Muscle Anlagen Research Articles

Complex expression pattern of tenascin during innervation of the posterior limb buds of the developing chicken

The histological localization of the extracellular matrix glycoprotein tenascin was studied during the formation of peripheral nerves in the developing chick hindlimb (embryonic stages 21.5 to 30) by light and electron microscopic immunological methods to obtain insights into the molecule's functional role in the pathway formation by motor and sensory nerves. At stages 21.5 and 23, nerve roots and plexus were surrounded by high tenascin-immunoreactivity, whereas the not yet innervated limb bud was not immunoreactive. During innervation of the limb bud at stages 24.5 and 25, tenascin was detectable at the limb bud base and restricted in its expression to the proximal nerve regions. The nerve tips did not contact areas of elevated tenascin-immunoreactivity. At stages 26 to 28 the dorsal and ventral trunks of the crural and sciatic nerves were surrounded by tenascin-immunoreactivity, which was localized between Schwann and mesenchymal cells. The tips of the growing nerve had now reached the tenascin-positive interface between bone and muscle anlagen. This interface was contacted tangentially rather than penetrated by the nerve tips. The medial and lateral femoral cutaneous nerves were surrounded by high and weak tenascin-immunoreactivity, respectively. In both nerves, tenascin-immunoreactivity was absent where the nerves branched extensively to innervate the skin. The cutaneous nerves diverging from the sciatic nerve were of very low tenascin-immunoreactivity or tenascin-negative at all developmental stages tested. At stages 29 and 30, muscle nerves, having just entered the tenascin-negative muscles, exhibited strong immunoreactivity, whereas the more proximally situated trunks of the sciatic nerve were weakly and discontinuously labeled, particularly at sites where smaller nerves were branching off. Since the cutaneous branches of the sciatic nerve were always of low tenascin-immunoreactivity, the question was raised whether tenascin expression in the sciatic nerve depended on the presence of motor axons. Spinal cords of stage 19 or 20 embryos were therefore removed and tenascin expression was investigated at stages 26 and 27. Some of the residual nerves were weakly tenascin-immunoreactive, whereas others were tenascin-negative. Our observations suggest that tenascin is not involved in the initial guidance of peripheral nerves to their targets. Rather, neuron-induced tenascin appears to stabilize the proximal nerve trunks during a transient time period, possibly by preventing axons and Schwann cells from intermingling with the surrounding mesenchyme, thus contributing to nerve fiber compaction. Conversely, nerve branching may be elicited by reduced levels of tenascin. Furthermore, tenascin may divert growth cones from the developing bone tissue and direct muscle afferents to their appropriate targets.

Immunohistochemical study on the development of extraocular muscles. I. Rat.

Development of extraocular muscles of rat was studied immunohistochemically using antibodies against three molecular markers of muscle differentiation: brain type (CK-B) and muscle type (CK-M) creatine kinase isoenzymes and muscle type enolase isoenzyme (β-enolase). The time of innervation of extraocular muscles was also studied immunohistochemically using antibody against neuron specific enolase (NSE). Periodic acid-Schiff (PAS) and PAS after diastase digestion stainings was used for the demonstration of glycogen. At embryonic day (E) 15, when muscle primordium of each extraocular muscle appears, β-enolase and glycogen were observed in all muscle primordia, while CK-B was immunoreactive only in lateral rectus (LR), superior rectus (SR), inferior rectus (IR) and inferior oblique (IO) muscle primordia. CK-B immunoreactivity appeared in superior oblique (SO) and medial rectus (MR) muscles at E17 and E18, respectively. By E18-19, CK-M immunoreactivity became positive in all muscles. NSE immunoreactive nerve fibers were first observed in LR, SR, IR and IO at E15, in SO at E16, and in MR at E17. Consequently, β-enolase immunoreactivity and glycogen appeared in all extraocular muscles at E15, while CK-M at E18. LR, SR, IR and IO muscles became immunoreactive to CK-B antibody at E15; however, SO and MR muscles became immunoreactive at E17 and E18, respectively. The sequence and time of appearance of CK-B in extraocular muscles were similar to those of NSE-immunoreactive nerve fibers. These findings suggest that in rats the expression of CK-B in each extraocular muscle coincides with the innervation of that muscle, while CK-M is expressed only after innervation.

The nerve growth factor receptor gene is expressed in both neuronal and non-neuronal tissues in the human fetus

In situ hybridization was used to study expression of β-nerve growth factor receptor (NGF-R) mRNA in the early human fetus. In 8- to 12-week old fetuses, high labelling was found over motoneurons along the entire length of the lateral motor column. High levels of NGF-R mRNA were also seen over most developing nerve cell bodies in both the dorsomedial and ventrolateral part of the dorsal root ganglia. Lower, but clearly specific labelling was detected over a subpopulation of cells in Auerbach's plexus in the intestines. Evidence for a non-neuronal expression of NGF-R mRNA came from labelling over a subpopulation of cells in glomeruli of the kidney in a 12-week old human embryo. Myoblasts in skeletal muscle anlagen were labelled as well as cells along peripheral nerve. The wide-spread expression of NGF-R mRNA in the human fetus suggests that the NGF-R is important for development of a variety of different tissues of both neuronal and non-neuronal origin.

The formation of premuscle masses during chick wing bud development

The skeletal musculature of chick limb buds is derived from somitic cells that migrate into the somatopleure of the future limb regions. These cells become organized into the earliest muscle primordia, the dorsal and ventral premuscle masses, prior to myogenic differentiation. Therefore, skeletal-muscle specific markers cannot be used to observe myogenic cells during the process of premuscle mass formation. In this study, an alternative marking method was used to determine the specific stages during which this process occurs. Quail somite strips were fluorescently labeled and implanted into chick hosts. Paraffin sections of the resulting chimeric wing buds were stained with the monoclonal antibody QH1 in order to identify graft-derived endothelium. Non-endothelial graft-derived cells present in the wing mesenchyme were assumed to be myogenic. At Hamburger and Hamilton stage 20, myogenic cells were distributed throughout the central region of the limb, including the future dorsal and ventral premuscle mass regions and the prechondrogenic core region. By stage 21, the myogenic cells were present at greater density in dorsal and ventral regions than in the core. By stage 23, nearly all myogenic cells were located in the dorsal and ventral premuscle masses. Therefore, the two premuscle masses become established by stage 21 and premuscle mass formation is not complete until stage 23 or later. Premuscle mass formation occurs concurrently with early chondrogenic events, as observed with the marker peanut agglutinin. To facilitate the investigation of possible underlying mechanisms of premuscle mass formation, the micromass culture system was evaluated, to determine whether or not it can serve as an accurate in vitro model system. The initially randomly distributed myogenic cells were observed to segregate from prechondrogenic regions prior to myogenic differentiation. This is similar to myogenic patterning in vivo.

Development and innervation of the abdominal muscle in embryonic Xenopus laevis

The morphogenesis and innervation of the ventral abdominal musculature in Xenopus embryos was examined using microscopic techniques. Muscle development begins at Nieuwkoop and Faber Stage 31, when aggregates of undifferentiated cells form on the ventrolateral margins of rostral trunk myotomes. During subsequent stages, aggregates form and detach from progressively more caudal myotomes to form a series of seven discrete cell clusters (anlagen). The anlagen migrate ventrally in a cell-free space between the epidermis and a subepidermal layer of pigment cells. Extracellular aggregates of 30-nm granules are evident transiently between the migrating anlagen and the epidermis. During stages 39 and 40, each anlage transforms into a sheet of myotubes which attaches rostrally and caudally to adjacent sheets to form a seven-segmented muscle. The series of broad segments, approximately one fiber thick, extends from the pericardium to the level of the proctodeum. The embryonic muscle is innervated by the ventral rami of spinal nerves 2 to 9. The major nerve trunks to the muscle develop between stages 35/36 and 40. Axons initially grow ventrally along the paths taken by the muscle anlagen. When the anlagen become muscle segments, the nerves are deep to the narrow boundaries between the segments. Spinal nerve 2 ramifies in the first muscle segment and sends fibers rostrally to the geniohyoid muscle. The findings represent the first description of the development of this muscle in Xenopus and the first account of the development of the abdominal motor nerves in an amphibian embryo.

Structure and developmental expression of the chicken NGF receptor

The nucleotide and deduced amino acid sequence of a cDNA clone of the chicken NGF receptor (NGFR) is reported and is compared with sequences of mammalian NGF receptors. A model is presented in which monodentate or bidentate binding of NGF dimers to repeated cysteine-rich sequence elements of the receptor yields low- or high-affinity NGF binding, respectively. In situ hybridization is used to characterize expression of NGFR in developing chick from 40 hr to 10 days of embryogenesis. NGFR mRNA expression is detected in premigratory neural crest cells, in epibranchial placode cells, and in all sensory, sympathetic and parasympathetic derivatives of these structures. In the embryonic CNS, NGFR mRNA is detected in the mantle zone but not the periventricular germinal zone throughout most of the neural tube. By Embryonic Day 8, NGFR mRNA is detected in a substantial fraction of cells in every brain region, with highest levels present in developing motor neurons. NGFR mRNA also is transiently expressed in many mesenchymal cell populations including cells in branchial arch, sclerotome, muscle anlagen, and feather follicles. The functional significance of wide-spread embryonic expression of the NGF receptor is discussed.

Acetylcholinesterase Activity of Developing Muscles in the Lower Limb of the Rat

A cytochemical study of acetylcholinesterase was done in the lower limb of the prenatal rat and in the gastrocnemius muscle of the postnatal rat. Between 15 and 17 days of gestation, mesenchymal cells constituting the muscle primordia are characterized by the presence of enzyme activity in their rough endoplasmic cisterns and nuclear envelopes, while those involved in the formation of the neocapillary and cartilage do not show enzyme activity. This suggests that mesenchymal cells destined to myogenic cells actively produce acetylcholinesterase in a limited period, which may play a role in cellular aggregation and fusion during the muscular morphogenesis. Cytochemical findings as to extensive networks of secondary synaptic folds of the neuromuscular junctions and invaginations of the sarcolemma in the extrasynaptic regions are also illustrated in the differentiating gastrocnemius muscles.

A hierarchy of determining factors controls motoneuron innervation. Experimental studies on the development of the plantaris muscle (PL) in avian chimeras.

Quail leg buds were grafted in place of chick leg buds or chick wing buds and vice versa at stages 18 to 21 after colonization by muscle precursor cells had been completed. Motor endplate pattern in the plantaris muscle of the grafts was analyzed before hatching by means of esterase and acetylcholinesterase staining techniques. Muscle fibre types were made visual using the myosin ATPase reaction. Investigations are based on the species-specific endplate pattern of the plantaris muscle: multiply innervated fibres in the chick and focally innervated fibres in the quail. Muscle pieces isolated from the adjacent medial gastrocnemius muscle of the grafted legs were histologically examined to judge their species-specific composition. Horseradish peroxidase was injected into the plantaris muscles of both the grafted and the opposite leg as well as in the plantaris muscle of normal quail embryos, in order to be sure that the plantaris muscle of the grafts is innervated by appropriate motoneurons. This procedural design offers for the first time a possibility to test experimentally the influences of motoneurons on endplate pattern formation under conditions corresponding to those in normal ontogenesis. It is shown that such appropriate motoneurons of one species which project to the plantaris muscle of the other species dictate the endplate pattern. When the plantaris muscle is innervated by inappropriate motoneurons, the endplate pattern inherent in the muscle primordium itself becomes realized. A sequence of hierarchically acting factors is proposed to bring different results in line. According to this, the neuronally set programme has priority compared with that set in the muscle. This is true for the normal development and might generate the high neuro-muscular specificity. If under experimental conditions the neuronal programme and the peripheral programme differ, the axons and muscle fibres selectively interact with respect to their inherent characteristics and the muscle-specific programme becomes expressed. If there is a lack of a certain axon type, muscle fibres might become innervated by non-corresponding motoneurons which alter the muscle fibre type.

Slow myosin in developing rat skeletal muscle.

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)

Nerve growth factor (NGF) receptor expression in chicken cranial development.

In order to map the expression of receptors for nerve growth factor (NGF) during brain and cranial ganglia development, iodinated NGF (125I beta NGF) was used as a probe in an autoradiographical analysis performed between embryonic day 3 (E3) and posthatching day 3 (P3) of chicken development. Heavy autoradiographic labelling was observed at the classical NGF target sites, the proximal cranial sensory ganglia and the sympathetic superior cervical ganglion, throughout development and after hatching. In contrast, only weak labelling could be detected during a restricted time span in the vestibulocochlear (E4-E8) and the distal cranial sensory ganglia (E4-E10), the neurons of which originate from the otic and epibranchial placodes. Specific 125I beta NGF binding was also observed in various brain regions during early brain development. NGF receptor expression there followed a characteristic pattern. The neuroepithelial layer displayed very low levels of specific 125I beta NGF binding, while strong 125I beta NGF labelling was found in the mantle layer. Brainstem somatomotor nuclei, visceromotor columns, brainstem alar plate, cerebellar anlage, tectum, and basal forebrain (epithalamus, striatum) were found to be transiently labelled by 125I beta NGF in early development (E4-E12). Non-nervous tissues such as parts of the otic vesicle epithelium and skeletal muscle anlagen of the head were also labelled. These results, showing specific binding of 125I beta NGF to cranial cells of different origin (neural tube, neural crest, placode, and possibly mesoderm) strengthen the concept that NGF may have diverse functions in growth and differentiation of various tissues and cell types.

Morphogenesis of the human gluteus maximus muscle arising from two muscle primordia

In human embryos and fetuses a supernumerary muscle was found situated on the distal margin of the gluteus maximus muscle and supplied by the most distal main branch of the inferior gluteus nerve. According to its origin and insertion it is being named the coccygeofemoralis muscle. In embryos and fetuses of up to 40 mm in CR length the coccygeofemoralis muscle is separated by loose connective tissue from neighbouring fetal muscles. Later on, close contact between the coccygeofemoralis and the distal margin of the fetal gluteus maximus muscle develops, and during the prenatal period both fetal muscles gradually fuse. Postnatally, the coccygeofemoralis muscle is incorporated into the gluteus maximus muscle of which the pars sacroiliaca corresponds to the fetal gluteus maximus itself and the pars coccygea represents the fetal coccygeofemoralis muscle. With respect to the general process of muscle morphogenesis, the developmental pattern described for the gluteus maximus muscle demonstrates that adult muscles may be formed by a fusion of several fetal muscles.

Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.

Development of the vascular supply in rat skeletal muscles.

The vascular development in the extensor muscles of rat forelegs from 12-day-old embryos up to 9-day-old animals was investigated with a light microscope using semithin plastic sections. On late day 12, blood vessels are distributed over the whole cross sectional area of the foreleg. On early day 13, a rearrangement of the blood vessels is visible. Now an avascular centre is enclosed by a vascularized tissue mantle, in which the muscle anlage starts developing. Few capillaries are visible within the muscle anlage. After differentiation of the muscle anlage into individual muscles the epimysium is passed by a netlike capillary plexus enwrapping the first avascular muscles. With the establishment of the perimysium on day 16 of embryonic development some capillaries are also visible within the individual muscles. The diameter of the capillaries is by far larger than the diameter of the myotubes. In older embryos the perimysium shows an increasing number of such large capillaries, whereas in the epimysium the capillaries are less frequent. In the perimysium a few additional capillaries with a small diameter of about 5 micron occur. On day 3 postpartum, small capillaries are also visible within the muscle fibre bundles. In the perimysium many large capillaries are still obvious. The vascular supply on day 9 postpartum, resembles that in adult muscles. The vascular supply of the muscles progresses from their periphery to more and more smaller compartments within the muscles, in close correlation to the establishment of the muscular connective tissue.

The striated urogenital sphincter muscle in the female

Striated muscle associated with the female urethra and vagina constitute a continuous mass which appropriately may be called the urogenital sphincter. Though continuous, the muscle may be separated into two parts--one that surrounds the urethra, and the other surrounding the urethra and vagina. The individual muscle fibers are small and are embedded in connective tissue and infiltrated with smooth muscle which obscures the visibility of the muscle to gross dissection. Developmentally the muscle primordium is laid down around the urogenital sinus and urethra early, and foreshadows the anatomical arrangement that is maintained in the adult with little change. The urogenital sphincter muscle extends from the base of the bladder where it lies within the pelvic cavity and continues through the urogenital hiatus of the pelvic diaphragm to expand around the vagina in the perineum. Additional fibers attach to the ischiopubic rami and constitute a compressor of the urethra. As a result there is no superior fascia of the so-called "urogenital diaphragm" which closes off a deep perineal compartment or forms a floor of the urogenital hiatus.

On the origin and development of the ventrolateral abdominal muscles in the avian embryo. An experimental and ultrastructural study.

In avian embryos the formation of ventrolateral abdominal muscles was studied by (1) heterospecific grafting experiments between chick and quail embryos and (2) ultrastructural examinations of cells having part in this process. The results demonstrate that the muscle cells are of somitic origin while the connective tissue derives from the somatopleure. Somatopleural cells do not differentiate into myocytes, and somite cells which have entered the ventrolateral abdominal wall, do not contribute to the connective tissue. It is concluded that both dermatome and myotome cells undergo muscular differentiation. The formation of muscles is found to take place in four characteristic steps. During the 4th day of development, epithelially structured ventral somite buds enter the somatopleure. The light cells of the inner myotome layer are elongated in a cranio-caudal direction and contain randomly distributed microfilaments. On the 5th day, the buds lose their epithelial arrangement and change into compact processes in which cells intermingle. The myotome cells show short bundles of thin and thick microfilaments. The third step can be characterized by the appearance of intercellular spaces and the disaggregation of processes becoming invaded by somatopleural cells. Thus, subdivision in single muscle blastemata begins to occur. In 7-day embryos, the muscle anlagen are distinctly separated and the first myotubes containing regularly arranged myofibrils are found. Coincidentally, signs of cell death are observed. Up to the 10th day, the tendons being of somatopleural origin become plainly outlined and the muscle anlagen move to their definitive positions. It is assumed that the formation of muscle pattern is controlled by the somatopleure.

Temporal and spatial distribution of fibronectin during development of the embryonic chick limb bud

Indirect immunofluorescence has been used to study the distribution of fibronectin during the course of embryonic chick limb morphogenesis and differentiation. At all stages of development from 19 through 25, fibronectin is distributed throughout the non-differentiating mesenchymal tissue directly subjacent to the apical ectodermal ridge (AER), i.e. the mesenchyme extending 0.15 mm or so from the AER. Fibronectin is also distributed throughout the proximal condensing central core of the limb during the early stages of cartilage differentiation. In fact, fibronectin persists as a major component of the intercellular matrix in the central core of the limb following overt chondrogenic differentiation, since it is present as late as stage 27 throughout the well-differentiated cartilage rudiments of the radius, ulna, and humerus. The detection of fibronectin in differentiated cartilage is facilitated by pre-treatment of the sections with testicular hyaluronidase prior to immunofluorescent staining. In contrast to the chondrogenic central core of the limb, in the peripheral dorsal and ventral (myogenic) regions in which muscle differentiation is progressing, there is a progressive and striking diminution in fibronectin staining. By stage 27, little, if any, is present in the well-differentiated muscle primordia. Finally, at all stages of development, there is an accumulation of fibronectin at the ectodermal—mesenchymal interface, suggesting it is a component of embryonic limb basement membranes. On the basis of these observations, the possible role of fibronectin in limb cartilage and muscle differentiation and in other aspects of limb morphogenesis is discussed.

Muscle morphogenesis: Evidence for an organizing function of exogenous fibronectin

Chicken myoblasts and myotubes attach to, and elongate upon, solid substrates only in the presence of exogenous fibronectin derived from, for example, serum or fibroblasts ( M. Chiquet, E. C. Puri, and D. C. Turner, 1979, J. Biol. Chem. 254, 5475–5482). We have sought answers to two questions: (1) whether myoblasts depend on exogenous fibronectin for attachment because they themselves synthesize none or too little of this glycoprotein; and (2) whether interaction of myogenic cells with fibroblast-derived fibronectin might guide muscle morphogenesis. Under conditions in which fibroblasts synthesized fibronectin and released it into the medium, myoblasts and myotubes synthesized and accumulated only small amounts of fibronectin. Although proliferating myogenic cells treated with 5-bromodeoxyuridine accumulate fibronectin on their surfaces, other evidence suggests that normal myogenic precursor cells, like myoblasts and myotubes, synthesize little fibronectin: (1) the accumulation of newly synthesized fibronectin in untreated primary myogenic cultures is attributable, at least in part, to the fibroblasts present; and (2) 1-week-old myogenic clones fail to form a fibronectin network, in contrast to fibrogenic clones of the same age and cell density. In cryosections of chick embryos, less fibronectin was found in the premuscle masses than in the surrounding mesenchyme. The splitting of the muscle anlagen in the limb bud was correlated with the appearance of fibronectin-accumulating cells in the cleavage furrows. In vitro, myoblasts displayed a marked tendency to align themselves along oriented fibrils or streaks of purified fibronectin that had been deposited on the substrate; this myoblast alignment influenced shape and branching of the developing myotubes. These results suggest that formation of myotubes in a certain spatial arrangement during muscle morphogenesis may be regulated by a fibronectin-containing matrix produced by connective tissue cells.

Differentiation of the avian latissimus dorsi primordium: Analysis of fiber type expression using the myosin ATPase histochemical reaction

AbstractTypically, fast twitch fibers of the adult avian posterior latissimus dorsi (PLD) muscle express alkali stable myosin ATPase histochemical activity, whereas slow tonic fibers of the anterior latissimus dorsi (ALD) muscle exhibit dual acid and alkali stability. Since PLD fibers cross‐reinnervated by the nerve of the ALD muscle also demonstrate dual stability, we sought to determine whether the ALD nerve induces this new phenotype de novo or de‐represses an embryonic phenotype repressed during further development in ovo. Chick embryos from Day 2 in ovo onward were frozen in toto, and the development of myosin ATPase histochemical profiles following alkali and acid preincubation was determined for myo‐genic cells destined to form the ALD and PLD muscles. In addition, the silver cholinesterase reaction was employed on serial sections to investigate early muscle‐nerve interaction in ovo. Our results indicate that all myogenic cells of the primary dorsal muscle mass, including those of the future ALD and PLD muscles, demonstrate their first myosin ATPase response at Stage (St) 25. At this time (4.5 days in ovo), only alkali stable enzymic activity is expressed. By St 28 (5.5 days) a distinct latissimus dorsi (LD) primordium has cleaved from the dorsal muscle mass and the entire primordium still exhibits only alkali stability. As development proceeds and the LD primordium separates into recognizable anterior and posterior regions (St 29, 6.0 days through St 30, 6.5 days), the future PLD muscle continues to express only alkali stability, whereas the future ALD muscle now demonstrates dual stability. By St 32 (7.5 days), these two muscles are completely separated, and during the remainder of in ovo development the profiles characteristic of adult slow tonic versus fast twitch fibers remain unaltered. Thus, our results indicate that, during cross‐reinnervation ex ovo, the ALD nerve induces the expression of a new phenotype never exhibited by potential fast twitch PLD fibers during the entire embryonic period, including their incipient formation. Moreover, our results indicate that the histochemical reaction for myosin ATPase activity provides an excellent enzymic marker both for the identification of myogenic cells and individual muscle primordia, and, for the detection of muscle fiber type differentiation during the first week of development in ovo.

The urethral sphincter muscle in the male.

The male urethral sphincter is a striated muscle in contact with the urethra from the base of the bladder to the perineal membrane. The individual muscle fibers are 25 to 30% smaller than fibers of associated muscles and are embedded in connective tissue which obscures the visibility of the whole muscle. The muscle primordium is laid down around the urethra prior to the development of the prostate. Subsequently, the prostate develops as a diverticulum of the urethra and grows into the developing sphincter, thinning the overlying musculature. With the onset of puberty, accelerated growth of the prostate displaces the sphincter, with atrophy of the overlying muscle, resulting in what may appear to be isolated segments of the sphincter muscle distributed around the prostate. The prostate overgrows the anterior portion of the urethra and the associated sphincter muscle. There is no distinct superior fascia of the so-called urogenital diaphragm separating the sphincter muscle from the prostate. The fascia of the sphincter muscle is inseparable from the prostatic sheath, is oriented vertically, and passes through the urogenital hiatus to unite with the fascia of the pudendal canals at the isochiopubic rami. Thus, the sphincter muscle is a component of a bladder-urethra-prostate-sphincter unit which lies within the pelvis, in the urogenital hiatus, and rests upon the perineal membrane. The concept of a urogenital diaphragm is not borne out by this study.

A Sequential Ranking System for Developmental Stages of an Acanthocephalan, Leptorhynchoides thecatus, in Its Intermediate Host, Hyalella azteca

Based on morphological features, 22 stages were defined in the development of Leptorhyn- choides thecatus in its intermediate host, Hyalella azteca. The ranking system allows close comparison of development and growth within or among amphipods, and is amenable to nonparametric statistics. Certain aspects of L. thecatus development are redescribed. Apical nuclei arise from the central, nuclear mass, rather than a cortical nucleus. Lateral, nucleated bands, previously thought to be somatic muscle anlagen, are actually the median longitudinal canals. The body cavity develops in a single step as muscles of the body wall and the median longitudinal canals separate from the proboscis receptacle and genital strand. Developmental anomalies (short, conical proboscis, misaligned hooks, and ventrally shortened trunk) were observed and are thought to be induced by high temperatures. Many factors, such as temperature, rearing conditions, and intraspecific competition, may affect size or developmental rates of parasites. These effects can be identified and character- ized only if developmentally equivalent in- dividuals can be recognized and ranked ac- cording to their developmental condition without regard to their chronological age. Pre- vious descriptions of acanthocephalan devel- opmental stages (Meyer, 1931; Kates, 1943; Van Cleave, 1947; Butterworth, 1969) recog- nized no more than nine ranks and were based on acquisition of major structural fea- tures (e.g., body cavity). They were not suf- ficiently sensitive to changes within these structures for close comparisons. Develop- mental descriptions are usually based on time-specific morphology. Individual varia- tion cannot be quantified, and effects of ex- perimental manipulation cannot be simply or precisely described. A morphologically based, sequential ranking system is desirable. DeGiusti (1949) provided a well-illustrated, comprehensive account of day-to-day changes during development of Leptorhynchoides thecatus in its intermediate host, Hyalella az- teca. Based on this description and examina- tion of many additional specimens, we devel- oped a ranking system which recognizes 22 stages. Each stage is defined by morphologi- cal features, and is assigned a rank number.