Redneck, a new mutant of the axolotl (Ambystoma mexicanum) likely affects the development of cranial neural crest.

A novel developmental mutant in the Mexican axolotl is described. Designated redneck (rn), the mutant gene is inherited as a simple Mendelian recessive. In homozygotes, rn causes massive haemorrhage in the posterior head, rostrocaudal compression of the craniovisceral skeleton, abnormal differentiation of vertebral cartilage, micrognathia, aglossia, microphthalmia and abnormal hepatic development. Affected larvae become evident at the onset of feeding, and eventually die of starvation. Based on the tissues affected, we propose that rn affects later developmental events in the differentiation and morphogenesis of a subset of cranial neural crest cells. Thus, rn may prove a valuable model system for examining the role of neural crest cells in the development of cranial and endodermal derivatives.

The cardiac neural crest in Ambystoma mexicanum.

To establish whether a region of the cranial neural crest contributes cells to the developing heart of Ambystoma mexicanum (axolotl), as it does in many other vertebrates, we constructed a fate map for the neural crest in late neurula stage (stage 19-20) embryos. The fluorescent vital dye, Dil, was used as the lineage label. The various regions of the cranial neural folds were identified in relation to such landmarks as the developing forebrain, midbrain and hindbrain, and the appearance and extent of emerging somites. Labelled cells originating in the rhombencephalic region were found in the aortic arches and in the truncus arteriosus, and occasionally in the walls of the conus arteriosus. Cells were also found in the third and fourth branchial arches. Labelled neural crest from the adjacent anterior trunk region appeared neither in the heart nor the visceral skeleton, whereas those from the mesencephalic region contributed to the first hypobranchial cartilage and to the first three branchial arches, but not to the heart. No labelled cells from any of the regions were seen in the ventricle or auricle.

Heart development and regeneration in urodeles.

Classical fate mapping and transplantation studies have yielded a rich embryological understanding of heart development in urodeles. Recent advances in understanding the molecular nature of many early developmental events can be applied to urodele heart development. In this review we examine urodele heart development from both morphological and molecular viewpoints. We focus primarily on cardiac induction, early cardiogenesis, and heart regeneration.

Development of an attitude scale to measure attitudes toward humans' use of nonhuman animals.

The objectives of this investigation were to develop a scale to measure attitudes towards animal use, decide upon the number of dimensions or scales needed, and test the reliability and validity of the dimension(s) obtained. Analysis showed the final scale to be unidimensional with evidence of reliability (Cronbach alpha = .99) and criterion validity via measurement of known groups. The known groups were derived from a random sample of members of the Animal Rights Information and Education Service (representing those opposed to humans' use of animals) and members of National Animal Damage Control Association (representing those supporting animal use). The final scale contained 48 items and was readable at Grade 6 which facilitates administration across a broad range of educational backgrounds and has utility for a variety of animal-use issues.

Heart specification in the Mexican axolotl (Ambystoma mexicanum).

The concept of the morphogenetic field has been used extensively in developmental biology. However, little is known about the mechanisms that partition these broad areas of tissue into the smaller areas which actually form the corresponding structures, and the remaining tissue. In the Mexican axolotl, the heart field forms as the anterior lateral plate mesoderm migrates over the underlying pharyngeal endoderm between stages 14 and 28. We have previously shown that both the mid-ventral and lateral walls of the pharyngeal cavity have considerable inductive capacity at stage 14. If this inductive capability, and the competence of the mesoderm to respond, is retained between stages 14 and 28, a much broader area of mesoderm would be induced than actually participates in heart development. In this paper, we use explant cultures to establish that pharyngeal endoderm retains its inductive activity, and that both pre-cardiac mesoderm and lateral plate mesoderm caudal to the pharyngeal cavity remain competent to respond to the induction throughout this period. We also map the specified region of the antero-lateral mesoderm between stages 14 and 28 by placing carefully measured areas of mesoderm in culture without inductive endoderm. We found that the region capable of initiating a spontaneous beat approximately doubles in size during this period. Since the specified region is larger than the actual heart primordium, some mechanism must exist to partition "induced" mesoderm into heart-forming and non-heart-forming areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Computations of post-inductive dynamics in axolotl heart formation.

This paper reports modelling of heart localization in the axolotl (Ambystoma mexicanum). The region of heart specification in the mesoderm defined by classical induction from the endoderm is larger than the area of final myocardial differentiation. For localizing the area of differentiation within the area of specification, we postulate a reaction-diffusion system that arises within the mesoderm in response to induction from the endoderm. This mechanism generates a spatial pattern for two chemicals, an activator and an inhibitor, corresponding to the area of myocardial differentiation. We postulate a diffusible chemical rescuer, which is absent in the cardiac lethal mutant, and which is a precursor to the reaction-diffusion mechanism. The activator, inhibitor, rescuer, and product of endodermal induction are presented in an enzyme mechanism with rate equations similar to the Gierer-Meinhardt equations. These equations were solved numerically in both one and two spatial dimensions. We have attained quantitative agreement with the experimental data for sizes of tissue regions and for times to heartbeat. Experiments modelled include wild-type heart localization as well as both in vitro and in vivo rescue of cardiac lethal mesoderm with wild-type mesoderm. Based upon the parameters necessary to model heart localization, we make a series of predictions. We predict: a specific profile for the endodermal inducer gradient; the possibility of producing multiple hearts in vivo; and a greater contribution to the heart from the wild-type mesoderm for in vivo transplants with cardiac lethal mesoderm. We make some suggestions as to the possible chemical nature of the substances in the model. We indicate that the inhibitory field and mechanochemical theories are probably not as promising as reaction-diffusion for the mechanism of heart localization.

Reaction-diffusion control of heart development: evidence for activation and inhibition in precardiac mesoderm.

In axolotl embryos homozygous for the cardiac-lethal (c) gene, the heart never begins to beat. Recently, we demonstrated that the c gene affects the heart mesoderm directly, making the latter incapable of responding to normal inductive stimuli. Based on these results, we proposed that a reaction-diffusion mechanism controls the later stages of vertebrate cardiogenesis. In the present study, we use a series of transplantations to examine the precardiac mesoderm of wild-type and cardiac-lethal mutant axolotl embryos for the activation and inhibition predicted by our model. When one or both heart primordia in c/c embryos were replaced with +/+ tissue at early stages, functional hearts developed, indicating that an activator-like activity is present in wild-type heart mesoderm. At later stages, replacement of the entire heart-field mesoderm also rescued mutant embryos, but replacement of only the heart-forming mesoderm did not, indicating that the mutant heart field has inhibitory properties. These results demonstrate that activator- and inhibitor-like factors are present in pre-cardiac mesoderm and thus provide evidence for reaction-diffusion control of postinductive heart development.

Heart development in normal and cardiac-lethal mutant axolotls: a model for the control of vertebrate cardiogenesis.

The mechanisms which regulate myocardial differentiation are poorly understood. The cardiac-lethal (c) mutant of Ambystoma mexicanum, in which the heart never begins to beat, provides a valuable model system for studying this process. Using an in vitro assay, we examine the nature of the defect in c/c embryos and find (contrary to previous reports) that the inductive endoderm is not affected by the mutation. Rather, the pre-cardiac mesoderm is directly affected by the c gene and is incapable of responding to normal inductive influences. Furthermore, we find that mutant mesoderm can complete its differentiation into functional cardiomyocytes when co-cultured with wild-type heart mesoderm. With this evidence, we propose a model for the regulation of heart differentiation based on the migration of the heart mesoderm over a gradient of inducer, and the subsequent establishment of a two-component reaction-diffusion system within the mesoderm itself. This model has the potential to explain several poorly understood aspects of cardiogenesis, including the gradual nature of heart induction, the restriction of the heart field, and possibly the early morphogenesis of the heart tube.

Ectopic expression of a genomic fragment containing a homeobox causes neural defects in the axolotl.

An axolotl (Ambystoma mexicanum) genomic fragment containing the Ahox1 homeobox was placed under the control of the mouse hsp68 promoter, which seems to function constitutively in the axolotl. The resulting construct was injected into fertilized axolotl eggs to see if it would perturb development. Of the injected embryos, 20% showed severe reduction of the anterior neural plate. Later in development, these embryos had small heads, no eyes, and appeared to lack the normal regionalization of the brain. An additional 35% of the embryos were less severely affected, but had reduced or missing eyes. Control embryos, including ones injected with a construct missing the DNA recognition helix of the homeobox, developed normally.

Heart induction in wild-type and cardiac mutant axolotls (Ambystoma mexicanum).

We have re-examined some of the factors affecting the induction of heart-forming mesoderm in the axolotl. The formation of functional, rhythmically contracting myocardial tissue was used as an assay. We have found that heart-forming mesoderm is fully induced and capable of completing its developmental repertoire by the end of neurulation. As has been previously reported, pharyngeal endoderm appears to be the major inductor of heart mesoderm. Unlike previous workers, we have found that the inducing activity appears to be highly localized in the mid-ventral pharyngeal endoderm. The endoderm retains its inductive properties, and the mesoderm retains at least some capacity to respond, long after the heart-forming mesoderm is apparently fully induced. We have also found that RNA extracts from pharyngeal endoderm, which are capable of causing cardiac-lethal (c/c) mutant axolotl hearts to begin beating, are not capable of inducing early wild-type heart-forming mesoderm. Based on these results, we speculate that induction of heart-forming mesoderm is a two-step process. The first signal, occurring during neurulation, directs the mesoderm to begin differentiating into cardiomyocytes, and the second, beginning in mid- to late neurulation and continuing until just prior to the onset of heartbeat, causes myofibrillogenesis and the initiation of rhythmic contractions. The latter signal, which is lacking in c/c mutant embryos, appears to be necessary to override an inhibition present in the embryonic milieu.

Isolation and characterization of a developmentally regulated homeobox sequence in the Mexican axolotl Ambystoma mexicanum.

A homeobox-containing genomic DNA fragment was isolated from the Mexican axolotl. This clone was obtained from a partial genomic library enriched for sequences that cross-hybridized with the Drosophila Antp homeobox under low stringency hybridization conditions. DNA sequence analysis revealed that this sequence (Ahox1) was 66% homologous to the Antp homeobox sequence and was most closely related to the mouse Hox-1.6 (84% identity) and Drosophila lab (79% identity) homeobox sequences. Several cross-hybridizing fragments to Ahox1 were detected in both mouse and axolotl genomic DNA. This sequence was also shown to be conserved in other Ambystoma species. Northern blot analysis revealed that genes containing this sequence are developmentally regulated. Transcripts hybridizing to the Ahox1 homeobox probe were detected during the neurula and tail bud stages of development.

Development of the mechanoreceptive lateral-line system in the axolotl: placode specification, guidance of migration, and the origin of neuromast polarity.

The mechanosensory lateral-line system offers a unique opportunity to study a wide variety of developmental phenomena, including cell migration, the origin of polarity, and pattern formation. In this study, we use a series of transplantation experiments to examine some of the factors affecting the origin of the lateral-line placodes, the establishment of sensory organ polarity and placement, and the guidance of cell migration in the Mexican axolotl (Ambystoma mexicanum). We find that placode-forming ectoderm is at least partially specified as early as the beginning of neurulation, and we suggest that this may be a result of early processes involved in neural induction. Furthermore, we find that the migration of the primordia on the body depends on the presence of both the ectoderm and the subjacent mesoderm for guidance. Sensory organ polarity on the body appears to be the result of an interaction between the primordia, which deposit organs of set polarity relative to the direction of migration, and the substrate, which determines the direction of migration. Spacing of the organs is independent of the substrate, and may be due to an intrinsic property of either the primordia or the emerging organs themselves. Finally, we suggest that the lateral-line primordia are guided, as they migrate, by a contact guidance mechanism.

An apparatus to retain the spatial orientation of breast biopsies.

An apparatus has been designed to retain the spatial orientation of breast biopsies throughout surgery, transport and processing in the pathology laboratory. It does not interfere with regular surgical procedures and simplifies the surgeon's task of marking the orientation of a specimen. As the use of the apparatus does not involve either chemical or physical substances it is an appropriate handling procedure for those specimens requiring sensitive testing procedures such as cell culture.

The unstable urethra in the female.

The clinical and urodynamic characteristics of 54 women with unstable urethras (falls of urethral pressures of at least 20 cm H2O) were compared with those of 171 patients with stable urethras. The unstable-urethra patients were younger and presented with an increased incidence of marked urgency and a lower incidence of genuine stress incontinence. The average urethral pressure fall was preceded by a 6-cm increase in maximum urethral pressure. The mean (+/- SD) fall of urethral pressure was 30 +/- 12 cm H2O and the average duration of the event was 8 seconds. The level of the urethral pressure fall was not influenced by bladder volume but correlated positively with higher maximum urethral pressures. The present findings suggest that the unstable urethra plays a limited role in genuine stress incontinence but may be linked with the unstable detrusor.

A rapid method for observing the internal morphology of amphibian embryos.

A simple, rapid method for visualizing the internal morphology of amphibian embryos is described. Fixed embryos of Ambystoma mexicanum are surrounded with commercial embedding material and are frozen. Internal structure is revealed by cutting the embryos with a cooled scalpel or razor blade, removing the frozen embedment with an aqueous buffer, and processing the halved embryos for scanning electron microscopy (SEM). This technique allows the internal anatomy of the embryo to be viewed but is much simpler and more rapid than previously described techniques. It should therefore prove useful for understanding and teaching the three-dimensional relationship between tissues in developing embryos. In addition, we believe that this technique could be used, with minor changes, as a rapid method for viewing the internal morphology of a variety of specimens.

Estrogen receptor distribution in the peripheral, intermediate and central regions of breast cancers.

The central, intermediate and peripheral regions of 25 breast tumors were analyzed for estrogen receptors (ER) using a combined biochemical (BC)/immunohistochemical (IHC) micromethod. To optimize the regional comparison, the percentage carcinoma per sample (PCS) was evaluated and incorporated into the quantification of the ER. Correction of the measured ER for the PCS eliminated differences in receptor levels between the central and peripheral regions but not the intermediate region. Although the corrected BC-ER level in this region was found to be about 20% higher, the IHC method did not detect such a difference. Determination of the ER status at the actual growth front of a tumor, rather than in the intermediate region where ER levels appear to be highest, may be of greater clinical relevance. Intra-regional ER heterogeneity appears, however, to be particularly pronounced in this area of a tumor.

Compartmentalization of motor units in the cat neck muscle, biventer cervicis.

1. The neck muscle biventer cervicis is supplied by five separate nerve bundles that originate from segments C2-C5 and enter the muscle at different rostrocaudal levels. We have used the glycogen-depletion method to investigate the distribution of muscle fibers supplied by each nerve bundle and also the extent of motor-unit territories supplied by single motoneurons in the C3 segment. 2. Prolonged intermittent stimulation of each nerve bundle produced glycogen depletion in a compartment of muscle fibers that ran only a fraction of the whole-muscle length. The depleted compartment was separated by tendinous inscriptions from adjacent, serially arranged compartments that were supplied by different nerve bundles. Thus the muscle was divided into five in-series compartments, arranged in the same rostrocaudal sequence as the nerves by which they were supplied. 3. Six fast, glycolytic (FG) and five fast, oxidative-glycolytic (FOG) motor units were depleted by repetitive intracellular stimulation of their antidromically identified motoneurons in the C3 segment. The fibers of each motor unit were confined to a striplike subvolume whose cross-sectional area was only 20-40% of that for the whole compartment in which it was located. Single motor units contained an average of 408 extrafusal fibers (range: 262-582 fibers), and these were distributed with an average density of 20 fibers/mm2 in cross sections through their motor domains. No significant differences were found between the numbers or densities of fibers in FG and FOG motor units. 4. The specialized in-series organization of compartments has functional implications because the forces generated by one compartment of motor units must be transmitted through other in-series compartments of muscle fibers rather than directly onto skeletal attachments. The confined distribution of muscle fibers belonging to a single motor unit suggests that an additional level of organization may exist within individual compartments. The implications of these features for the physiological behavior and neural control of biventer cervicis are discussed.

Fiber architecture and histochemistry in the cat neck muscle, biventer cervicis.

1. Biventer cervicis (BC) is an anatomically complex muscle that is divided by tendinous inscriptions into five in-series compartments of motor units. We have analyzed the fiber architecture and fiber-type composition of these different compartments using microdissection and histochemical methods. 2. BC narrows as it runs rostrally, but its in-series compartments have similar cross-sectional areas. The tapered shape of BC comes about because tendinous inscriptions and the tendon of insertion are oriented obliquely and muscle fibers attach in a progressively offset fashion from the medial to the lateral muscle edge. 3. Individual compartments of BC differ from one another in their architecture. The rostral two compartments (1 and 2) contain fibers of similar length that run between two plates of tendinous tissue. Compartments 3 and 4 are divided into two or three in-parallel subvolumes whose fiber bundles differ in their lengths and sites of attachment. Compartment 5 is the most variable in its structure. In some cats it is separated from compartment 4 by a tendinous inscription, but in other cats, it blends with a dorsomedial part of compartment 4 to form a single subvolume. 4. The relative lengths of fibers in different compartments were analyzed when the head and neck were held in different postures. Fibers in rostromedial regions were stretched more effectively when the head was flexed at suboccipital joints, and appeared to be less sensitive to movements at lower cervical joints. Movements across lower cervical joints produced substantial length changes in caudolateral parts of BC. 5. Muscle fibers of different histochemical types were not distributed evenly within each muscle compartment. Slow, oxidative (SO) fibers accounted for the majority of fibers near the nuchal midline but for only 30%-45% of fibers in lateral muscle regions. Proportions of fast, glycolytic (FG) fibers were greatest in lateral regions. Fast, oxidative-glycolytic (FOG) fibers were distributed quite uniformly throughout each compartment. 6. The specialized architecture of BC may shape its physiological capabilities. The complex internal structures of different compartments may alter the length-tension properties of BC.(ABSTRACT TRUNCATED AT 400 WORDS)

Progressive patterning precedes somite segmentation in the Mexican axolotl (Ambystoma mexicanum).

Beginning at mid-neurulation, a wave of somite segmentation passes down the axolotl body axis in a cephalocaudal direction. At 20 degrees C a somite forms every 2.57 hr. Fate-mapping of the presomitic mesoderm indicates that the primordia for the next few somites occupy nearly the same space that they will after segmentation, but that the remaining somites are densely packed in tip of the tail bud. Brief heat shocks at 37 and 38.5 degrees C reveal that within the first of these two zones, there is a graded sensitivity to the shock, with the primordia closest to the last-formed somite showing the greatest resistance. However, primordia within the densely packed tip (the packing zone) also appear resistant, or have sufficient time to repair the damage. We propose that once cells have left the packing zone, they undergo progressive patterning which renders them increasingly insensitive to the disruptive effects of heat shock, and culminates in rosette formation.