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In this report, we describe the dysmorphologic phenotype associated with the transgenic insertional mutation legless. This autosomal recessive, perinatally lethal mutation results in an interesting pleiotropic array of congenital malformations. The phenotype of the legless mutation in homozygous perinatal mutants is compared to wild-type nontransgenic and heterozygous siblings. Skeletal, craniofacial, and visceral malformations are characterized. We have observed by skeletal analysis a consistent loss of distal hindlimb structures, as well as the loss of distal forelimb structures with a predilection for the preaxial side of the developing forelimb. Craniofacial malformations commonly observed appear to represent a range of severity of affect, with the mildest manifestation evident as apparently shallow lateral clefts of the upper lip and mild midfacial clefts accompanied by clefts of the secondary palate. At the severe end of the spectrum, the midline clefts of the face (and secondary palate) are very wide, with obvious accompanying frontonasal encephaloceles and overt lateral clefts of the upper lip. Examination of the mutant brain has demonstrated marked defects in the anterior structures, particularly the olfactory lobes and cerebrum, in greater than 90% of the brains studied. Observation of the internal viscera has identified transposition of thoracic and abdominal organs in approximately 50% of the mutant offspring. The limb, head, and visceral defects were not observed in the wild-type nontransgenic or heterozygous siblings. Transgenic insertional mutations leading to congenital malformations are useful because the transgene sequence may serve as a tag to facilitate molecular retrieval. Analysis of the flanking DNA sequences will allow the identification of the interrupted gene. A complete description of the mutant phenotype will assist in the understanding of this genetic locus.

Cordarone was examined for its effects on alpha- and beta-adrenergic receptors and adenylate cyclase (AC) of some tissues. Cordarone was shown to suppress the binding of [3H]-clonidine with alpha 2-receptors of the rabbit brain (Ki = 4 microM) and that of [3H]-prazosin with alpha 1-receptors of the rat liver (Ki = 22 microK), but not to displace [3H]-dihydroalprenolol from rabbit cardiac and pulmonary beta 1-receptors and from beta 2-receptors of rat reticulocytes and human lungs. Cordarone failed to affect the activity of rabbit heart and lung AC, as well as that of thrombocytes and human lungs, but showed a 80% inhibition of the activating effect of isoproterenol on reticulocyte AC. It was suggested that the effect of cordarone on reticulocytes was not mediated by beta-receptors and was tissue specific and dependent on the characteristics of AC regulation in these cells. Cordarone's antiadrenergic effect found in vitro may be one of the causes of its coronary dilating and antiarrhythmic effects.

In this work we separated homogeneous embryoid bodies (EB); this population is composed of only endodermal vesicles of a visceral type. These EB were used to immunize rabbits. After absorption, the antisera were analyzed immunohistochemically in sections of 7.5- and 9-day-old mouse embryos, which showed a selective reaction to visceral endoderm. The cytodifferentiation of the visceral endoderm was studied.

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.

Hoechst 33342 was injected either intravenously or intraperitoneally into mice which were killed 1 or 24 hr or 7, 14 or 28 days later. Various organs were fixed and paraffin embedded. Visual inspection showed that independently of the route of dye administration or survival time, distinct fluorescence of nuclei was observed in organs other than cerebral cortex. Even formic acid decalcification of bone failed to abolish the fluorescence of osteocytes. In vivo staining with Hoechst 33342 is proposed as an alternative for staining after sectioning. Cells from spleens of Hoechst 33342-injected mice or stained in vitro were injected intramuscularly into mice. Hoechst 33342-stained splenocytes could be found in deparaffinized sections at the site of injection 24 hr later.

The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.

Slow outward currents were recorded from voltage-clamped neurones in nodose ganglia excised from rabbits. In the majority of Type C neurones, a short depolarizing command pulse evoked a slow outward tail current (ISAH) with a decay time constant ranging from 0.5 to 2 s. The ISAH was due to an increase in membrane conductance to K+ because its reversal potential was approximately equal to the Nernst potential for K+. The ISAH was reversibly blocked by removal of external Ca2+ or by Ca2+ antagonists. A Ca2+ ionophore, A23187, produced an outward current which was similar to the ISAH. The ISAH was resistant to tetraethylammonium and depressed by Ba2+, whereas it was not affected by Cs+ and 4-aminopyridine. The ISAH was initially augmented and subsequently depressed by apamin (1-10 nM) and (+)-tubocurarine (100-600 microM). It is concluded that the ISAH in visceral primary neurones may be due to a long-lasting increase in K+ conductance caused by an increase in the concentration of intracellular Ca2+, resulting from Ca2+ entry during the depolarizing command pulse.

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.