (Zeiss EM 9-DFG grant LE 69–8) during part of this work and Prof. Dr. H. Kroeger, Institut für Genetik, Saarbrücken for the supply withChironomus larvae.     

Heterogeneity of tight junctions along the collecting duct in the renal medulla

Summary The tight junctions along the medullary collecting duct in the kidneys of the rat and the rabbit were studied with freeze-fracture electron microscopy and quantitated according to the number of strands and the apico-basal depth (nm) of the junctions.The most elaborate tight junctions were found in the inner stripe of the outer medulla; rat: 10.6±0.8 strands and 205±24nm; rabbit: 11.6±2.4 strands and 291±55 nm.The elaboration of the tight junctions decreased continuously towards the papillary tip. Inner zone I; rat: 9.3±2.6 strands and 186±38nm, rabbit: 9.5±2.3 strands and 247±59nm. Inner zone II; rat: 7.1±2.2 strands and 129±32nm, rabbit: 8.5±1.4 strands and 199±26nm. Inner zone III; rat: 6.0±1.6 strands and 111 + 19 nm, rabbit: 7.0±1.5 strands and 183±43 nm. In the inner zone III comprising the papillary tip tight junctions with only 1–3 strands were not infrequently seen. Preliminary findings in the kidney of the golden hamster indicate a similar decline of junctional tightness along the collecting duct.These morphological observations suggest that the permeability of the paracellular pathway of the medullary collecting duct increases towards the tip of the papilla, especially in the rat. The functional implications for the medullary recycling of urea and electrolytes, and for the urinary concentrating mechanism are discussed.In addition, the tight junctions of the papillary epithelium are described.     

A freeze-fracture study of tight junctions in the pars convoluta and pars recta of the renal proximal tubule

Summary The morphology of tight junctions of the renal proximal tubule was studied comparing the pars convoluta and pars recta of rat, golden hamster, rabbit, cat, dog and tupaia. Though some interspecies variations were observed, the convoluted portions of the proximal tubules revealed quite uniformly very leaky tight junctions with mainly 1–2 strands.Along the whole proximal tubule of the rabbit kidney including the pars recta only minor differences of the zonulae occludentes were found. By contrast, the tight junctions of the pars recta in other species were much more elaborate, especially in cat and tupaia, having up to 6 strands and an overall depth of more than 150 nm. The implications of these findings are discussed with special regard to the functional differences between the pars convoluta and pars recta of the proximal tubule.This work was supported by the Deutsche Forschungsgemeinschaft     

Two types of neuromuscular junctions in the pharyngeal retractor muscle ofHelix lucorum

The pharyngeal retractor muscle of the snailHelix lucorum is innervated by a pair of nerves containing axons of two types, for which there are two corresponding types of myoneural junctions with the muscle cells. The junctions of type I correspond to the thick axons. The terminals of these axons, which contain numerous spherical transparent vesicles (41±5 nm) and fewer vesicles of the dense-core type (67±3 nm), make contact mainly with noncontracting sarcoplasmic projections of the muscle cells. Junctions of type II correspond to thin axons, containing many granules. The terminals of these axons make contact with contractile parts of the muscle cells and they contain a heterogeneous population of vesicles: small spherical clear vesicles (44±2 nm), granules with fine-grained contents (135±5 nm), and a few spherical dense-core vesicles. The distance between the muscle cells is usually great — over 50 nm, but in the region of the sarcoplasmic processes the surface membranes come together to form a gap which in some areas does not exceed 10 nm.N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 5, pp. 539–542, September–October, 1977.     

MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.     

Small granulated cell types in rat superior cervical and coeliac-mesenteric ganglia

Summary In the rat superior cervical and coeliac-mesenteric ganglia we have observed three types of small granulated (SG) cell: Type I cells are characterised by membrane-bounded cytoplasmic granules with a core of variable, moderate to low electron-density, whose limiting membranes are rounded in profile ranging from 50–150 nm in diameter. Type II SG cells contain numerous highly electron-dense, polymorphic cytoplasmic granules ranging from 100–300 nm in diameter. The haloes of Type II cell granules are variable in shape, and the core is often eccentrically located or fragmented. Type III SG cells contain membrane-bounded granules with a core of variable moderate to low electron-density. In profile these granules appear oblong or circular with average dimensions of 170 × 50 nm. All three SG cell types receive cholinergic-type pre-ganglionic terminals whose afferent nature is confirmed by their degeneration following pre-ganglionic neurectomy. Only Type I cells have been observed to donate efferent synapses to dendrites of principal ganglionic neurones and are thus interneuronal.This work was in part supported by a grant from the Medical Research Council. We wish to thank Mr. T.T. Lee for valuable technical assistance and Mr. P.F. Hire and Mr. K. Twohigg for illustrative help     

The fine structure of polychaete septate junctions

Summary Epidermal septate junctions of Nereis sp. and Cirriformia sp. fixed with OsO₄ or glutaraldehyde/OsO₄ display variable structure in electron micrographs. In transverse section the septa are often indistinct and obscured by opaque material that fills the junctional cleft. Septa (spaced at 180–280 Å) are more clearly defined in slightly oblique transverse section; they exhibit an electron lucent center and appear to be linked by arms. En face views of the junction show a honeycomb pattern. Cytoplasmic faces of junctional membranes are backed with plaques opposite the septa. Lanthanum used as a tracer delineates junctional structure in negative contrast. In transverse section a chain-like lattice is present in the junctional cleft. En face views show parallel rows of pleated elements often linked by arms into honeycomb arrays. Oblique sections demonstrate that these pleated elements are continuous with the chain-like lattice seen in transverse sections. Lanthanum does not pass entirely through the junction. Lanthanum reveals that the septa have a very intricate substructure, but it is difficult to visualize the architecture that could generate the various images presented by these junctions when seen in different orientations. However, it is clear that these junctions possess some features that are diagnostic of several supposedly different types of septate junctions in invertebrates.Supported by USPHS grants NIH 5 P01 NS-07512, NIH 2701 GM-00102, and NB-00840, and by a grant from the Pomona College Research CommitteeI thank Sarah Wurzelmann, Stanley Brown, Nancy Kelly, and Gerhard Ott for excellent technical assistance. Portions of this study were carried out while I was a Postdoctoral Fellow in the Department of Anatomy, Albert Einstein College of Medicine. I dedicate this article to Berta Scharrer as a token of appreciation and affection for her guidance, encouragement, inspiration, and example of excellence     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Electrophysiological evidence for rod and cone-based vision in the nocturnal flying squirrel

Summary The flying squirrel (Glaucomys volans) is a strongly nocturnal rodent. Previous anatomical observations suggested that the retina of this animal contains some cone-like receptors in addition to large numbers of rods. Evidence for duplicity of function in this visual system was obtained from an examination of three indices of visual activity: the electroretinogram (ERG), the isolated PIII retinal response, and the visually evoked cortical potential (VECP). The spectral sensitivity of the dark-adapted flying squirrel is similar to that of other mammals — it has a 500 nm peak (Figs. 3, 8). Responses of the ERG and isolated PIII to flickering light indicate the operation of two processes (Figs. 4, 7), one of which is unable to follow flickering light at repetition rates above 10–15 Hz. Spectral sensitivity measurements reveal that these two processes have different spectral sensitivities. The photopic mechanism in the flying squirrel visual system has peak sensitivity at about 520 nm (Figs. 5, 7, 9). The effects of steady light adaptation are much more obvious in the cortical potentials than they are in the retinal potentials.We thank David Birch for his advice and assistance. This research was supported by a Grant from the National Eye Institute (EY-00105).     

Structural changes at the myogenic cell surface during the formation of myotendinous junctions

Summary Myotendinous junctions are sites which are morphologically and molecularly specialized for force transmission between intracellular and extracellular structural proteins. In the present investigation, the formation of these specialized junctions is studied in chicken embryos from 9 days following fertilization to 1 day posthatching, using light and electron microscopy. Observations indicate that the first discernible event in myotendinous junction formation is the appearance of basement membrane at the incipient junction at 9–10 days postfertilization, concomitant with the aggregation of fibroblasts at the junctional regions of myogenic cells. Subsequently, subsarcolemmal densities appear at sites opposite basement membrane locations by 13 days postfertilization. Myofibrils insert into subsarcolemmal densities by day 15 and invaginations of the cell membrane are initiated at those insertions. Type I collagen fibers appear at the cell surface at day 17. Junctional structure at day 17 qualitatively resembles that of adult myotendinous junctions. Changes in junctional structure following day 17 are primarily increases in the amount of subsarcolemmal densities, myofibril-membrane associations, and amount of junctional membrane folding.